Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 03/10/2025 Aceptado: 08/01/2026 Publicado: 29/01/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 7 Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Effects of lactaon period on some blood parameters and ANAE/AcP-ase enzyme acvies in domesc Donkeys Efectos del período de lactancia sobre algunos parámetros sanguíneos y acvidades de la enzima ANAE/AcP-asa en asnos doméscos Mehmet Faruk Aydin 1,* , İhsan Kisadere 2 ¹Department of Histology and Embryology, Faculty of Veterinary Medicine, Balıkesir University, Balıkesir 10050, Türkiye ²Department of Physiology, Faculty of Veterinary Medicine, Balıkesir University, Balıkesir 10050, Türkiye *Corresponding Author: mfaydin@balikesir.edu.tr. ABSTRACT It was aimed to research the influences of the lactaon period on some hematological (red blood cells, white blood cells, and its subtypes, hematocrit, hemoglobin, mean erythrocyte hemoglobin, mean corpuscular hemoglobin concentraon, mean erythrocyte volume, and blood clot count) and biochemical parameters (alanine amino transferase, aspartate amino transferase, gamma glutamyl transferase, blood urea nitrogen, and creanine) as well as α - naphthyl acetate esterase and acvity of the enzymes acid phosphatase posive lymphocytes percentages in domesc donkeys. For this purpose, 20 female donkeys were selected and divided into two equal groups as control (non-lactang, n = 10) and lactang (n = 10). They were in the middle of lactaon (three to six months old), ranging in age from five to fourteen. Following the roune clinical examinaon, blood samples were taken from the animals in order to measure the acvies of the α - naphthyl acetate esterase / acvity of the enzymes acid phosphatase enzyme as well as several blood parameters. As a result, red blood cells, hematocrit, and hemoglobin values were higher in lactang donkeys than non-lactang donkeys (P < 0.05). Serum aspartate amino transferase and gamma glutamyl transferase values were found to be stascally higher in lactang animals (P < 0.05). Although enzyme acvies were high in lactang donkeys, α - naphthyl acetate esterase and acvity of the enzymes acid phosphatase posive lymphocyte percentages were found to be similar between the groups (P > 0.05). In conclusion, the middle-lactaon phase in female donkeys was found to have an impact on several blood parameters, but not on the acvity of enzymes. Key words: biochemistry; Equus asinus; hematology; lactaon RESUMEN El objevo fue invesgar la influencia del período de lactancia en algunos parámetros hematológicos (glóbulos rojos, leucocitos y sus subpos, hematocrito, hemoglobina, hemoglobina media, hemoglobina media, volumen volumétrico medio y plaquetas) y bioquímicos (alanina aminotransferasa, nitrógeno ureico en sangre y creanina sérica), así como en el porcentaje de linfocitos posivos para en asnos hembras doméscas. Para ello, se seleccionaron 20 burras y se dividieron en dos grupos iguales: control (no lactantes, n = 10) y lactantes (n = 10). Se encontraban en la mitad de su período de lactancia (de tres a seis meses de edad), con edades comprendidas entre los cinco y los catorce años. Tras el examen clínico de runa, se tomaron muestras de sangre de las animales para medir la acvidad de la enzima, así como diversos parámetros sanguíneos. Como resultado, los valores de glóbulo rojo, hematocrito y hemoglobina fueron mayores en las asnos hembras lactantes que en las no lactantes (P < 0,05). Se observó que los valores séricos de fueron estadíscamente superiores en animales lactantes (P < 0,05). Si bien la acvidad enzimáca fue alta en asnos hembras lactantes, los porcentajes de linfocitos posivos para fueron similares entre los grupos (P > 0,05). En conclusión, se observó que la fase media de la lactancia en asnos hembras influyó en varios parámetros sanguíneos, pero no en la acvidad enzimáca. Palabras clave: Bioquímica; Equus asinus; hematología; lactancia https://doi.org/10.52973/rcfcv-e361815

Effects of lactaon period on female donkeys / Aydin and Kisadere UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Historical processes indicate that humans domescated donkeys (Equus asinus) about 5,000 years ago. The donkey is one of the domescated animals of the equine family, as is widely known. They have been significant throughout human history and all phases of evoluon [1]. Donkeys were mostly used as load carriers and as riding animals in the past. The growing popularity of donkeys is aributed to the possible health benefits of donkey milk, even though they are sll ulized for onotherapy, sports, labor, and rural transportaon [2],3]. Their populaon has increased globally as a result of this phenomenon, parcularly in Asia and Europe. Donkey meat is eaten by humans like other meats in several regions of the world, parcularly in Asian sociees [4 ,5]. Nearly all female mammals, including horses and donkeys, nurse their offspring aſter parturion by secreng milk from their mammary glands. This period is called as lactaon. In general, the lactaon period is divided into three periods, especially for donkeys and horses, as early, middle, and late lactaon [6]. Previous studies on donkeys and horses have showen that the lactaon period significantly affects many physiological, hematological, and biochemical parameters [7 , 8 ,9]. The primary causes of these changes can be idenfied as the mother’s (a mule or female donkey) increased energy requirements during nursing, hormonal and immunological changes, stress, and environmental factors [10]. The health condion of the donkeys and also horses (alongside other animals) is frequently evaluated by using hematological markers. These markers help Veterinaries, specialists, and also researchers to diagnosis and prognosis of many equine diseases. The count of white blood cells (WBC) and its subtypes, hemoglobin (HGB) concentraon, red blood cells count (RBC), hematocrit (HCT) value, mean erythrocyte volume (MCV), mean erythrocyte hemoglobin (MCH) and its concentraon (MCHC), and blood clot count (PLT) are most frequently used hematological parameters in this area. Furthermore, a wide range of variables including species status, age, breed, diet, sex, disease, and seasonal fluctuaons have been documented to have an impact on the paern of these parameters [11 , 12]. In both clinical treatment and fundamental research, the evaluaon of an animal’s status can be facilitated by measuring its blood biochemical characteriscs. Variaons in blood biochemical markers are a useful tool for evaluang the health, welfare, and stress level of an animal since they are frequently suggesve of alteraons in the animal’s physiological state (e.g., gestaon, parturion, and lactaon). In this sense, it is crical to determine some enzyme levels to reveal the general condion of the liver and kidneys, especially in a different metabolic phenomenon such as lactaon [13]. Gamma glutamyl transferase (GGT), blood urea nitrogen (BUN), alanine amino transferase (ALT), aspartate amino transferase (AST), and creanine (CREAT) can be given as samples of these important enzyme types [14]. Based on the data gathered from earlier research, using enzyme histochemical techniques seems to be an easy and affordable process. The main purpose of enzyme histochemical techniques is to have crucial data regarding the identy, locaon, and quanty of an enzyme. Determining the acvity of the enzymes acid phosphatase (AcP - ase) and α - Naphthyl acetate esterase (ANAE) is one of these important histochemical techniques. The primary purpose of these techniques is to categorize lymphocyte subsets. According to reports, ANAE appears later in the thymus during the T - lymphocyte maturaon phase, while AcP - ase occurs early on [15 , 16]. Naphthyl acetate esterase belongs to the nonspecific esterase family, which is extensively present in different kinds of cells. According to research, both adult and immature T lymphocytes exhibit ANAE, a lymphocyte lysosomal enzyme that is specifically localized in lysosome membranes. Numerous research on the topic has highlighted how crucial ANAE is for differenang peripheral blood samples, parcularly those containing T and B cells and monocytes [16 , 17]. Moreover, AcP - ase, like ANAE, is a lysosomal enzyme. For many cell types, especially mammalian T-lymphocytes, this enzyme is unique. It has also been noted in earlier research that fetal thymocytes exhibit AcP - ase enzyme acvity [18]. Important illnesses, including T cell lymphoproliferaon, have also been diagnosed with this technic [19]. According to earlier research, a variety of physical and metabolic acvies can influence the acvies of the ANAE and AcP - ase enzymes in living organisms including donkeys and horses. The rates of these indicators have also been found to be influenced by species, age, sex, and stress levels [11 , 12 , 20 , 21]. The purpose of the study was to examine how the lactaon period affected the percentages of ANAE and AcP-ase posive lymphocytes as well as certain hematological (RBC, WBC, HCT, HGB, MCH, MCHC, MCV, and PLT) and biochemical (ALT, AST, GGT, BUN, and CREAT) parameters in domesc donkeys. MATERIAL AND METHODS Animals and sample collecon Twenty female donkeys were chosen as animal material and split into two equal groups: 10 lactang (n = 10) and 10 non- lactang (control, n = 10). Animals were from 5 to 14 years old, and into the middle lactaon period (~ 3 - 6 months). Donkeys were housed at the Aegean Donkey Farm in Edremit District of Balikesir Province. They were parally grazed and fed ad libitum with hay produced on the farm. No mineral or vitamin supplements were given during the study. Before the applicaons, general clinical examinaon techniques were ulized to idenfy the healthy state, and the animals did not exhibit any abnormal symptoms. A 1.2 mm × 38 mm needle was used to draw blood samples from the jugular vein into both heparinized and non-heparinized tubes. Then, the samples were quickly moved by the cold chain to the laboratory for analysis. For every animal, four smears were done by using blood samples [ANAE (2) and AcP - ase (2) stainings]. Hematological parameters Abacus Junior Vet - 5 (Vienna, Austria) hematology analyzer was used to idenfy parameters (WBC and its subtypes, HGB, RBC, PLT, MCV, HCT, MCH, and MCHC) in female donkeys. Biochemical parameters Using an automated biochemical analyzer (BS - 400 PLUS, Mindray, China), the levels of serum ALT, AST, GGT, BUN, and CREAT were determined. 2 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico α - Naphthyl acetate esterase acvity Naphthyl acetate esterase was demonstrated with some modificaons ulizing Aydin’s approach no mineral or vitamin supplements were given during the study [12]. The produced smears were first fixed for three min at - 10 °C in a glutaraldehyde acetone soluon. They were then air-dried at room temperature (24 °C) aſter being cleaned three mes in dislled water. In order to prepare the ANAE soluon, 0.8 mL of acetone (Merck, Germany) was used to dissolve 20 mg of α – naphthyl - acetate (N - 8505; Sigma, Germany). The afore menoned soluon was then supplemented with 4.8 mL of hexazozed pararosaniline. Subsequently, 80 mL of buffered phosphate saline (pH = 5) was filled with dissolved α-naphthyl-acetate and hexazozed pararosaniline mix soluon. Aſter adding 1 N NaOH to the produced ANAE soluon, the pH was finally adjusted to 5.8 and filtered. Following a 2 - hour (h) incubaon period at 37 °C, the produced smears underwent three rinses with distilled water. Then, leukocyte nuclei were stained for 20 min using 1 % methyl green (Sigma, Germany) that had been produced in an acetate buffer with a pH of 4.2. enzymes acid phosphatase acvity With minor modificaons, AcP - ase acvity was demonstrated ulizing Donmez and Sur’s [16] methodology. The ready samples were fixed for ten min at 4 °C in formal - Ca. They were then cleaned with dislled water. The AcP - ase soluon was made as follows: 1 mL of N,N - dimethyl formamide (Sigma, Germany), 13 mL of dislled water, 1.6 mL of hexazozed pararosaniline, and 5 mL of veronal acetate buffer (Michaelin’s, pH = 5) were used to dissolve 10 mg of naphthol AS-BI phosphate (N-2125, Sigma, Germany). The produced AcP - ase soluon was filtered, and its pH was adjusted to five using one N sodium hydroxide. Following a 1 - h incubaon period at 37 °C, the blood smears underwent three rounds of rinsing with distilled water. Leukocyte nuclei were then stained for 20 min with 1 % methyl green (Sigma, Germany) in acetate buffer (pH = 4.2). Using a light microscope (Leica DM 2500, Wetzlar, Germany), all smear samples were examined. Two hundred lymphocytes were counted in each of the prepared smears showing ANAE and AcP - ase posives, and the percentage of cells exhibing an enzyme-posive lymphocyte rao was announced. Stascal analysis The SPSS 22.0 soſtware was ulized to analyze the differenc- es between the groups by an independent samples T-test. The suitability of the data for normal distribuon was examined with the Shapiro - Wilk test. Since the data regarding ANAE and AcP enzyme acvies did not comply with normal distribuon, the Mann - Whitney U test was used to compare the two groups. (SPSS, Inc, Chicago, IL). P values less than 0.05 were regarded as significant. RESULTS AND DISCUSSIONS Hematological parameters It was not detected any stascal alteraons between groups according to WBC, lymphocyte (lym), monocyte (mon), and granulocyte (gran) counts in this study (P > 0.05). In addion, lym, mon, and gran percentages were not affected from the lactaon period in the present study (P > 0.05). Conversely, HGB, HCT, and RBC values were higher in lactang donkeys than non- lactang donkeys (P < 0.05). Other hematological parameters such as MCV, MCH, MCHC, and PLT also were not influenced by the lactaon period in the present study (P > 0.05), as shown in TABLE I. TABLE I Hematological parameters in different (lactang / non-lactang) donkey groups Parameters / Groups n Mean ± SE WBC (10 9 / L) lactaon 10 11.14 ± 1.32 control 10 14.17 ± 1.95 lym (%) lactaon 10 38.22 ± 3.78 control 10 39.68 ± 3.00 mon (%) lactaon 10 5.18 ± 0.58 control 10 4.56 ± 0.37 gran (%) lactaon 10 56.60 ± 4.00 control 10 55.76 ± 3.03 lym (#) lactaon 10 4.30 ± 0.68 control 10 5.77 ± 1.03 mon (#) lactaon 10 0.54 ± 0.09 control 10 0.58 ± 0.05 gran (#) lactaon 10 6.30 ± 0.79 control 10 7.79 ± 1.07 RBC (10 12 / L) lactaon 10 7.20 ± 1.14a control 10 5.69 ± 0.63b MCV(fL) lactaon 10 70.25 ± 1.24 control 10 68.14 ± 1.22 HCT (%) lactaon 10 50.04 ± 7.76a control 10 39.03 ± 4.78b MCH(pg) lactaon 10 19.13 ± 0.33 control 10 17.93 ± 0.31 MCHC(pg) lactaon 10 27.32 ± 0.31 control 10 26.39 ± 0.25 HGB (g / dL) lactaon 10 13.89 ± 2.31a control 10 10.35 ± 1.30b PLT (10 9 / L) lactaon 10 110.70 ± 13.03 control 10 150.00 ± 23.23 a-b Values within a column with no common superscripts are different, P < 0.05. U/L: Units per liter, mg: milligram, dL: deciliter, fL: femtoliters, (g/L): grams per liter, pg: picograms, (%): percentage, (#): count, WBC: White blood cell, Lym: Lymphocyte, Neu: Neutrophil, Mono: Monocyte, Eos: Eosinophil, RBC: Red blood cell, HGB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentraon, PLT: Platelet count, SE: Standart Error 3 of 7

Effects of lactaon period on female donkeys / Aydin and Kisadere UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Biochemical parameters In this study, there is no difference was determined when serum ALT values were compared between groups (P > 0.05). On the other hand, lactang animals had stascally increased serum AST and GGT values (P < 0.05). BUN and CREAT values of the donkeys were also not affected by the lactaon period in the present study (P > 0.05), as shown in TABLE II. TABLE II Biochemical parameters in different (lactang / non-lactang) donkey groups Parameters / Groups n Mean ± SE ALT (U / L) lactaon 10 7.45 ± 0.05 control 10 7.32 ± 0.29 AST (U / L) lactaon 10 176.33 ± 11.97a control 10 142.60 ± 7.34b GGT (U / L) lactaon 10 19.19 ± 1.45a control 10 14.04 ± 1.41b BUN (mg / dL) lactaon 10 37.53 ± 2.01 control 10 35.51 ± 2.69 CREAT (μmol / L) lactaon 10 58.21 ± 2.87 control 10 57.78 ± 3.39 a-b Values within a column with no common superscripts are different, P < 0.05. ALT: alanine amino transferase, AST: aspartate amino transferase, GGT: Gamma glutamyl transferase, BUN: blood urea nitrogen, CREAT: creanine, U/L: Units per liter, mg: milligram, dL: deciliter, μmol: micromol, SE: Standart Error α-Naphthyl acetate esterase and enzymes acid phosphatase posive lymphocyte percentages Lactang donkeys had elevated enzyme acvity, although the percentages of ANAE and AcP-ase-posive lymphocytes were similar among the groups. They were also not influenced by the lactaon period in donkeys (P > 0.05), as shown in TABLE III. In addion, ANAE and AcP - ase posive lymphocytes were presented in FIGS. 1, 2, 3, 4. TABLE III ANAE and AcP - ase enzyme acvies in different (lactang / non - lactang) donkey groups Parameters / Groups n Mean ± SE ANAE lactaon 10 74.17 ± 1.05 control 10 73.04 ± 1.18 AcP - ase lactaon 10 65.13 ± 1,59 control 10 62.91 ± 1.89 ANAE: α - naphthyl acetate esterase, AcP – ase: acid phosphatase enzyme, SE: Standart error FIGURE 1. α - Naphthyl acetate esterase posive lymphocyte in non - lactang donkeys. Arrow: ANAE posive lymphocyte FIGURE 2. Acid phosphatase posive lymphocyte in non - lactang donkeys. Arrow: AcP posive lymphocyte FIGURE 3. α - Naphthyl acetate esterase posive lymphocytes in lactang donkeys. Arrow: ANAE posive lymphocytes 4 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 4. Acid phosphatase posive lymphocyte in lactang donkeys. Arrow : AcP posive lymphocyte Assessing hematological parameters is a useful step toward figuring out a living being’s overall health. In the present study, WBC counts were not influenced by the middle lactaon period in donkeys. Besides, the number and percentages of WBC subgroups (lym, mon, and gran) were also found to be similar between both lactang and non-lactang donkeys. On the other hand, Gul et al. [6] suggested that WBC counts increased due to the lactaon period in donkeys. In addion, neutrophil and eosinophil percentages decreased, however mon, lym, and basophil percentages increased in non-lactang donkeys compared to lactang donkeys in the same research. Dezzuo et al. [8] also wanted to evaluate the changes in the hematological, electro-phorec, and hemato-chemical parameters in different lactang periods of donkeys. As a result, they revealed that the WBC count was higher in early lactaon than in middle and late lactaon, and the rate of WBC subgroups (lym, mon, and gran) was not affected by different lactaon periods which was consistent with this study’s results. Following earlier research on mares, Bonelli et al. [9] also found that donkeys had a higher WBC count at foaling than throughout late pregnancy and lactaon [22 ,[23]. Salari et al. [7] suggested that the three phases of the lactaon period (early, middle, and late) did not affect the WBC counts in lactang donkeys. Hormonal fluctuaons (especially the release of corsol and catecholamine) occurring during different periods of lactaon may have caused these different results [24]. Although the RBC count was found to be high in lactang donkeys, no change was observed between the groups regarding MCV, MCHC, and MCH values in this study. Gul et al. [6] demonstrated the decreased RBC counts in lactang donkeys when compared to non-lactang. In a previous study, Dezzuo et al. [8] determined that RBC and MCHC values were not affected by different lactaon periods in donkeys, but MCH values were found the highest in the early lactaon period. Salari et al. [7] also reported that RBC and MCV values were not influenced by the three phases of the lactaon, but MCH and MCHC values tended to increase in the second phase (middle) of lactaon in donkeys. In another study, results for MCHC, MCV, and MCH showed a constant trend over different periods of lactaon in donkeys, as reported for mares [25]. Furthermore, HGB values were determined higher in lactang donkeys than non-lactang ones in this study. These results were consistent with a previous study which was conducted by Gul et al. [6]. In the previously conducted limited studies on this subject, it was determined that HGB values did not alter at any stage of lactaon in donkeys [7 , 8 ,9]. Different environmental condions (altude), raon, or lactaon period may have caused different results. PTL values also did not change due to the lactaon period in donkeys in the present study. Dezzuo et al. [8] also stated that PLT values were not affected by any type of lactaon period in donkeys. According to Bonelli et al. [9] and Salari et al. [7], the PLT values were higher in the middle of lactaon than in other periods. These results were not compable with previous studies on mares where PLT was constant over lactaon me. Furthermore, Gul et al. [6] found higher fibrinogen values in non- lactang donkeys than lactang in a previous study. Prolacn is one of the main hormones that increases during pregnancy and lactaon [26]. Furthermore, prolacn was acknowledged as a platelet coacvator in earlier research on the topic, where it improved ADP - induced platelet acvaon [27]. It is well known that unl the final two weeks of pregnancy, mares’ plasma prolacn concentraons are generally low. However, at this me, levels rise significantly as the mare’s mammary gland develops and lactogenesis begins. The last two weeks of pregnancy see an increase in plasma prolacn concentraon, which stays high during the early stages of nursing and returns to normal 1 - 2 months following delivery [28]. It is believed that, among other things, variaons in outcomes are especially influenced by this hormone’s amount. In the literature studies, a limited number of studies were found on the relaonships between PLT numbers and different periods of lactaon in donkeys [13]. In the present study, while ALT values, beer known as liver ssue enzymes, were not affected by lactaon, AST and GGT values considerably increased in female donkeys. These obtained results were corresponding with Gul et al. [6] according to ALT and AST values. Dezzuo et al. [8] detected similar AST and GGT values in the different periods of lactaon in donkeys. Addionally, it was proposed by Bonelli et al. [9] that the GGT acvity trend in donkeys was lower during lactaon and at birth than the late gestaon. Conversely, the AST acvity trend was observed an increase near to parturion and early lactaon in donkeys in the same research. These findings could be explained by the fact that the liver responds differently to various animal species, dietary regimens, and stages of lactaon. In this study, BUN and CREAT values, which are mostly used as kidney funcon tests, were not affected by the lactaon period in donkeys. Also, Dezzuo et al. [8] showed that BUN and CREAT values were not affected by any lactaon period in donkeys. Bonelli et al. [9] suggested that the amount of BUN increased near birth (2 weeks) and then remained constant during the birth and breaseeding period in donkeys. Moreover, 5 of 7

Effects of lactaon period on female donkeys / Aydin and Kisadere UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico CREAT values were determined stable, within normal ranges for adult species [22], throughout the study period in above menoned research. The evaluaon of this study’s findings along with those of other research leads to the conclusion that the energy requirements associated with lactaon do not affect on the BUN and CREAT levels of nursing female donkeys [22]. ANAE and AcP - ase enzyme acvies (Two of the enzyme histochemical techniques) were found to be similar in both lactang and non-lactang donkeys in the present study. In a previous study, the mean ANAE - posive lymphocyte percentage of Kyrgyz donkeys was determined as 42.90 ± 1.18 % [11]. Besides, Krumrych et al. [29] detected the ANAE and AcP - ase enzyme acvies in horses as 67 % and 27 %, respecvely. Furthermore, Ozaydın et al. [30] demonstrated the mean ANAE posive peripheral blood lymphocytes (PBL) as 67.70, 73.10, and 49.20 % in female horses; 64.00, 70.53, and 50.60 % in males 1 d, 3 d, and 1 year old, respecvely. They also suggested the AcP - ase posive PBL as 27.33, 32.83, and 37.40 % in female horses; 29.67, 31.67, and 38.40% in males 1 d, 3 d, and 1 year old, respecvely. In addion, Oruç et al. [31] determined the ANAE - posive lymphocyte rates (%) before and also aſter the race in jumping horses as 54.90 ± 1.60 % and 38.90 ± 1.20 %, respecvely. Moreover, mean percentages of ANAE - posive PBL were found as 76 %, whereas mean percentages of AcP - posive PBL were detected as 28.46 % in Arabian horses by Aydin et al. [12]. Although ANAE - posive lymphocyte rates of donkeys have been found in literature studies, no informaon on the AcP acvies of donkeys both in lactang and non - lactang has been found. Accordingly, we can express it as the first report. Aſter analyzing the data, it may be suggested that the middle - lactaon phase does not affect the acvies of ANAE and AcP in female donkeys in parcular. CONCLUSION In female donkeys, the middle lactaon phase was observed to affect a number of blood parameters, but not the acvity of enzymes (AcP and ANAE). Large-scale studies on this subject are required. Funding The Balıkesir University Scienfic Research Co-ordinatorship provided funding for this study (Project no: 2021 / 116). Conflict of interest The authors declared that there is no conflict of interest. Ethics The Balikesir University Experimental Animal Ethics Commiee’s Experimental Medicine Research and Applicaon Center (Approval no: 2022 / 10 - 6) authorized all animal handling and procedures. BIBLIOGRAPHIC REFERENCES [1] Burden F, Thiemann A. Donkeys are different. J. Equine. Vet. Sci. [Internet]. 2015; 35(5):376–382. doi: hps://doi. org/f7cs76 [2] Fantuz F, Ferraro S, Todini L, Piloni R, Mariani P, Salimei E. 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