Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 16/12/2024 Aceptado: 19/02/2025 Publicado: 09/04/2025 hps://doi.org/10.52973/rcfcv-e35589 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 10 Effects of orally administraon free and Liposomal Levamisole on hematological and biochemical parameters in Sheep Efecto de la administración oral de levamisol libre y liposomal sobre parámetros hematológicos y bioquímicos en ovejas Hasan Susar 1, * , Mehmet Özüi�li 2 , Murat Çelebi 3 , Çağla Çelebi 1 , İzzet Karahan 1 ¹Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Balikesir University, Balıkesir, Türkiye. ²Department of Parasitology, Faculty of Veterinary Medicine, Balikesir University, Balıkesir, Türkiye. ³Department of Laboratory and Veterinary Health, Savastepe Vocaonal School, Balikesir University, Balıkesir, Türkiye. *Corresponding Author: mehmet.ozuicli@balikesir.edu.tr ABSTRACT Analysis of haematological and biochemical parameters monitors animal health and guides diagnosis and treatment. This study compared the haematological and biochemical effects of free and liposomal levamisole given to sheep orally. The study included twenty-one female Curly breed sheep. The animals were divided into three groups: free levamisole (7.5 mg/kg), liposomal levamisole (7.5 mg/kg), and control (physiological serum 7.5 mL/kg). Blood samples were obtained on day (d) 0 (control), d 1, and d 3. Haematological parameters (WBC, RBC, HGB, HCT, MCV, MCH, MCHC, RDW-CV, RDW-SD, and PCT) were assessed ulizing a haematology analyzer, while biochemical parameters (urea, creanine, AST, ALT, BUN) were evaluated using an autoanalyzer. On d 1, the liposome group exhibited the highest white blood cell measurements. Despite a reducon in PCT values on d 1 within the liposome group, an increase was observed again on d 3. Urea levels on d 1 and 3 were elevated in the control, liposomal levamisole, and free levamisole groups. On d 3, creanine measurements indicated that levels in the control group were significantly elevated compared to those in the liposomal levamisole and free levamisole groups. On d 3, BUN measurements indicated that the mean for the control group was significantly elevated compared to the liposomal levamisole and free levamisole groups. The Neutrophil, Lymphocyte, and Monocyte counts in the liposomal and free levamisole groups of animals were significantly elevated compared to other measurements recorded on the d 3. This study’s findings demonstrate that liposomes affect haematological and biochemical parameters. The results demonstrate that liposomal levamisole causes no harmful effects on animals. It produces advantageous results. Further invesgaon is necessary to elucidate the effects of Liposomal Levamisole on hematological and biochemical parameters among various animal species. Key words: Biochemical; haematological; levamisole; liposome; sheep RESUMEN El análisis de los parámetros hematológicos y bioquímicos facilita el monitoreo de la salud animal y guía el diagnósco y tratamiento. Este estudio comparó los efectos hematológicos y bioquímicos del levamisol libre y liposomal administrado por vía oral an ovejas. Veinuna ovejas hembras de la raza Curly parciparon en el estudio. Los animales fueron clasificados en tres grupos: levamisol libre (7,5 mg/kg), levamisol liposomal (7,5 mg/kg) y control (solución salina 7,5 mL/kg). Se recolectaron muestras de sangre en el día (d) 0 (control), el d 1 y el d 3. Los parámetros hematológicos (WBC, RBC, HGB, HCT, MCV, MCH, MCHC, RDW-CV, RDW-SD y PCT) fueron analizados mediante un analizador hematológico, mientras que, los parámetros bioquímicos (urea, creanina, AST, ALT, BUN) fueron evaluados con un autoanalizador. En el primer día, el grupo del liposoma exhibió las mediciones más elevadas de Leucocitos. A pesar de una disminución en los valores de PCT en el d 1 en el grupo de liposomas, se registró nuevamente un incremento en el d 3. Los niveles de urea en los d 1 y 3 fueron elevados en los grupos de control, levamisol liposomal y levamisol libre. En el tercer d, las mediciones de creanina revelaron que los niveles en el grupo de control eran significavamente más altos en comparación con los grupos de levamisol liposomal y levamisol libre. El d 3, las mediciones de BUN revelaron que la media del grupo de control era significavamente superior en comparación con los grupos de levamisol liposomal y levamisol libre. Los recuentos de neutrófilos, linfocitos y monocitos en los grupos de animales tratados con levamisol liposomal y libre fueron significavamente superiores en comparación con otras mediciones registradas el tercer día. Las conclusiones de este estudio evidencian que los liposomas influyen en los parámetros hematológicos y bioquímicos. Los resultados evidencian que el levamisol liposomal no produce efectos adversos en los animales. Generar resultados favorables. Es imperavo connuar la invesgación para esclarecer los efectos del levamisol liposomal en los parámetros hematológicos y bioquímicos de diversas especies animales. Palabras clave: Bioquímica; hematológica; levamisol; liposoma; oveja

Levamisole effect over Hematological and Biochemical Parameters in sheep / Susar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Levamisole is an anthelminc that is used against in the treatment of lung and gastrointesnal nematode infecons in both humans and animals [1]. Levamisole can be administered orally, intramuscularly, subcutaneously, parenterally, and transdermally. A variety of formulaons has been developed to deliver the drug through mulple routes. These comprise tablets, oblets, weable powder, and soluons for injecon, pouring, or dripping. Upon oral administraon, the drug is swiſtly absorbed from the gastrointesnal tract and disseminated throughout the body. Approximately 95% of levamisole undergoes biotransformaon in the body and is excreted predominantly in feces and urine. The plasma half-life of the drug is 4 to 6 hours (h) in ruminants and 1.8 to 2 h in canines [2]. Approximately 40% of the drug is eliminated in the urine within 12 h post-oral administraon. Thereaſter, the urinary excreon rate diminishes, with roughly 8% excreted within an 8-h meframe [2]. The eliminaon of the drug via feces is a process that exceeds eight d. Approximately 40% of the drug is eliminated from the body in this manner, with a substanal poron occurring within the first 12-24 h [2]. Approximately one percent of the administered levamisole is detected in the kidneys and liver within the inial 12 to 24 h. Levamisole is administered to cale (Bos taurus), sheep (Ovis aries), and goats (Capra hircus) at doses of 7.5 mg/kg for oral use and 3.5-8 mg/kg for intramuscular and subcutaneous administraon [3 , 4]. The immunomodulatory and smulatory effects of levamisole on the immune system are well recognized; however, the exact mechanism by which this occurs remains uncertain. Research indicates that smulang phagocytes sensizes them to mitogens and angens, subsequently augmenng the quanty of T cells. Levamisole reportedly does not directly affect B lymphocytes; however, it indirectly smulates the humoral immune response by influencing T lymphocytes and phagocyc cells, leading to elevated anbody levels [5]. Liposomes represent a substanal advancement in drug delivery systems. Considering that liposomes are deemed the premier carriers for the administraon of various agents, such as ancancer drugs, anbiocs, an-inflammatory, anparasic, and anfungal agents, research on liposomes has gained prominence in the pharmaceucal, biological, and medical fields. Liposomes are the first closed microscopic phospholipid sheet systems, which were approved in 1965 [6 , 7 ,[8]. Liposomes possess considerable potenal as drug delivery systems owing to their biocompability and modular characteriscs. Moreover, they provide the significant benefit of diminishing the risk of systemic toxicity associated with the encapsulated therapeuc agent. Specifically, liposomes can engage with plasma proteins, resulng in opsonizaon and consequently modifying the healthy cells they encounter during circulaon and eliminaon [9 , 10]. Moreover, the pharmacokinecs of circulang liposomes may lead to the sequestraon of drugs within the organs of the mononuclear phagocyte system, potenally affecng liver and spleen funconality. Liposomal agents can either smulate or suppress the immune system, depending on their physicochemical properes such as size, lipid composion, pegylaon, and surface charge [11 , 12]. Biochemical and hematological blood parameters are essenal indicators for evaluang the health status of humans and animals. Certain biological parameters, detected in blood or other biological fluids, are essenal for the inial evaluaon of potenal ssue and organ damage. Haematological parameters, such as leukocytes, erythrocytes, haemoglobin, haematocrit, and platelets, are primarily linked to bone marrow funcon but are also evaluated as indicators of infecon and electrolyte imbalances [13 , 14]. The assessment of aspartate aminotransferase and alanine aminotransferase levels determines the degree of damage to the liver and bile ducts. The assessment of blood urea nitrogen and creanine levels is ulized to determine the degree of damage to the kidneys [15 , 16]. There is no literature available regarding the impact of free and liposomal levamisole on hematological parameters, specifically (white blood cells (WBC) count, The red blood cells (RBC) count, hemoglobin (HGB) concentraon, Hematocrit Value (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentraon (MCHC), Red Cell Distrubion (RDW-CV), Platelet Value (PLT), Mean platelet volume (MPV), Platelet Distribuon Width (PDW), Procalcitonin (PCT), Platelet Large Cell Rao (P-LCR), platelet acvaon (P-LCC)) and biochemical parameters (plasma alanine transaminase (ALT), aspartate transaminase (AST), creanine (Cr), urea (U), blood urea nitrogen (BUN)) in sheep. The aim of this study was to determine the effects of free and liposomal levamisole on hematological and biochemical parameters in sheep at a standard dosage. MATERIALS AND METHODS Animals The research involved 21 female Merino of the origin of Türkiye (Ovis aries), aged one to two years, with an average weight of 60 kg, selected randomLy. The sheep were relocated to various pens ten days before the study began to aid their acclimazaon to the research environment. They were housed in these pens for the duraon of the study. The animals received specialized feed tailored (Energy Sources (Cereals): 50-60%, Protein Sources: 12-16%, Fibre Sources (Roughage): 30-40%, Mineral and Vitamin Premixes: 1-2%, Metabolic Energy (ME): 2.5-3 Mcal/kg, Crude Protein (HP): 12-14%, Calcium: 0.8-1%, Phosphorus: 0.4-0.6%, Salt: 0.5-1%, Vitamin A: 4000-5000 IU/ kg, Vitamin D: 800-1000 IU/kg, Vitamin E: 20-40 IU/kg, Crude Cellulose: 15-20%, Fat (Crude Oil): 3-5%) to their weight and age. Hay and water were provided ad libitum. Experimental protocol In this study, liposomes were prepared with special techniques and equipment [17]. A total of 21 female sheep, each weighing 60 kg, were randomly allocated to one of three groups, comprising seven animals per group. In the first group (n=7), a dose of free levamisole at 7.5 mg/kg was administered orally to the sheep; in the second group (n=7), liposomal levamisole at 7.5 mg/kg was administered orally; and in the third group (n=7), serum physiological at 7.5 mL/kg was administered orally to the sheep. The drug was administered as a single dose. Prior to administraon, 5 mL blood samples were obtained in Tripotassium Ethylenediaminetetraacec acid (K3EDTA) and gel tubes on d 0 (Control) and subsequently on d 1 and 3. The analysis of haematological parameters was conducted 2 of 10

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico promptly (15 min) aſter the collecon of blood samples. Blood samples designated for biochemical analysis were subjected to centrifugaon (ALLEGRA–X64R, USA, 3500 G for 15 min), aſter which the serum was isolated and transferred into 2 mL Eppendorf tubes, subsequently stored in a deep freezer (Arçelik 270530EB, Türkiye) at -80°C unl the analysis date. Analysis of haematological and biochemical parameters Hematological parameters were assessed using a Veterinary blood counng device (HASVET VH3 Automac Hematology Analyzer, Türkiye) from samples collected in K3EDTA tubes. Biochemical analyses were conducted using the RX Daytona+ fully automated veterinary biochemistry apparatus (HASVET, Türkiye) on serum samples thawed at ambient temperature. Ethical aspects of the research The research procedure was conducted with the approval of the Balıkesir University Animal Experiments Local Ethics Commiee (Date: 28.11.2024, Decision Number: 2024/11-1). Stascal analysis Friedman’s test was applied for intra-group comparisons, while the Kruskal-Wallis test was applied for inter-group comparisons in the stascal analysis of hematological and biochemical parameter results. A p-value below 0.05 indicates that the difference is stascally significant [18]. RESULTS AND DISCUSSIONS In this study, the effects of different forms of levamisole on hematological and biochemical parameters were examined in detail. The findings indicate that levamisole formulaon leads to significant differences in certain parameters. This study reported that oral administraon of free and liposomal levamisole to sheep at a dosage of 7.5 mg/kg did not result in any adverse effects in the animals. The reference intervals of haematological and biochemical parameters in sheep are presented in TABLE I [19]. The haematological and biochemical parameters of sheep are displayed in TABLE II and III, respecvely. A stascally significant difference was observed between the groups regarding the d 1 WBC measurements (P<0.05). The mean WBC on d 1 in the control group (0.83±0.24) was significantly lower compared to that in the liposomal levamisole group (4.63±4.34) and the free levamisole group (1.46±0.37). In the liposomal levamisole group, white blood cell (WBC) values were observed to increase significantly on the d 1. This may be related to the potenal effects of liposomal carrier systems on immune modulaon. However, by the d 3, a balancing in WBC values was observed, suggesng that the immune system is regulated by a homeostac mechanism. On d 1, a stascally significant difference in PCT values was observed between the groups (P<0.05). The mean PCT of the control group animals on d 1 (0.38±0.10) was significantly higher compared to that of the animals in the liposomal levamisole group (0.23±0.13) and the free levamisole group (0.24±0.08). On d 3, a stascally significant difference was observed between the groups regarding PCT measurements (P<0.05). The mean PCT values on d 3 for the control group (0.30±0.07) were higher compared to those for the liposomal levamisole group (0.29±0.07) and the free levamisole group (0.20±0.07). Regarding PCT values, although a decrease was observed in the liposomal levamisole group on the d 1, an increase was recorded again on the d 3. This change may be associated with an inflammatory response and requires further invesgaon. A stascally significant difference was idenfied in the MCV values recorded on d 0, 1, and 3 for the control group animals (P<0.05). The value recorded on d 3 was significantly higher compared to the other values. A stascally significant difference was idenfied in the MCV values recorded on d 0, 1, and 3 for the animals in the liposomal levamisole group (P<0.05). The value on d 3 demonstrated a significant increase relave to the other values. The results indicated no stascally significant difference in intra and inter-group comparisons concerning the remaining variables (P>0.05). TABLE I. Reference values of some haematological and biochemical parameters in sheep [19] Parameters Reference range WBC (10^9/L) 5,10-15,80 Lym% 40-75 Neu% 10-50 Mono% 0-6 Eos% 0-10 RBC (10^12/L) 5,50-14,20 HGB (g/L) 63- 132 HCT 0,200- 0,390 MCV (fL) 25,0- 41,0 MCH (pg) 8,0-12,3 MCHC (g/L) 290- 360 RDW-CV (%) 11,0-17,0 RDW-SD (fL) 20,0-35,0 PLT (10^9/L) 100 -800 MPV (fL) 3,5 -6,0 PDW-CV (%) 0,133- 0,185 PDW-SD (fL) 12,0- 17,5 PCT (%) 0,50- 4,20 P-LCC (10^9/L) 15 -278 P-LCR (%) 0,150- 0,650 Urea (mg/dL) 8-30 Creanine (mg/dL) 0,6-1,2 BUN (mg/dL) 10-30 ALT (U/L) 26-34 AST (U/L) 60-280 U/L: Units per liter, mg: milligram, dL: deciliter, fL: femtoliters, (g/L): grams per liter, pg: pico- grams, WBC: White blood cell, Lym: Lymphocyte, Neu: Neutrophil, Mono: Monocyte, Eos: Eosinophil, RBC: Red blood cell, HGB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpus- cular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentraon, RDW-CV: Red cell distribuon width-coefficient of variaon, RDW-SD: Red cell distribuon width-standard deviaon, PLT: Platelet count, MPV: Mean platelet volume, PDW-CV: Platelet distribuon width-coefficient of variaon, PDW-SD: Platelet distribuon width-standard deviaon, PCT; Procalcitonin, P-LCC: Platelet large cell coefficient, P-LCR: Platelet large cell rao, BUN: Blood urea nitrogen, ALT: Alanine aminotransferase, AST: As- partate aminotransferase. 3 of 10

Levamisole effect over Hematological and Biochemical Parameters in sheep / Susar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE II. Intragroup and intergroup comparisons of hemogram values Control Liposomal Levamisole Free Levamisole Mean±SD Median (Min-Max) Mean±SD Median (Min-Max) Mean±SD Median (Min-Max) H p WBC (10^9/L) Day 0 1.13±0.77 0.90 (0.50-2.70) 1.53±0.88 1.30 (0.60-3.10) 0.89±0.35 10 (0.40-1.30) 2.433 0.296 Day 1 0.83±0.24 0.80 (0.50-1.10) 4.63±4.34 3.30 (0.10-11.7) 1.46±0.37 1.50 (0.90-1.80) 7.897 0.019 Day 3 1.61±0.85 1.70 (0.70-2.70) 1.81±0.75 1.60 (1.30-3.50) 1.70±0.79 1.30 (0.70-2.90) 0.237 0.888 F=2.741 p=0.254 F=1.143 p=0.565 F=5.429 p=0.066 RBC (10^12/L) Day 0 9.38±1.25 9.69 (7.88-11.22) 9.48±1.56 9.39 (8.03-12.4) 9.48±0.52 9.34 (8.75-10.3) 0.096 0.953 Day 1 9.02±1.21 9.18 (7.15-10.81) 9.76±3.53 9.29 (3.36-13.7) 9.98±0.82 9.91 (9.18-11.3) 2.111 0.348 Day 3 9.26±0.95 9.53 (7.91-10.32) 8.26±0.88 8.32 (7.07-9.75) 9.36±1.09 9.18 (7.74-11.1) 4.219 0.121 F=1.143 p=0.565 F=3.714 p=0.156 F=3.714 p=0.156 HGB (g/L) Day 0 9.60±1.56 9.50 (7.80-11.60) 10.09±2.24 9.90 (7.80-14.4) 9.91±0.70 10.10 (9.10-10.7) 0.424 0.809 Day 1 9.35±1.51 9.40 (7.20-12) 11.11±4.56 9.90 (3.40-16.2) 10.76±1.07 11.10 (9.40-12) 2.291 0.318 Day 3 9.76±1.09 10.10 (8.50-11.30) 9.06±1.13 8.90 (7.30-10.8) 10.09±1.19 9.90 (8.50-11.7) 2.498 0.287 F=2.000 p=0.368 F=1.143 p=0.565 F=3.714 p=0.156 HCT Day 0 33.36±4.45 34.30 (28.20-39.0) 35.21±6.88 35.30 (28.1-48.9) 33.93±2.21 34.10 (30.9-37.4) 0.067 0.967 Day 1 32.64±4.92 33.30 (25.60-40.1) 37.94±15.55 33.20 (12-55.20) 36.70±3.77 37.80 (31.6-40.6) 1.244 1.785 Day 3 34.60±3.98 36 (29.90-39.1) 32.17±3.35 31.60 (27.3-36.6) 34.90±3.66 35.40 (30.1-40.5) 1.785 0.410 F=0.857 p=0.651 F=0.667 p=0.717 F=1.143 p=0.565 MCV (fL) Day 0 35.60±0.84 35.50 (34.50-37) 37.07±1.35 36.90 (35.1-39.4) 35.49±1.33 35.30 (33.9-37.9) 5.138 0.077 Day 1 36.20±1.26 35.90 (34.4-38.1) 38.34±3.28 37.80 (35.2-44.3) 36.86±3.07 36.10 (34.1-43.1) 1.806 0.405 Day 3 37.40±1.14 37.20 (35.9-39.4) 39.07±2.64 38.70 (36.4-44.1) 37.39±1.79 37.10 (35.1-40.5) 2.601 0.272 F=7.185 p=0.028 F=8.000 p=0.018 F=5.429 p=0.066 MCH (pg) Day 0 10.16±0.58 10 (9.80-11.40) 10.51±0.60 10.40 (9.7-11.5) 10.23±0.25 10.30 (9.9-10.6) 2.560 0.278 Day 1 10.31±0.46 10.30 (9.70-11.10) 11.13±0.98 11.10 (10.1-12.9) 10.74±0.83 10.40 (10-12.40) 3.381 0.184 Day 3 10.47±0.26 10.50 (10.2-10.9) 10.89±0.50 10.70 (10.3-11.8) 10.71±0.42 10.50 (10.30-11.40) 3.369 0.186 F=2.571 p=0.276 F=4.692 p=0.096 F=4.000 p=0.135 4 of 10

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico MCHC (g/L) Day 0 28.67±1.35 27.80 (27.6-31.0) 28.49±1.18 28 (27.7-30.8) 29.11±0.73 29.40 (28-29.90) 2.723 0.256 Day 1 28.66±1.03 29.10 (27.2-29.9) 29.20±0.48 29.30 (28.3-29.8) 29.29±0.41 29.30 (28.8-29.8) 1.269 0.530 Day 3 28.19±0.67 28.40 (26.9-28.9) 28.06±1.15 27.80 (26.7-29.7) 28.84±1.06 28.30 (27.9-30.7) 1.496 0.473 F=1.407 p=0.495 F=6.000 p=0.050 F=2.000 p=0.368 RDW-CV (%) Day 0 15.89±0.65 16.10 (14.8-16.7) 16.07±0.56 16 (15.3-16.9) 15.70±0.71 15.80 (14.7-16.4) 1.013 0.603 Day 1 15.81±0.67 15.60 (15-16.80) 15.21±0.42 15.10 (14.9-16.1) 15.97±0.92 16.20 (14.5-17.1) 4.945 0.084 Day 3 15.91±0.60 15.70 (15.20-17) 15.66±0.50 15.90 (14.7-16.1) 16.26±0.81 15.90 (15.3-17.8) 1.484 0.476 F=1.556 p=0.459 F=6.000 p=0.050 F=2.000 p=0.368 PLT (10^9/L) Day 0 693.57±181.88 704 (417-903) 652.29±153.14 652 (442-818) 515.57±189.98 538 (197-716) 3.058 0.217 Day 1 786±207.42 800 (485-1086) 530±313.28 533 (209-1040) 493.43±207.79 529 (259-757) 4.695 0.095 Day 3 664.29±121.21 685 (495-771) 641.29±219.80 692 (342-946) 443.14±159.89 418 (244-715) 5.450 0.066 F=1.143 p=0.565 F=2.571 p=0.276 F=0.286 p=0.867 MPV (fL) Day 0 4.90±0.73 4.90 (3.90-6) 4.54±0.25 4.50 (4.30-5) 4.87±0.98 4.60 (4.10-7) 0.788 0.674 Day 1 4.97±0.58 5 (4.10-5.90) 4.63±0.41 4.50 (4.20-5.40) 5.23±1.05 4.90 (4-6.90) 1.815 0.403 Day 3 4.66±0.44 4.50 (4.10-5.20) 4.79±0.50 4.80 (4.10-5.40) 4.64±0.25 4.60 (4.30-5) 0.275 0.872 F=1.143 p=0.565 F=0.308 p=0.857 F=2.296 p=0.317 PDW (fL) Day 0 6.81±1.50 6.80 (4.90-8.90) 5.97±0.59 5.80 (5.50-7.10) 6.37±1.61 5.80 (5.20-9.90) 0.919 0.632 Day 1 6.77±1.34 6.80 (4.90-8.90) 6.31±0.99 5.80 (5.20-7.70) 6.30±2.12 6.80 (3.10-9.90) 0.510 0.487 Day 3 6.10±0.92 5.80 (4.90-7.40) 6.54±1.58 5.80 (4.90-9.60) 6.40±0.67 6.50 (5.20-7.40) 0.487 0.784 F=0.286 p=0.867 F=1.000 p=0.607 F=0.857 p=0.651 PCT (%) Day 0 0.33±0.09 0.37 (0.20-0.44) 0.29±0.07 0.31 (0.20-0.37) 0.25±0.08 0.28 (0.08-0.32) 2.249 0.325 Day 1 0.38±0.10 0.42 (0.24-0.50) 0.23±0.13 0.23 (0.10-0.43) 0.24±0.08 0.24 (0.15-0.39) 7.046 0.030 Day 3 0.30±0.07 0.32 (0.21-0.38) 0.29±0.07 0.32 (0.18-0.38) 0.20±0.07 0.20 (0.12-0.32) 7.069 0.029 F=1.143 p=0.565 F=2.571 p=0.276 F=1.143 p=0.565 P-LCR (%) Day 0 0.27±0.72 0 (0-1.90) 0.00±0.00 0 (0-0) 2.51±6.65 0 (0-17.60) 1.057 0.589 Day 1 0.39±0.69 0 (0-1.70) 0.69±1.42 0 (0-3.80) 5.21±8.41 0 (0-18.70) 0.959 0.619 Day 3 0.16±0.42 0 (0-1.10) 1.46±2.17 0 (0-5.70) 0.00±0.00 0 (0-0) 4.594 0.101 F=0.500 p=0.779 F=2.800 p=0.247 F=3.500 p=0.174 P-LCC (10^9/L) Day 0 1.86±4.91 0 (0-13) 0.00±0.00 0 (0-0) 8.29±21.92 0 (0-58) 1.057 0.589 Day 1 2.71±5.09 0 (0-13) 2.00±3.61 0 (0-9) 16.86±25.24 0 (0-58) 1.161 0.560 Day 3 1±2.65 0 (0-7) 6.43±9.24 0 (0-24) 0.00±0.00 0 (0-0) 4.594 0.101 5 of 10

Levamisole effect over Hematological and Biochemical Parameters in sheep / Susar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico F=0.500 p=0.779 F=2.800 p=0.247 F=3.500 p=0.174 Neutrophills Day 0 20.70±1.26 20.60 (19.3-22.4) 21.50±1.37 21.40 (19.6-22.3) 22±001.10 20.00 (20.3-24.1) 4.124 0.088 Day 1 20.50±1.14 20.30 (20.1-22.3) 31.60±1.24 31.10 (28.7-33.4) 20.60±1.66 20.70 (20.1-21.9) 1.223 0.030 Day 3 20.40±1.35 20.10 (18.5-22.6) 32.70±1.64 32.30 (31.8-32.6) 20.50±1.14 22.40 (19.5-22.1) 2.401 0.028 F=1.164 p=0.578 F=6.000 p=0.017 F=2.644 p=0.032 Lympho- cytes Day 0 52.40±1.73 52.10 (48.7-55.2) 53.50±1.61 53.10 (48.7-56.2) 52.50±2.04 53.40 (52.6-56.4) 3.126 0.125 Day 1 52.50±1.64 52.40 (50.3-53.8) 65.40±2.10 65.50 (58.3-69.6) 53.30±2.11 53.50 (52.1-55.8) 5.154 0.043 Day 3 50.10±1.26 50.40 (50.1-51.6) 70.60±1.36 70.20 (66.8-73.4) 53.60±1.68 52.40 (50.2-53.5) 5.076 0.035 F=3.195 p=0.074 F=2.441 p=0.021 F=1.144 p=0.043 Monocytes Day 0 1.30±0.62 1.20 (0.90-2.10) 1.60±1.01 1.40 (1.20-2) 2.40±0.86 2.30 (1.50-3.30) 2.129 0.136 Day 1 1.80±0.81 1.60 (1.20-1.90) 4.20±1.04 2.30 (1.60-3.10) 1.10±1.24 1.70 (1.60-2.30) 6.074 0.038 Day 3 1.60±1.03 1.50 (1-1.60) 6.00±1.14 4.70 (3.40-6.60) 3.70±0.86 2.70 (1.90-4.20) 5.854 0.026 F=4.574 p=0.765 F=2.741 p=0.028 F=3.496 p=0.035 Eosinophils Day 0 4.40±1.37 4.20 (3.10-5.60) 6.60±1.05 6.30 (5.80-7.40) 6.50±0.74 6.40 (5.70-7.70) 3.805 0.064 Day 1 4.80±1.58 4.50 (3.90-6.10) 4.50±0.74 4.10 (4-5.30) 5.00±1.23 5.00 (4.80-6.20) 2.259 0.191 Day 3 3.30±0.84 3.20 (3.10-4.80) 3.60±0.86 3.50 (3.30-4.20) 3.70±0.84 3.80 (3.50-4.90) 3.716 0.225 F=2.000 p=0.373 F=1.153 p=0.675 F=2.364 p=0.125 F: Firedman Test, H: Kruskal-Wallis H Test, P<0.05, SD: Standart deviaon, Min: Minumum, Max: Maximum, WBC: White blood cell, RBC: Red blood cell, HGB: Haemoglobin, HCT: Hematoc- rit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentraon, RDW-CV: Red blood cell distribuon width, PLT: Platelet count, MPV: Mean platelet volume, PDW: Platelet distribuon width, PCT: Procalcitonin, P-LCR: Platelet large cell rao, P-LCC: Platelet large cell coefficient The results from d 1 of urea measurements demonstrated a stascally significant difference between the groups (P<0.05). The mean urea level on d 1 was significantly elevated in the control group (52.43±7.55) compared to the liposomal levamisole group (43.71±4.82) and the free levamisole group (46.14±5.34). On d 3 of the study, a stascally significant difference was found between the groups (P<0.05). The mean urea level on d 3 was significantly elevated in the control group (52.43±6.45) compared to the liposomal levamisole group (50.00±3.11) and the free levamisole group (45.43±2.37). A stascally significant difference was found in the urea values of the animals in the liposomal levamisole group on d 0, 1, and 3 (P<0.05). On d 1 the recorded value was markedly lower than the subsequent tests. On d 3, a stascally significant difference in creanine values was found between the groups (P<0.05). The mean creanine level on d 3 of the study was significantly elevated in the control group (0.67±0.05) in comparison to both the liposomal levamisole group (0.56±0.05) and the free levamisole group (0.57±0.02). The analysis of the intragroup values demonstrated a stascally significant disparity among the three measurements across all three groups (P<0.05). The minimum creanine levels were recorded in all three groups on d 3. Similarly, significant differences were detected in urea and creanine levels between the groups. In parcular, the significantly higher creanine levels in the control group on the d 3 suggest the potenal effects of levamisole on kidney funcon. This finding indicates the need for further research on the eliminaon mechanisms of levamisole. On d 3 of the study, a stascally significant difference was observed between the groups regarding BUN values (P<0.05). The mean BUN value on d 3 for the control group (23.88±2.57) was significantly higher than that recorded for the liposomal levamisole group (23.29±1.28) and the free levamisole group (21.32±1.12). A stascally significant difference was idenfied in the BUN values of the animals in the liposomal levamisole group on d 0, 1, and 3 (P<0.05). On d 1, a markedly lower value was recorded. A stascally significant difference was found in the AST values of the control group animals on d 0, 1, and 3 (P<0.05). The data collected on day 0 exhibited markedly higher levels than those recorded on d 1 and 3. A stascally significant difference was observed between the groups in the ALT values on d 1 (P<0.05). The mean ALT value on the inial observaon d for the control group (14.57±3.46) was significantly lower than that 6 of 10

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico of the liposomal levamisole group (20.14±2.85) and the free levamisole group (17.43±4.43). A stascally significant difference was found among d 0, d 1, and d 3 counts of neutrophils, lymphocytes, and monocytes in both the liposomal and free levamisole groups (P<0.05). The measurement on d 3 demonstrated a significantly higher value compared to the other measurements. No stascally significant differences were found in the other variables when comparing intra- and inter-group data (P>0.05) (TABLE III). Significant increases were observed in neutrophil, lymphocyte, and monocyte counts in the liposomal and free levamisole groups. This increase is thought to be associated with the smulatory effects on the immune system. In this context, analyzing similar parameters in different animal species will contribute to a beer understanding of the biological effects of levamisole. TABLE III. Intragroup and intergroup comparisons of biochemical parameters Control Liposomal Levamisole Free Levamisole Mean±SD Median (Min-Max) Mean±SD Median (Min-Max) Mean±SD Median (Min-Max) H p Urea Day 0 45.71±3.99 45 (40-52) 50.29±7.36 52 (40-62) 47.71±5.65 49 (37-53) 2.265 0.322 Day 1 52.43±7.55 51 (42-67) 43.71±4.82 45 (35-49) 46.14±5.34 46 (38-56) 6.714 0.035 Day 3 52.43±6.45 49 (47-63) 50.00±3.11 49 (46-55) 45.43±2.37 46 (42-49) 8.922 0.012 F=4.667 p=0.097 F=8.074 p=0.018 F=0.963 p=0.618 Creanine Day 0 0.78±0.08 0.76 (0.99-0.91) 0.78±0.06 0.78 (0.67-0.84) 0.83±0.06 0.84 (0.76-0.89) 2.051 0.359 Day 1 0.81±0.06 0.81 (0.73-0.92) 0.73±0.11 0.76 (0.62-0.94) 0.76±0.09 0.74 (0.65-0.92) 3.588 0.166 Day 3 0.67±0.05 0.66 (0.62-0.77) 0.56±0.05 0.55 (0.51-0.65) 0.57±0.02 0.57 (0.55-0.61) 11.890 0.003 F=8.000 p=0.018 F=10.296 p=0.006 F=11.143 p=0.004 BUN Day 0 21.97±2.23 22.65 (18.69-24.6) 23.72±3.58 24.77 (18.7-28.97) 22.08±2.54 22.08 (17.3-24.8) 1.732 0.421 Day 1 23.50±3.93 23.36 (19.6-31.3) 20.04±2.18 19.76 (16.36-22.9) 21.69±2.58 21.96 (17.8-26.2) 3.770 0.152 Day 3 23.88±2.57 22.90 (21.9-29.4) 23.29±1.28 22.90 (21.5-25.04) 21.32±1.12 21.50 (19.63-22.9) 8.543 0.014 F=0.667 p=0.717 F=8.000 p=0.018 F=0.963 p=0.618 AST Day 0 120.29±56.79 107 (77-245) 129.29±9.81 130 (113-141) 131.86±38.30 118 (91-191) 3.684 0.159 Day 1 110.29±21.95 99 (85-144) 108.43±19.64 101 (86-146) 113.71±20.01 111 (87-143) 0.387 0.824 Day 3 89.71±10.63 88 (78-109) 112.29±21.85 108 (81-150) 103.29±22.69 94 (88-152) 5.286 0.071 F=7.143 p=0.028 F=2.296 p=0.317 F=3.714 p=0.156 ALT Day 0 16.57±4.65 16 (11-24) 22.57±4.24 20 (18-28) 22.29±4.64 22 (14-29) 5.549 0.062 Day 1 14.57±3.46 16 (10-18) 20.14±2.85 20 (17-23) 17.43±4.43 18 (11-23) 6.137 0.046 Day 3 14.57±3.78 16 (8-19) 17.71±3.90 17 (12-23) 19.29±4.31 19 (13-24) 3.916 0.141 F=0.538 p=0.764 F=3.769 p=0.152 F=5.429 p=0.066 F: Firedman Test, H: Kruskal-Wallis H Test, P<0.05, SD: Standart deviaon, Min: Minumum, Max: Maximum, BUN: Blood urea nitrogen, AST: Aspartate aminotransferase, ALT: Alanine transaminase Besides its anthelminc properes, levamisole has been shown to possess immunosmulant effects. The advised dosage is 7.5 mg/kg administered orally once. Adverse effects are linked to a narrow therapeuc index resulng from overdose and acvaon of niconic acetylcholine receptors, leading to a reduced convulsion threshold, respiratory muscle paralysis, and asphyxia [20 , 21]. No adverse effects were observed in this study; however, symptoms related to levamisole toxicity were documented in some animals [22]. Addionally, symptoms linked to depression, anorexia, seizures, ataxia, and frothy salivaon in canines were documented [23]. Furthermore, the occurrence of severe niconic-type symptoms, such as hypersalivaon, oral foaming, muscle tremors, recumbency, and tachypnea in Friesian calves was recorded [24]. A study was performed to examine the effects of levamisole on the haematobiochemical profiles of gastrointesnal nematodosis in sheep. The results indicated a substanal 7 of 10

Levamisole effect over Hematological and Biochemical Parameters in sheep / Susar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico elevaon in the average values of hemoglobin (HGB), packed cell volume (PCV), glucose, iron (Fe), and calcium (Ca) ten days post- treatment. A notable reducon was observed in the average values of total leucocyte count (TLC), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) [25]. Other researchers assessed the therapeuc efficacy of the triclabendazole- levamisole combinaon in endoparasic infecons in sheep. In hematological findings, it was established that HCT, HGB, and eosinophil raos were significantly reduced compared to the control group on d 0. Nonetheless, they indicated that these values ascended to the levels of the control group by the 14th d post-treatment, and no stascally significant differences were observed in other hematological parameters when compared to the control group and the baseline (d 0) [26]. In this study, contrary to previous studies, HGB, HCT, eosineophil values were not affected and AST was found to be different in the control group. The impact of therapeuc and toxic doses of levamisole on thyroid hormone levels and certain biochemical parameters in Akkaraman breed sheep was studied. Administraon of levamisole orally at doses of 7.5 mg/kg and 40 mg/kg resulted in a decrease in ALP acvies, while acvies of AST, ALT, lactate dehydrogenase (LDH), and creanine kinase (CK) increased significantly [27]. A study was performed on certain haemato- biochemical parameters of sheep subjected to levamisole administraon during transport. Aſter the administraon of 15 mg/kg intramuscular levamisole, sheep were transported over a distance of 260 km within 30 min. PCV decreased, while total serum protein, total leucocyte count, absolute lymphocyte count, and absolute neutrophil count remained relavely unchanged; however, serum albumin levels increased significantly. The researchers determined that intramuscular administraon of levamisole at a dosage of 15 mg/kg, 30 min prior to the transportaon of Yankasa sheep, elevated serum albumin levels and the neutrophil-to-lymphocyte rao [28]. This study yielded results consistent with [27], indicang that ALT acvies were elevated in both free and liposomal levamisole groups relave to the control. Likewise, the neutrophil and lymphocyte raos in the liposomal and free levamisole groups of animals were markedly elevated compared to other measurements recorded on the third day. A study revealed that the percentages of monocytes, erythrocyte sedimentaon rate, and packed cell volume at 1 and 24 h, as well as MCV and MCHC concentraons, increased aſter Sahiwal heifers were administered 7.5 mg/kg of levamisole [29]. Others study was performed on the haematological and serum biochemistry parameters of levamisole in buffaloes (Bubalus bubalis), with oral administraon of levamisole at a dosage of 15 mg/kg resulted in a significant increase in monocytes, PCV, MCV, and MCHC aſter day 1 to d 7. On d 1, both WBC and RBC levels rose, but subsequently declined. Significant increases in total serum protein, albumin, and globulin concentraons were observed on d 1 and 7, while serum urea concentraon significantly increased on d 14 [30]. The impact of levamisole administraon on the immune system of cale vaccinated for anthrax was studied. They determined that there was no disparity between the groups in neutrophil, eosinophil, monocyte, and lymphocyte counts on d 0; however, the elevaon in neutrophil and monocyte counts in the levamisole group relave to the vaccine group was stascally significant, while the increase in lymphocyte counts on d 7 was not stascally significant. On d 14, the levels of neutrophils, monocytes, and lymphocytes in the levamisole-vaccine group exhibited a significant increase compared to the vaccine group. The findings of this study align with the literature regarding certain parameters [31]. A study was carried out on the performance and hematology of dietary levamisole in juvenile Piaractus mesopotamicus. It was determined that haematological parameters, including HGB, plasma glucose, WBC, and differenal leukocyte count, were influenced by levamisole. They reported that the counts of WBC, lymphocytes, neutrophils, monocytes, eosinophils, and specialized granulocyc cells significantly decreased aſter 15 d, and that prolonged levamisole administraon beyond 15 d exhibited toxicity to lymphopoiec ssues [32]. A study was performed to evaluate the toxicological, hematological, and immunological impacts of dietary levamisole and ivermecn on Colossoma macropomum (Serrasalmidae). The study demonstrated that levamisole did not induce mortality or behavioral alteraons in fish. Ivermecn was determined to induce 100% mortality at specific feeding concentraons. Aſter 24, 96, and 240 h of administraon, there were no changes in erythrocyte parameters or albumin levels in any treatment group administered levamisole. Levamisole, at doses of 900 and 1200 mg/kg, resulted in reducons in the albumin-globulin rao relave to the control group and the 300 and 600 mg/kg treatment groups. Fish administered diets of 600, 900, and 1200 mg/kg exhibited elevated glucose and total plasma protein levels relave to the control and 300 mg/kg diet groups, alongside an increase in leucocyte acvity. The researchers determined that levamisole is effecve in the diet of C. macropomum for anparasic intervenons against helminth species and that dietary administraon of levamisole can enhance elements of the innate immune system [33]. A study assessed the impact of single and triple levamisole administraon on hematological parameters in rats with experimental pleuris. Three doses of levamisole resulted in a notable reducon in RBC and an increase in MCV 48 h post- administraon. The administraon of a single dose of levamisole resulted in a notable elevaon in hematocrit and neutrophil count at 72 h, as well as an increase in white blood cell count at both 24 and 72 h. The study determined that both single and triple doses of levamisole administraon resulted in stascally significant alteraons in certain hematological parameters, thereby influencing the inflammatory process [34]. A study on the haematological and biochemical parameters of levamisole in broiler chickens was conducted. Broilers administered levamisole exhibited elevated anbody tres in contrast to those not treated with levamisole. No differences were observed in TEC, HGB, and PCV, and creanine levels remained within the normal range among the treated groups. ALT and AST levels were elevated in birds treated with a high dose (10 mg/ kg) of levamisole. Serum total cholesterol, LDL cholesterol, and triglycerides were elevated, while HDL cholesterol remained unchanged [35]. This study aligns with the findings of exisng literature. The variaons in certain parameters may be aributable to the differences among animal species. To acquire more comprehensive informaon, liposomal levamisole should be sourced from various animal species, including fish, broiler chickens, and cale. 8 of 10

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico CONCLUSIONS In this study, a liposomal levamisole formulaon was prepared for use in sheep. Aſter characterisaon, it was administered orally. It was established that liposomal levamisole has the potenal to induce alteraons in haematological and biochemical parameters. A limitaon of this study is that it was conducted using only sheep as the subject populaon. Thanks to this research, studies on liposomal levamisole in other animal species should be carried out and the results should be evaluated comprehensively. If the findings demonstrate posive outcomes, the producon of commercially available drugs containing liposomal levamisole for the treatment of parasites and immunological purposes in animals within the domain of veterinary medicine will become a viable opon. In conclusion, this study is an important invesgaon evaluang the effects of different formulaons of levamisole on hematological and biochemical parameters. Liposomal levamisole was observed to have the potenal to modulate the immune response. Accordingly, liposomal levamisole should be evaluated in large-scale clinical studies, and its long-term effects should be determined by applying different doses. In this regard, the present study will serve as a example for future research in this field. Funding This research was conducted with the support of the Balıkesir University Scienfic Research Coordinaon Office (BAP Project No: 2022 / 084). Conflict of Interest The authors have stated that they do not have any compet- ing interests. BIBLIOGRAPHIC REFERENCES [1] Sajid MS, Iqbal Z, Muhammad G, Iqbal MU. Immunomodulatory effect of various an-parasics: a review. Parasitol. [Internet]. 2006; 132(3):301-313. doi: hps://doi.org/c2sx3r [2] McKellar QA, Gokbulut C. 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