Effect of Tramadol on Rohu (Labeo rohita); A Histopathological and Hematological study

Effect of Tramadol on Rohu (Labeo rohita); A Histopathological and Hematological study | 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 Effect of Tramadol on Rohu (Labeo rohita); A Histopathological and Hematological study Nadeem Bukhash, Muhammad Alfahad, Muhammad Shoaib, Mishal Zahra, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8150296/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 Tramadol is a weak synthetic opioid primarily used to treat moderate to severe pain. It can persist in the aquatic environment through wastewater, posing a significant environmental threat. As a result, aquatic organisms, particularly fish, face the risk of toxicological effects. This study aimed to investigate the toxicological impacts of tramadol on the hematology, biochemistry, and histology of Rohu ( Labeo rohita ). A total of one hundred and fifty healthy fingerlings of the freshwater fish L. rohita with a total mean length of 20 ± 5 cm and weight of 14 ± 0.5 g was used for this research. The 48-hour exposure rates of L. rohita exposed to tramadol were 1.25 mg/L for low-dose and 2.5 mg/L for high-dose. The hematological analysis showed increased WBCs, MCV, RDW, RDW-SD, PLT, PDW, neutrophils, lymphocytes, monocytes, and eosinocytes. In contrast, Hgb, RBC, HCT, MCH, MCHC, and MPV decreased significantly. Biochemical analysis showed an increase in cholesterol, triglycerides, HDL, ALT, AST, total protein, glucose, T4, TSH, urea, BUN, and creatinine, while others like LDL, VLDL, albumin, globulin, and T3 were decreased. Fish exposed to tramadol showed some prominent abnormalities, including abnormal blood congestion, bone cell deformities, lamellar aneurism, edema, fusion of primary lamellae, hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, necrosis, melanomacrophages, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion with increments in concentrations and treatment intervals, except for the untreated group. The findings demonstrate that tramadol is toxic to fish, therefore its presence in aquatic ecosystems should be examined for non-target organisms. Rohu (Labeo rohita) Toxicology Histology Hematology Tramadol Pollutants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Pharmaceutical pollutants, especially analgesics like tramadol, are 2-(dimethyl amino)-methyl-1-(3-methoxyphenyl) cyclohexanol hydrochloride. It is the 4-phenyl-piperidine analogue of the opioid drug codeine (Patterson, 2017 ). Besides being used as an analgesic for mild to severe pain, tramadol is also used as an antidepressant, anesthetic, and anxiolytic. (Vazzana et al., 2015 ). Tramadol poisoning or toxic exposure results in an elevated concentration in the blood and critical outcomes such as cardiovascular problems and mortality (Belin et al., 2017 ; Cole et al., 2012 ). Tramadol gets into aquatic ecosystems through extensive usage by mankind, may persist in the aquatic environment, and pose a major environmental threat (Clara et al., 2005 ; Halling-Sørensen et al., 1998 ). Among aquatic organisms, aquatic vertebrates may face considerable physiological and histological alterations due to long-term exposure to small concentrations of pharmaceuticals (Kondera et al., 2020 ; Nowakowska et al., 2020 ). In the initial developmental phases of carp and zebrafish, tramadol has deleterious impacts on embryogenesis and hatching and also resulted in oxidative stress (Sehonova et al., 2016 ; Sehonova et al., 2017 ). Fish can serve as major indicators of a particular aquatic ecosystem because they are highly sensitive to changes in their immediate surroundings (Nikalje et al., 2012 ). To assess the well-being of fish, hematological profiles act as pathophysiological indicators because fish hematological profiles indicate changes in the aquatic environment brought about by environmental pollutants (Eriegha et al., 2017 ; Raza et al., 2021 ). For determining preliminary cues of contaminants stress over measurement procedures, assessment of biochemical and histopathological abnormalities in fish blood and hepatorenal tissues serves as responsive markers (Akter et al., 2020 ; Renieri et al., 2017 ). Previous studies have demonstrated that medications may be nephrotoxic, hepatotoxic, or toxic to the gills in non-target organisms (Hema et al., 2023 ; Kazmi et al., 2023 ; Kumar et al., 2023 ). Therefore, aquatic organisms, especially fish, are at risk (Ikeogu et al., 2010 ). Despite extensive research on the effects of tramadol on human health, there is a lack of data on its toxic effects on non-target aquatic organisms. Roho (Labeo rohita) , a freshwater fish commonly farmed in Asia (Sarker et al., 2020 ), is an excellent example for analyzing the sub-lethal effects of tramadol, as its exposure to environmental contaminants can cause serious disturbances. This study aimed to investigate the biochemical, histological, and hematological effects of tramadol on Roho (Labeo rohita). This will help to learn more about pharmaceutical pollution and the potential risks it poses to aquatic life. To assess the damaging impacts of a low tramadol concentration on the hematological, biochemical, and histological parameters of the fish L. rohita , the current experiment was carried out. The primary objectives of this study to evaluate the toxicological impact of tramadol on L. rohita by variations in hematological parameter, histological structure, and biochemical markers. The secondary objective was investigated the physiological and biochemical impact of tramadol exposure on Rohu ( L. rohita ) health; and also grasped the potential environmental risk related to pharmaceutical contamination. 2. Materials and methods Tramadol (C 16 H 25 NO 2 ), a chemical utilized as an analgesic, was employed in this study. It was purchased from City Pharmacy, Renala Khurd, Pakistan. Tramadol stock solution was prepared using a commercial formulation of Tonoflex manufactured by Sami Pharm (Pvt) Limited, Karachi, Pakistan. Each uncoated capsule contained 50 mg of tramadol as the principal component. 2.1 Acclimatization of Fish As an experimental organism, L. rohita , each with a weight of 14 ± 0.5 g and a length of 20 ± 5 cm, was used. It is the most cultivable fish in Pakistan, India, and Bangladesh and is crucial to human nutrition. 150 fish, which were devoid of external illnesses, were brought from the Head Balloki Fish Hatchery, District Qasur, Pakistan. The entire fish collection was transported in plastic bags that had hatchery water that was supplied with enough oxygen. During their transportation, no fish deaths were recorded from the Head Balloki Fish Hatchery to the University Fish Laboratory. The Scientific Ethic Committee's recommended protocol was followed in keeping them in regulated lab settings. Before the experiment, all subjects were acclimatized in a large water tank for seven days. 2.2 Experimental Design After acclimatizing for a week, the fish were divided into three groups, with each glass aquarium containing 50 fish. The aquarium measured approximately 40 cm in length, 20 cm in height, and 20 cm in width with de-chlorinated tap water. Under typical environmental circumstances (pH 7.4–8.2, dissolved oxygen 6.3 mg/L, temperature 19–22°C, total ammonia 0.04 mg/L, and chemical oxygen requirement 1.1–1.3 mg/L), the fish were kept in glass aquariums holding 40 liters of water each. The National Institute of Health's (NIH) recommendations for the handling and use of laboratory animals in research were followed when conducting the experiments. Prior to the experiment, glass aquariums were cleaned in a 1% KMnO 4 solution and then sun-dried. The pathogenic diseases of the fish were investigated. Group 1 was designated as the control group, while groups 2 and 3 were regarded as treatment groups. During the experiment, daily changes to the medium were made to eliminate feces and food particles, while the fish were fed commercial food the second upcoming day. We replaced 90% of the aquarium's water after every 2-day interval, added fresh water, and then added the chemical again. The treatment groups, group 2 and group 3 of fish L. rohita were exposed to 1.25 mg/L and 2.5 mg/L of tramadol, respectively, for 25 days. 2.3 Hematological analysis Using 26-gauge hypodermic needles, blood was drained from the caudal vein of five fish from each group. In an EDTA tube, blood was kept for hematological examination. Before analysis, each specimen was kept at -20°C. The hematological analyzer conducted the hematological analysis. Monocyte counts, lymphocytes, neutrophils, hemoglobin concentration, total leukocyte counts, hematocrit, and total erythrocyte counts were determined shortly after retrieval (Sarwar et al., 2019 ). 2.4 Biochemical Studies Using 26-gauge hypodermic needles, 3 millilitres (mL) of blood were drained from the caudal vein of each group of fish. After a 2-day treatment interval, all blood specimens were obtained without an anticoagulant. Thereafter, serum was separated on ice and stored at -20°C for further processing. Using commercially available kits, triglycerides, cholesterol, lipid peroxidation product (MDA), kidney function tests, and liver function tests were determined from serum specimens (Ghaffar, Hussain, Abbas, Ali, Saleem, et al., 2017). A clinical chemistry analyzer (M/S Randox Company) was employed to estimate serum levels of AST, ALT, LDH, and ALP using kits supplied by the manufacturer (Faheem et al., 2019 ; Ghaffar, Hussain, Abbas, Ali, Ahmad, et al., 2017). 2.5 Histological Examination Five fish from each group were dissected at the end of the experiment. Prior to their preservation in 10% neutral formalin and Bouin’s for 24 hours and 48 hours, respectively, tissues were preliminarily cleansed in fish saline and then blot dried. Then, using a rotatory microtome, tissue blocks were made and slices were cut at a thickness of 6µm. After being subjected to various ethanol treatments, Ehrlich hematoxylin and eosin staining, and mounting with dibutylphthalate polystyrene xylene (DPX), the specimens were finally processed. Lastly, an imaging microscope was used to analyze and take pictures of the slides. 2.6 Statistical Analysis Statistics were used to express all results. GraphPad Prism 9.5.1 (733), a statistical tool, was employed for evaluation. The statistical program GraphPad Prism 9.5.1 (733) ran a one-way Anova test. Mean and S.E.M. were used to express the results. Turkey's test was used after one-way analysis of variance (ANOVA) to determine how homogeneous the groups were. P-value and Graph Pad Prism Software Version 9.5.1 (733) were used for all analyses. 3. Results 3.1 Hematological Analysis L. rohita exposed to tramadol hydrochloride showed a significant increase in WBCs, MCV, RDW, RDW-SD, PLT, PDW, neutrophils, lymphocytes, monocytes, and eosinophils as compared to the tramadol-free group. While the values of other hematological parameters such as Hgb, RBCs, HCT, MCH, MCHC, and MPV decreased significantly. 3.2 Biochemical Analysis This study showed a significant rise in cholesterol, triglycerides, and HDL levels. While the levels of LDL and VLDL decreased in comparison with the tramadol-free groups (Table 1 ). The biochemical analysis of L. rohita-treated groups showed a significant rise in the levels of ALT, AST, total protein, and glucose, while albumin and globulin were significantly decreased as compared to the tramadol-free group (Table 2 ). The biochemical analysis of tramadol-treated groups shows a significant increase in the concentration of T3, T4, TSH, urea, BUN, and creatinine as compared to tramadol-free groups. (Table 3 ). Table 1 Lipid profile of rohu ( L. rohita ) prior and after Tramadol Exposure Parameters Groups Mean SD SE F P Cholesterol (mg/dl) Control 227.60 4.84 2.79 71.14 0.001*** Low 272.50 6.18 3.57 High 290.80 8.51 4.92 Triglyceride (mg/dl) Control 238.10 5.47 3.16 123.40 0.001*** Low 285.40 6.15 3.55 High 325.70 8.50 4.91 HDL (mg/dl) Control 46.67 1.53 0.88 7.92 0.021* Low 51.33 2.08 1.20 High 54.00 3.00 1.73 LDL (mg/dl) Control 110.70 6.90 3.98 14.25 0.005** Low 91.06 6.73 3.89 High 84.74 4.75 2.74 VLDL (mg/dl) Control 94.57 4.56 2.64 20.72 0.002** Low 72.77 6.27 3.62 High 67.13 5.58 3.22 ⁎ Statistically significant differences; NS = non-significant (p˃0.05), ⁎ =Significant (p˂0.05), ⁎⁎ =Highly Significant (p˂0.01), ⁎⁎⁎ = Very Highly Significant (p˂0.001) Table 2 Liver Enzymes, Blood Proteins and Blood Glucose of Rohu ( L. rohita ) Prior and after Tramadol Exposure Parameters Groups Mean SD SE F P ALT (U/L) Control 28.40 1.56 0.90 11.71 0.008** Low 31.28 1.80 1.04 High 36.59 2.76 1.59 AST (U/L) Control 156.90 4.82 2.78 10.31 0.011* Low 175.20 5.96 3.44 High 179.80 8.35 4.82 Total protein (g/dl) Control 5.40 0.20 0.12 6.29 0.034* Low 5.90 0.30 0.17 High 6.60 0.62 0.36 Albumin (g/dl) Control 1.95 0.07 0.04 18.39 0.003** Low 1.77 0.08 0.04 High 1.63 0.06 0.03 Globulin (g/dl) Control 6.70 0.20 0.12 12.67 0.007** Low 6.10 0.30 0.17 High 5.33 0.45 0.45 Glucose (mg/dL) Control 97.67 3.51 2.03 29.74 0.001*** Low 109.70 4.51 2.60 High 127.70 6.03 3.48 Table 3 Thyroid and Renal Functioning Analysis of Rou (L. rohita) Prior and After Tramadol Exposure Parameters Groups Mean SD SE F P T3 (nmol/l) Control 1.35 0.05 0.03 41.00 0.001*** Low 1.83 0.08 0.04 High 2.00 0.13 0.08 T4 (nmol/l) Control 68.35 0.58 0.33 18.59 0.003** Low 73.42 0.76 0.44 High 75.35 2.33 1.34 TSH (nmol/l) Control 0.84 0.08 0.05 37.87 0.001*** Low 1.26 0.10 0.06 High 1.70 0.16 0.09 Urea (mg/dl) Control 12.85 0.17 0.10 22.99 0.002** Low 14.75 0.62 0.36 High 16.68 1.01 0.59 BUN (mg/dl) Control 30.40 1.08 0.62 14.15 0.005** Low 32.20 1.60 0.92 High 37.52 2.24 1.29 Creatinine (mg/dl) Control 2.08 0.08 0.05 3.25 0.110 NS Low 2.34 0.10 0.06 High 2.52 0.34 0.20 3.3 Histopathological Analysis The histological analysis of gills is shown in Fig. 2 ; the normal histology of gills was observed in the control group (2a). The alterations in gill histology of the treated group (2b & c) after exposure to tramadol gill filaments showed blood congestion, bone cell deformities, lamellar aneurism, edema, and fusion of primary lamellae. The histology of the liver is shown in Fig. 3 . The normal histology of the liver was observed in the control group (3a). The alterations in liver histology of the treated groups (3b & c) after exposure to tramadol showed hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, and necrosis. The histology of the kidney is shown in Fig. 4 . The normal histology of the kidney was observed in the control group (4a). The alterations in kidney histology of the treated groups (4b & c) after exposure to tramadol showed melanomacrophages, sinusoidal spaces, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion. 4. Discussion The research was carried out to evaluate the impacts of tramadol on the hematological, biochemical, and histological parameters of the Rohu fish L. rohita . In this study, we studied tramadol as a pollutant and not as an analgesic because there is an alarming increase in tramadol in aquatic environments. In environmental and aquaculture investigations, hematological parameters are highly helpful biomarkers of pollution and physiological dysfunction (Burgos-Aceves et al., 2019 ; Oluah et al., 2020 ). Our hematological results showed a significant decrease in HCT value, RBC count, and Hgb concentration after administration of different concentrations of tramadol (1.25 mg/L for low dose and 2.5 mg/L for high dose) for 25 days. We found results that were in agreement with Aldalou et al. ( 2014 ), who observed that there was a significant decline in Hgb content and RBC count in rabbits after exposure to tramadol (4 mg/kg/day) and sildenafil (1.40 mg/kg/day) for 25 days (Aldiwan et al., 2015 ). Diminished hematopoiesis or erythropoiesis may be a possible cause of the decline in RBCs and Hgb (Melefa et al., 2020 ). The toxicants get accumulated in the gill area, causing osmoregulation impairment in the gill epithelium, which in turn results in restricted erythropoiesis, which eventually leads to reduced hematopoiesis or erythropoiesis (Pereira et al., 2013 ; Saravanan et al., 2012 ). Environmental stress exposure in living organisms is indicated by variations in white blood cell differential counts (Dogan & Can, 2011 ). Functional immune system maintenance in fish is affected by variations in white blood cells (Liu et al., 2017 ). The response of the immune system to the toxicological effects of tramadol may be indicated by a significant increase in white blood cells in the treated group. Ogueji et al. ( 2018 ) showed similar results in diazepam-exposed C. gariepinus . Aldalou et al. ( 2014 ) revealed that the increase in WBCs was caused by a high lymphocyte percentage in male rats given a 30-day injection of tramadol (100 mg/kg). In the present study, the biochemical parameters were recorded in L. rohita after the treatment of tramadol. The treated groups exhibited have higher values of creatinine, BUN, AST, and ALT. These observations correspond to El-Gaafarawi ( 2006 ) who manifested that rise in serum concentrations of creatinine, BUN, ALT, and AST revealed the tramadol toxicological effects (40 and 80 mg/kg bw/day). Acute cellular necrosis may be coupled with intensified hepatic enzyme secretion. The enzymes are released into the bloodstream because liver cell membrane necrosis or injury caused by tramadol use in rats could result in an elevation of the plasma level of these enzymes (Loughrey et al., 2003 ). Similarly, Ajima et al. ( 2015 ) observed that chronic diclofenac exposure in fish resulted in an elevation in ALT and AST levels. Sera of tramadol-treated L. rohita showed prominent elevations in the levels of cholesterol and triglycerides. The accounts of other writers who treated with certain fungicides and had comparable effects supported this finding. It was stated that high-density lipoprotein cholesterol, total plasma cholesterol, and triacylglycerol levels rose while lipoprotein lipase (LPL) activity in adipose tissue lowered in afflicted rats due to acute thiram (tetramethyl-bis-thiocarbamyl disulfide) toxicity (Sakr & Abel-Samie, 2008 ). It has been hypothesized that acceleration of acetyl-CoA, recognized as the precursor of cholesterol biological production, enhanced tissue lipogenesis levels in metalaxyl-treated mice (Kackar et al., 1999 ). In fish, proteins have a variety of important roles, including serving as an energy reserve during times of prolonged stress, a source of nitrogenous metabolism, and a component of the structural and functional parts of cells (Ullah et al., 2014 ). To meet energy needs, proteins are used as a backup energy source in fish because they don’t have a lot of carbohydrates. Kidney or liver disorders after the treatment of tramadol might be caused in the tissues of Rohu ( L. rohita) by an increase in the tissues’ protein content. Inflammatory diseases or prolonged inflammation may also be linked to it. Chemical and toxicological risk evaluation analyses commonly utilize the level of blood glucose as a bioindicator. Deltamethrine-treated fish turned hyperglycemic after exposure, as manifested by an increase in blood glucose levels. Impaired glycogenolysis, or gluconeogenesis, might be a possible cause of blood glucose elevation. Hyperglycemia emerged when glucose and glycogen leaked into the blood due to disintegration caused by the released hyperglycemic hormones (glucocorticoids and catecholamines), which are caused by an impaired respiratory metabolism due to stress (Uddin et al., 2018 ). Preliminary indicators of disease and damage in organs, tissues, or cells and toxic agent impacts on fish well-being are recognized by toxic agent effect evaluation using a sensitive tool, histopathological investigation. A number of investigators have observed such structural variations in fish as indicators in several tissues from distinct species (Butchiram et al., 2013 ; Magar & Bias, 2013 ). In present study, melanomacrophages, sinusoidal spaces, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion in Rohu ( L. rohita) receiving tramadol can be considered evidence of renal damage. On the other hand, Rohu (L. rohita) receiving a low tramadol dose showed minimal histopathologic changes in the kidneys. The major target of the pollutants is believed to be the gills, as they are highly susceptible to fluctuations in water quality, contribute to various key processes in fish such as excretion, respiration, and osmoregulation, and maintain a close connection with the outside environment (Mazon et al., 2002 ; MN, 2003 ). In present study, alterations in gill histology showed lamellar aneurysms, blood congestion, bone cell deformities, edema, and fusion of primary lamellae. Similarly, the same changes have also been reported in the gills of fish exposed to organic toxicants (Rosety-Rodrı́guez et al., 2002 ) and metals and industrial effluent (Marchand et al., 2009 ; Reddy & Baghel, 2010 ). Resilient adjustments to stop contaminants from entering via the gill surface or responses to toxicants may be responsible for these pathological changes (MN, 2003 ; Poleksić & Mitrović-Tutundžić, 1994 ). Whenever fish experience a more extreme kind of stress, certain blood vessel changes may also take place. In this situation, injured pillar cells may cause higher blood circulation within the lamellae, which may cause the peripheral channel to enlarge, possibly causing an aneurysm or blood congestion (Laurén & Wails, 2018 ; Takashima & Hibiya, 1995 ). Liver histopathology has consistently been used as a biomarker of contamination, and several studies have demonstrated increased sensitivity of the liver to waterborne pollutants (Deore & Wagh, 2012 ; Reddy & Baghel, 2012 ) (Van der Oost et al., 2003 ). In present study, alterations in liver histology showed hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, and necrosis. Numerous researchers who investigated the impacts of various contaminants on the liver of fish agree with our findings (Fanta et al., 2003 ; Mohamed, 2001 ). Similarly, Olojo et al. ( 2005 ) discovered that C. gariepinus exposed to lead exhibited localized liver necrosis and hepatocyte degeneration. Congestion in the liver blood vessels, hepatocyte degeneration, and sinusoidal dilation are induced by copper sulfate exposure in Oncorhynchus mykiss (Atamanalp et al., 2008 ). The kidney is an important organ for excretion and osmoregulation, as well as maintaining homeostasis (Iqbal et al., 2005 ). The renal damage is indicated by the levels of serum creatinine and BUN (Dehkordi et al., 2012 ; Meyer et al., 1992 ). Kidney damage is indicated by the elevated levels of these biochemical substances (El-Gaafarawi, 2006 ; Meyer et al., 1992 ). In our study, alterations in kidney histology showed melanomacrophages, sinusoidal spaces, tubular cells with a hypertrophied nucleus, elongated tubules, and glomerulus expansion. This pharmaceutical drug is really impaired as tramadol exposure causes metabolic and structural damage to the kidneys, as proved by the large number of melanomacrophages in the kidneys of Rohu (L. rohita) . 5. Conclusion This study demonstrated tramadol-induced histological and hematological alterations in fish; The gills, liver, and kidneys were the most affected by tramadol exposure in L. rohita fish. Hematological, biochemical, and histological changes in these organs indicate that fish respond to both the direct and secondary effects of contaminants. Hematological, biochemical, and histological alterations are good biomarkers for drug assessment, particularly in fish that are good indicators of pollutants in aquatic life. However, more research is needed to determine long-term physiological consequences, recovery capability after exposure, and molecular components that influence toxicity. Extending the study to non-targeted fishes might reveal species-specific tramadol sensitivity. Declarations This study involving Rohu fish (Labeo rohita) was conducted in accordance with relevant guidelines and regulations. The experimental procedures were approved by the Department of Zoology, The University of Okara, Animal Care and Use Committee. Acknowledgements: None Funding: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors’ Contributions : NB and MA share the equal contribution and also share the first authorship in this manuscript. UR share senior authorship. MS and MZ share equal and last authorship. All authors contributed to the article and approved the submitted version. Ethical Approval: Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Consent to Participate/Publish : This is not applicable. Declaration of competing interest: The authors declare no competing interests. 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1","display":"","copyAsset":false,"role":"figure","size":190729,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract of hematobiochemical and histological parameters \u003cem\u003ein\u003c/em\u003e Rohu\u003cem\u003e (L. rohita).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/1c90153219cd9f153f44901a.jpeg"},{"id":96587180,"identity":"5298d6f6-b16a-4d84-9663-326137a6a621","added_by":"auto","created_at":"2025-11-24 05:10:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":623353,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of fish gills. (a) Control group showing normal gill structure. (b, c) Gill alterations observed after tramadol exposure; including blood congestion, bone cell deformities, lamellar aneurysm, edema, and fusion of primary lamellae\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/4c5c05baaea3eb2f2c5aa637.png"},{"id":96587175,"identity":"5616ea64-fe1b-4927-9f98-8cd3b9321e1b","added_by":"auto","created_at":"2025-11-24 05:10:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":544814,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of fish gills. (a) normal Gill structure in control group liver, (b, c) Histology of liver after tramadol exposure; including hepatocytes with irregular shape nucleus, sinusoidal spaces, nuclear degeneration, and necrosis\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/d9214bad1a40822ec948811e.png"},{"id":96587177,"identity":"41fdd7bc-f5af-4e5d-ae21-8bcb1d0918e7","added_by":"auto","created_at":"2025-11-24 05:10:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":634659,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of fish kidney. (a) control group showing normal structure of kidney. (b, c) Histology of kidney after tramadol exposure; including melanomacropages, sinusoidal spaces, tubular cells with hypertrophied nucleus, elongated tubules, and glomerulus expansion\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/8960069d78badad877851de2.png"},{"id":96587178,"identity":"13b70436-d504-4e2c-bc8d-d1e3a079200a","added_by":"auto","created_at":"2025-11-24 05:10:58","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73661,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameters in the control and experimental groups represents; (a) HGB levels, (b) White blood cells (WBC) count, (c) Red blood cells (RBC) count in control, low dose and high dose treated groups\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/3a2e5351b50b0054810505f0.jpeg"},{"id":96587184,"identity":"fb386ef0-5ddb-4759-94a3-76db7c26430e","added_by":"auto","created_at":"2025-11-24 05:10:58","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65052,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameters in the control and experimental groups represents; (a) HCT levels (b) Mean corpuscular volume (MCV), and (c) Mean corpuscular hemoglobin (MCH)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/f0decab5822048d8ec51efa2.jpeg"},{"id":96587173,"identity":"e0dad989-8ad7-45fc-b14d-ca9bd5709df9","added_by":"auto","created_at":"2025-11-24 05:10:57","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":66713,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameter in control and experimental groups represents; (a) Mean corpuscular hemoglobin concentration (MCHC) values, (b) Red cells distribution width (RDW), (c) Red cells distribution width- Standard Deviation (RDW-SD)\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/041374c5b51f98c27050db6c.jpeg"},{"id":96587179,"identity":"0f92d054-b877-49e3-bbb0-661744460c95","added_by":"auto","created_at":"2025-11-24 05:10:58","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":66209,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameter in control and experimental groups represents; (a) Platelet count (PLT) (b) Mean Platelet Volume (MPV) (c) Platelet distribution width (PDW)\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/df8111bcf6b2735fd81e8e2f.jpeg"},{"id":96605869,"identity":"ac999038-673d-46f3-a384-10d2c60e0bae","added_by":"auto","created_at":"2025-11-24 09:24:15","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":72602,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameter in control and experimental groups represents; (a) neutrophils values (b) lymphocytes values (c) monocytes values\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/726129cbb9196fcd5d518cf0.jpeg"},{"id":96605661,"identity":"f0871c65-b1b3-4567-aca7-a854159461ce","added_by":"auto","created_at":"2025-11-24 09:23:47","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":38117,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameter represents eosinocytes values in control and experimental groups\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/6c38cd44c9bf7d400c165e45.jpeg"},{"id":97139324,"identity":"39a7c546-441e-4cd2-9e97-1e5eb60c7980","added_by":"auto","created_at":"2025-12-01 10:00:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3688109,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8150296/v1/77cd2a74-4e1e-4698-81d6-3ea0ac3c0eb5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Tramadol on Rohu (Labeo rohita); A Histopathological and Hematological study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePharmaceutical pollutants, especially analgesics like tramadol, are 2-(dimethyl amino)-methyl-1-(3-methoxyphenyl) cyclohexanol hydrochloride. It is the 4-phenyl-piperidine analogue of the opioid drug codeine (Patterson, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides being used as an analgesic for mild to severe pain, tramadol is also used as an antidepressant, anesthetic, and anxiolytic. (Vazzana et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Tramadol poisoning or toxic exposure results in an elevated concentration in the blood and critical outcomes such as cardiovascular problems and mortality (Belin et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cole et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTramadol gets into aquatic ecosystems through extensive usage by mankind, may persist in the aquatic environment, and pose a major environmental threat (Clara et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Halling-S\u0026oslash;rensen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Among aquatic organisms, aquatic vertebrates may face considerable physiological and histological alterations due to long-term exposure to small concentrations of pharmaceuticals (Kondera et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nowakowska et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the initial developmental phases of carp and zebrafish, tramadol has deleterious impacts on embryogenesis and hatching and also resulted in oxidative stress (Sehonova et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sehonova et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFish can serve as major indicators of a particular aquatic ecosystem because they are highly sensitive to changes in their immediate surroundings (Nikalje et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To assess the well-being of fish, hematological profiles act as pathophysiological indicators because fish hematological profiles indicate changes in the aquatic environment brought about by environmental pollutants (Eriegha et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Raza et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For determining preliminary cues of contaminants stress over measurement procedures, assessment of biochemical and histopathological abnormalities in fish blood and hepatorenal tissues serves as responsive markers (Akter et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Renieri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that medications may be nephrotoxic, hepatotoxic, or toxic to the gills in non-target organisms (Hema et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kazmi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, aquatic organisms, especially fish, are at risk (Ikeogu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Despite extensive research on the effects of tramadol on human health, there is a lack of data on its toxic effects on non-target aquatic organisms. Roho \u003cem\u003e(Labeo rohita)\u003c/em\u003e, a freshwater fish commonly farmed in Asia (Sarker et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), is an excellent example for analyzing the sub-lethal effects of tramadol, as its exposure to environmental contaminants can cause serious disturbances. This study aimed to investigate the biochemical, histological, and hematological effects of tramadol on Roho \u003cem\u003e(Labeo rohita).\u003c/em\u003e This will help to learn more about pharmaceutical pollution and the potential risks it poses to aquatic life. To assess the damaging impacts of a low tramadol concentration on the hematological, biochemical, and histological parameters of the fish \u003cem\u003eL. rohita\u003c/em\u003e, the current experiment was carried out.\u003c/p\u003e\u003cp\u003eThe primary objectives of this study to evaluate the toxicological impact of tramadol on \u003cem\u003eL. rohita\u003c/em\u003e by variations in hematological parameter, histological structure, and biochemical markers. The secondary objective was investigated the physiological and biochemical impact of tramadol exposure on Rohu (\u003cem\u003eL. rohita\u003c/em\u003e) health; and also grasped the potential environmental risk related to pharmaceutical contamination.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eTramadol (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e), a chemical utilized as an analgesic, was employed in this study. It was purchased from City Pharmacy, Renala Khurd, Pakistan. Tramadol stock solution was prepared using a commercial formulation of Tonoflex manufactured by Sami Pharm (Pvt) Limited, Karachi, Pakistan. Each uncoated capsule contained 50 mg of tramadol as the principal component.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Acclimatization of Fish\u003c/h2\u003e\u003cp\u003eAs an experimental organism, \u003cem\u003eL. rohita\u003c/em\u003e, each with a weight of 14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g and a length of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;5 cm, was used. It is the most cultivable fish in Pakistan, India, and Bangladesh and is crucial to human nutrition. 150 fish, which were devoid of external illnesses, were brought from the Head Balloki Fish Hatchery, District Qasur, Pakistan. The entire fish collection was transported in plastic bags that had hatchery water that was supplied with enough oxygen. During their transportation, no fish deaths were recorded from the Head Balloki Fish Hatchery to the University Fish Laboratory. The Scientific Ethic Committee's recommended protocol was followed in keeping them in regulated lab settings. Before the experiment, all subjects were acclimatized in a large water tank for seven days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Experimental Design\u003c/h2\u003e\u003cp\u003eAfter acclimatizing for a week, the fish were divided into three groups, with each glass aquarium containing 50 fish. The aquarium measured approximately 40 cm in length, 20 cm in height, and 20 cm in width with de-chlorinated tap water. Under typical environmental circumstances (pH 7.4\u0026ndash;8.2, dissolved oxygen 6.3 mg/L, temperature 19\u0026ndash;22\u0026deg;C, total ammonia 0.04 mg/L, and chemical oxygen requirement 1.1\u0026ndash;1.3 mg/L), the fish were kept in glass aquariums holding 40 liters of water each. The National Institute of Health's (NIH) recommendations for the handling and use of laboratory animals in research were followed when conducting the experiments. Prior to the experiment, glass aquariums were cleaned in a 1% KMnO\u003csub\u003e4\u003c/sub\u003e solution and then sun-dried. The pathogenic diseases of the fish were investigated. Group 1 was designated as the control group, while groups 2 and 3 were regarded as treatment groups. During the experiment, daily changes to the medium were made to eliminate feces and food particles, while the fish were fed commercial food the second upcoming day. We replaced 90% of the aquarium's water after every 2-day interval, added fresh water, and then added the chemical again. The treatment groups, group 2 and group 3 of fish \u003cem\u003eL. rohita\u003c/em\u003e were exposed to 1.25 mg/L and 2.5 mg/L of tramadol, respectively, for 25 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Hematological analysis\u003c/h2\u003e\u003cp\u003eUsing 26-gauge hypodermic needles, blood was drained from the caudal vein of five fish from each group. In an EDTA tube, blood was kept for hematological examination. Before analysis, each specimen was kept at -20\u0026deg;C. The hematological analyzer conducted the hematological analysis. Monocyte counts, lymphocytes, neutrophils, hemoglobin concentration, total leukocyte counts, hematocrit, and total erythrocyte counts were determined shortly after retrieval (Sarwar et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Biochemical Studies\u003c/h2\u003e\u003cp\u003eUsing 26-gauge hypodermic needles, 3 millilitres (mL) of blood were drained from the caudal vein of each group of fish. After a 2-day treatment interval, all blood specimens were obtained without an anticoagulant. Thereafter, serum was separated on ice and stored at -20\u0026deg;C for further processing. Using commercially available kits, triglycerides, cholesterol, lipid peroxidation product (MDA), kidney function tests, and liver function tests were determined from serum specimens (Ghaffar, Hussain, Abbas, Ali, Saleem, et al., 2017). A clinical chemistry analyzer (M/S Randox Company) was employed to estimate serum levels of AST, ALT, LDH, and ALP using kits supplied by the manufacturer (Faheem et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghaffar, Hussain, Abbas, Ali, Ahmad, et al., 2017).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Histological Examination\u003c/h2\u003e\u003cp\u003eFive fish from each group were dissected at the end of the experiment. Prior to their preservation in 10% neutral formalin and Bouin\u0026rsquo;s for 24 hours and 48 hours, respectively, tissues were preliminarily cleansed in fish saline and then blot dried. Then, using a rotatory microtome, tissue blocks were made and slices were cut at a thickness of 6\u0026micro;m. After being subjected to various ethanol treatments, Ehrlich hematoxylin and eosin staining, and mounting with dibutylphthalate polystyrene xylene (DPX), the specimens were finally processed. Lastly, an imaging microscope was used to analyze and take pictures of the slides.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistics were used to express all results. GraphPad Prism 9.5.1 (733), a statistical tool, was employed for evaluation. The statistical program GraphPad Prism 9.5.1 (733) ran a one-way Anova test. Mean and S.E.M. were used to express the results. Turkey's test was used after one-way analysis of variance (ANOVA) to determine how homogeneous the groups were. P-value and Graph Pad Prism Software Version 9.5.1 (733) were used for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Hematological Analysis\u003c/h2\u003e\u003cp\u003e\u003cem\u003eL. rohita\u003c/em\u003e exposed to tramadol hydrochloride showed a significant increase in WBCs, MCV, RDW, RDW-SD, PLT, PDW, neutrophils, lymphocytes, monocytes, and eosinophils as compared to the tramadol-free group. While the values of other hematological parameters such as Hgb, RBCs, HCT, MCH, MCHC, and MPV decreased significantly.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Biochemical Analysis\u003c/h2\u003e\u003cp\u003eThis study showed a significant rise in cholesterol, triglycerides, and HDL levels. While the levels of LDL and VLDL decreased in comparison with the tramadol-free groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The biochemical analysis of \u003cem\u003eL. rohita-treated\u003c/em\u003e groups showed a significant rise in the levels of ALT, AST, total protein, and glucose, while albumin and globulin were significantly decreased as compared to the tramadol-free group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The biochemical analysis of tramadol-treated groups shows a significant increase in the concentration of T3, T4, TSH, urea, BUN, and creatinine as compared to tramadol-free groups. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLipid profile of rohu (\u003cem\u003eL. rohita\u003c/em\u003e) prior and after Tramadol Exposure\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eCholesterol (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e227.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e71.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e272.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e290.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eTriglyceride (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e238.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e123.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e285.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e325.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eHDL (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.021*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e51.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e54.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eLDL (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e110.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e14.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.005**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e91.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eVLDL (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e20.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.002**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e72.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e67.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e⁎\u003c/sup\u003eStatistically significant differences; NS\u0026thinsp;=\u0026thinsp;non-significant (p˃0.05), \u003csup\u003e⁎\u003c/sup\u003e=Significant (p˂0.05), \u003csup\u003e⁎⁎\u003c/sup\u003e=Highly Significant (p˂0.01), \u003csup\u003e⁎⁎⁎\u003c/sup\u003e= Very Highly Significant (p˂0.001)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLiver Enzymes, Blood Proteins and Blood Glucose of Rohu (\u003cem\u003eL. rohita\u003c/em\u003e) Prior and after Tramadol Exposure\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eALT (U/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e11.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.008**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e36.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eAST (U/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e156.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.011*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e175.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e179.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eTotal protein (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e6.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.034*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eAlbumin (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e18.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.003**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGlobulin (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e12.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.007**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGlucose (mg/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e29.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e109.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e127.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThyroid and Renal Functioning Analysis of Rou (L. rohita) Prior and After Tramadol Exposure\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eT3 (nmol/l)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e41.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eT4 (nmol/l)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e68.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e18.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.003**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e73.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e75.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eTSH (nmol/l)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e37.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eUrea (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e22.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.002**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eBUN (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e14.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.005**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eCreatinine (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e3.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.110\u003csup\u003eNS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Histopathological Analysis\u003c/h2\u003e\u003cp\u003eThe histological analysis of gills is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; the normal histology of gills was observed in the control group (2a). The alterations in gill histology of the treated group (2b \u0026amp; c) after exposure to tramadol gill filaments showed blood congestion, bone cell deformities, lamellar aneurism, edema, and fusion of primary lamellae. The histology of the liver is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The normal histology of the liver was observed in the control group (3a). The alterations in liver histology of the treated groups (3b \u0026amp; c) after exposure to tramadol showed hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, and necrosis. The histology of the kidney is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The normal histology of the kidney was observed in the control group (4a). The alterations in kidney histology of the treated groups (4b \u0026amp; c) after exposure to tramadol showed melanomacrophages, sinusoidal spaces, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe research was carried out to evaluate the impacts of tramadol on the hematological, biochemical, and histological parameters of the Rohu fish \u003cem\u003eL. rohita\u003c/em\u003e. In this study, we studied tramadol as a pollutant and not as an analgesic because there is an alarming increase in tramadol in aquatic environments. In environmental and aquaculture investigations, hematological parameters are highly helpful biomarkers of pollution and physiological dysfunction (Burgos-Aceves et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oluah et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur hematological results showed a significant decrease in HCT value, RBC count, and Hgb concentration after administration of different concentrations of tramadol (1.25 mg/L for low dose and 2.5 mg/L for high dose) for 25 days. We found results that were in agreement with Aldalou et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), who observed that there was a significant decline in Hgb content and RBC count in rabbits after exposure to tramadol (4 mg/kg/day) and sildenafil (1.40 mg/kg/day) for 25 days (Aldiwan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Diminished hematopoiesis or erythropoiesis may be a possible cause of the decline in RBCs and Hgb (Melefa et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The toxicants get accumulated in the gill area, causing osmoregulation impairment in the gill epithelium, which in turn results in restricted erythropoiesis, which eventually leads to reduced hematopoiesis or erythropoiesis (Pereira et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Saravanan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEnvironmental stress exposure in living organisms is indicated by variations in white blood cell differential counts (Dogan \u0026amp; Can, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Functional immune system maintenance in fish is affected by variations in white blood cells (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The response of the immune system to the toxicological effects of tramadol may be indicated by a significant increase in white blood cells in the treated group. Ogueji et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showed similar results in diazepam-exposed \u003cem\u003eC. gariepinus\u003c/em\u003e. Aldalou et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) revealed that the increase in WBCs was caused by a high lymphocyte percentage in male rats given a 30-day injection of tramadol (100 mg/kg).\u003c/p\u003e\u003cp\u003eIn the present study, the biochemical parameters were recorded in \u003cem\u003eL. rohita\u003c/em\u003e after the treatment of tramadol. The treated groups exhibited have higher values of creatinine, BUN, AST, and ALT. These observations correspond to El-Gaafarawi (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) who manifested that rise in serum concentrations of creatinine, BUN, ALT, and AST revealed the tramadol toxicological effects (40 and 80 mg/kg bw/day). Acute cellular necrosis may be coupled with intensified hepatic enzyme secretion. The enzymes are released into the bloodstream because liver cell membrane necrosis or injury caused by tramadol use in rats could result in an elevation of the plasma level of these enzymes (Loughrey et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Similarly, Ajima et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed that chronic diclofenac exposure in fish resulted in an elevation in ALT and AST levels.\u003c/p\u003e\u003cp\u003eSera of tramadol-treated \u003cem\u003eL. rohita\u003c/em\u003e showed prominent elevations in the levels of cholesterol and triglycerides. The accounts of other writers who treated with certain fungicides and had comparable effects supported this finding. It was stated that high-density lipoprotein cholesterol, total plasma cholesterol, and triacylglycerol levels rose while lipoprotein lipase (LPL) activity in adipose tissue lowered in afflicted rats due to acute thiram (tetramethyl-bis-thiocarbamyl disulfide) toxicity (Sakr \u0026amp; Abel-Samie, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It has been hypothesized that acceleration of acetyl-CoA, recognized as the precursor of cholesterol biological production, enhanced tissue lipogenesis levels in metalaxyl-treated mice (Kackar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn fish, proteins have a variety of important roles, including serving as an energy reserve during times of prolonged stress, a source of nitrogenous metabolism, and a component of the structural and functional parts of cells (Ullah et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To meet energy needs, proteins are used as a backup energy source in fish because they don\u0026rsquo;t have a lot of carbohydrates. Kidney or liver disorders after the treatment of tramadol might be caused in the tissues of Rohu (\u003cem\u003eL. rohita)\u003c/em\u003e by an increase in the tissues\u0026rsquo; protein content. Inflammatory diseases or prolonged inflammation may also be linked to it.\u003c/p\u003e\u003cp\u003eChemical and toxicological risk evaluation analyses commonly utilize the level of blood glucose as a bioindicator. Deltamethrine-treated fish turned hyperglycemic after exposure, as manifested by an increase in blood glucose levels. Impaired glycogenolysis, or gluconeogenesis, might be a possible cause of blood glucose elevation. Hyperglycemia emerged when glucose and glycogen leaked into the blood due to disintegration caused by the released hyperglycemic hormones (glucocorticoids and catecholamines), which are caused by an impaired respiratory metabolism due to stress (Uddin et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePreliminary indicators of disease and damage in organs, tissues, or cells and toxic agent impacts on fish well-being are recognized by toxic agent effect evaluation using a sensitive tool, histopathological investigation. A number of investigators have observed such structural variations in fish as indicators in several tissues from distinct species (Butchiram et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Magar \u0026amp; Bias, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In present study, melanomacrophages, sinusoidal spaces, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion in Rohu (\u003cem\u003eL. rohita)\u003c/em\u003e receiving tramadol can be considered evidence of renal damage. On the other hand, \u003cem\u003eRohu (L. rohita)\u003c/em\u003e receiving a low tramadol dose showed minimal histopathologic changes in the kidneys.\u003c/p\u003e\u003cp\u003eThe major target of the pollutants is believed to be the gills, as they are highly susceptible to fluctuations in water quality, contribute to various key processes in fish such as excretion, respiration, and osmoregulation, and maintain a close connection with the outside environment (Mazon et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; MN, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In present study, alterations in gill histology showed lamellar aneurysms, blood congestion, bone cell deformities, edema, and fusion of primary lamellae. Similarly, the same changes have also been reported in the gills of fish exposed to organic toxicants (Rosety-Rodrı́guez et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and metals and industrial effluent (Marchand et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Reddy \u0026amp; Baghel, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Resilient adjustments to stop contaminants from entering via the gill surface or responses to toxicants may be responsible for these pathological changes (MN, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Poleksić \u0026amp; Mitrović-Tutundžić, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Whenever fish experience a more extreme kind of stress, certain blood vessel changes may also take place. In this situation, injured pillar cells may cause higher blood circulation within the lamellae, which may cause the peripheral channel to enlarge, possibly causing an aneurysm or blood congestion (Laur\u0026eacute;n \u0026amp; Wails, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Takashima \u0026amp; Hibiya, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLiver histopathology has consistently been used as a biomarker of contamination, and several studies have demonstrated increased sensitivity of the liver to waterborne pollutants (Deore \u0026amp; Wagh, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Reddy \u0026amp; Baghel, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Van der Oost et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In present study, alterations in liver histology showed hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, and necrosis. Numerous researchers who investigated the impacts of various contaminants on the liver of fish agree with our findings (Fanta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mohamed, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Similarly, Olojo et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) discovered that \u003cem\u003eC. gariepinus\u003c/em\u003e exposed to lead exhibited localized liver necrosis and hepatocyte degeneration. Congestion in the liver blood vessels, hepatocyte degeneration, and sinusoidal dilation are induced by copper sulfate exposure in \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (Atamanalp et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe kidney is an important organ for excretion and osmoregulation, as well as maintaining homeostasis (Iqbal et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The renal damage is indicated by the levels of serum creatinine and BUN (Dehkordi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Meyer et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Kidney damage is indicated by the elevated levels of these biochemical substances (El-Gaafarawi, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Meyer et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). In our study, alterations in kidney histology showed melanomacrophages, sinusoidal spaces, tubular cells with a hypertrophied nucleus, elongated tubules, and glomerulus expansion. This pharmaceutical drug is really impaired as tramadol exposure causes metabolic and structural damage to the kidneys, as proved by the large number of melanomacrophages in the kidneys of \u003cem\u003eRohu (L. rohita)\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated tramadol-induced histological and hematological alterations in fish; The gills, liver, and kidneys were the most affected by tramadol exposure in \u003cem\u003eL. rohita\u003c/em\u003e fish. Hematological, biochemical, and histological changes in these organs indicate that fish respond to both the direct and secondary effects of contaminants. Hematological, biochemical, and histological alterations are good biomarkers for drug assessment, particularly in fish that are good indicators of pollutants in aquatic life. However, more research is needed to determine long-term physiological consequences, recovery capability after exposure, and molecular components that influence toxicity. Extending the study to non-targeted fishes might reveal species-specific tramadol sensitivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study involving Rohu fish (Labeo rohita) was conducted in accordance with relevant guidelines and regulations. The experimental procedures were approved by the Department of Zoology, The University of Okara, Animal Care and Use Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e: NB and MA share the equal contribution and also share the first authorship in this manuscript. UR share senior authorship. MS and MZ share equal and last authorship. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate/Publish\u003c/strong\u003e: This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData included in the manuscript\u003c/strong\u003e: All data generated or analyzed during this study are included in this article will be available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjima, M. N., Ogo, O. A., Audu, B. S., \u0026amp; Ugwoegbu, K. C. (2015). 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Tramadol hydrochloride: pharmacokinetics, pharmacodynamics, adverse side effects, co-administration of drugs and new drug delivery systems. \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy\u003c/em\u003e, \u003cem\u003e70\u003c/em\u003e, 234\u0026ndash;238.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Rohu (Labeo rohita), Toxicology, Histology, Hematology, Tramadol, Pollutants","lastPublishedDoi":"10.21203/rs.3.rs-8150296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8150296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTramadol is a weak synthetic opioid primarily used to treat moderate to severe pain. It can persist in the aquatic environment through wastewater, posing a significant environmental threat. As a result, aquatic organisms, particularly fish, face the risk of toxicological effects. This study aimed to investigate the toxicological impacts of tramadol on the hematology, biochemistry, and histology of Rohu (\u003cem\u003eLabeo rohita\u003c/em\u003e). A total of one hundred and fifty healthy fingerlings of the freshwater fish \u003cem\u003eL. rohita\u003c/em\u003e with a total mean length of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;5 cm and weight of 14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g was used for this research. The 48-hour exposure rates of \u003cem\u003eL. rohita\u003c/em\u003e exposed to tramadol were 1.25 mg/L for low-dose and 2.5 mg/L for high-dose. The hematological analysis showed increased WBCs, MCV, RDW, RDW-SD, PLT, PDW, neutrophils, lymphocytes, monocytes, and eosinocytes. In contrast, Hgb, RBC, HCT, MCH, MCHC, and MPV decreased significantly. Biochemical analysis showed an increase in cholesterol, triglycerides, HDL, ALT, AST, total protein, glucose, T4, TSH, urea, BUN, and creatinine, while others like LDL, VLDL, albumin, globulin, and T3 were decreased. Fish exposed to tramadol showed some prominent abnormalities, including abnormal blood congestion, bone cell deformities, lamellar aneurism, edema, fusion of primary lamellae, hepatocytes with an irregularly shaped nucleus, sinusoidal spaces, nuclear degeneration, necrosis, melanomacrophages, tubular cells with hypertrophied nuclei, elongated tubules, and glomerulus expansion with increments in concentrations and treatment intervals, except for the untreated group. The findings demonstrate that tramadol is toxic to fish, therefore its presence in aquatic ecosystems should be examined for non-target organisms.\u003c/p\u003e","manuscriptTitle":"Effect of Tramadol on Rohu (Labeo rohita); A Histopathological and Hematological study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 05:10:53","doi":"10.21203/rs.3.rs-8150296/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":"bc6baa3e-fb69-4c58-83f7-9ce08463e624","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-29T23:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 05:10:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8150296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8150296","identity":"rs-8150296","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}