Slov Vet Res | Vol 60 No 1 | 15 Characterisation of the Haematological Profile in the Posavje Horse Breed Key words autochthonous breeds, Posavje horse, haematology, age, sex Matjaž Mesarič1*, Marija Nemec1, Nina Čebulj Kadunc2 1Clinic for Reproduction and Large Animals, University of Ljubljana, Veterinary Faculty, Cesta v Mestni log 47, 1000 Ljubljana, 2Institute for Preclinical Sciences, University of Ljubljana, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia *Corresponding author: matjaz.mesaric@vf.uni-lj.si Abstract: The aim of this study was to investigate the influences of sex and age on hae- matological values in the Posavje Horse breed. A total of 163 healthy Posavje horses (30 foals, 94 mares and 39 stallions) were used in this study; their complete blood counts and a leucogram were obtained with a haematological analyser. The horses were classi- fied into five groups: foals (1 to 6 months, n = 30), 3 to 6 years (n = 8 stallions/21 mares), 7 to 9 years (n = 9 stallions/22 mares), 10 to 13 years (n = 8 stallions/20 mares), 14 to 15 years (n = 6 stallions/10 mares) and 16 and over (n = 8 stallions/21 mares). The results obtained show an influence of sex on haematological parameters, with red blood cell count (RBC), haematocrit (HCT) and haemoglobin concentration (HGB) being higher in stallions (P < 0.001) and white blood cell count (WBC) being higher in mares. Differences between the age groups of the Posavje horses examined indicate a decrease in RBC and HGB with a compensatory increase in mean corpuscular volume and mean corpuscu- lar haemoglobin, a decrease in WBC and platelet counts (PLT) and proportion of lym- phocytes, and an increase of neutrophil to lymphocyte ratio (N/L) with age (P < 0.001). Although the Posavje horse is classified as a draft horse breed, its haematological pa- rameters show characteristics common to warm-blooded breeds, with the exception of the N/L ratio. One of the most important findings of this study is a higher neutrophil count in reproductively active breeding stallions. Higher levels of RBC, HGB, HCT and neutrophil count in the Posavje stallions suggest an effect of androgens (testosterone), which may be an effective mechanism to prevent infections, that can affect the survival of the stallions and thus the evolution of the species. Received: 10 May 2022 Accepted: 15 March 2023 DOI 10.26873/SVR-1508-2023 UDC 636.1.09:616.15-074 Pages: 15–23 Original Research Article Introduction Intensive artificial and natural selection have shaped sub- stantial variation among horse breeds, which are reflected in the differences of haematological and biochemical pa- rameters. In addition to geographical origin, horse breeds can be divided into phenotypic or performance categories. Due to the great diversity of breeds, horses are most often classified as “warm-blooded” breeds including light hors- es of Arabian descent (such as Arabians, Thoroughbreds, Standardbreds and Quarter Horses), and “cold-blooded” breeds essentially including heavy draft horses (such as the Belgian Horse, the Slovenian Cold Blooded Horse and the Posavje Horse). Several differences in haematological parameters were found between these two groups, such as a lower haematocrit in cold-blooded horses and higher erythrogram values in warm-blooded horses, which should be considered when determining reference values of blood parameters and interpreting blood tests (1-4). In contrast, light horse breeds have a higher red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit value (HCT) and blood volume than draft horses (2, 5, 6). In ad- dition to horse type, haematological parameters may also vary due to numerous internal and external factors, includ- ing breed, sex, age, reproductive status, fitness and train- ing status, exercise load, feeding and, circadian variations. SloVetRes_NOTRANJOST ver2.indd 15 09/05/2023 10:14 16 | Slov Vet Res | Vol 60 No 1 Moreover, handling procedures during blood withdrawal, operating conditions, criteria for selecting healthy subjects, preparation of the subjects for the procedures, level of ex- citement and health status are also important factors that affect haematological values in horses (1, 6, 7, 8). Differences in the cellular constituents of the blood are the result of specific changes in an organ or organ system, or a general response of the individual to certain physiologi- cal or pathological conditions (6). For example, the total leukocyte count (WBC) and differential leukocyte count in healthy horses are dependent on age, which is associated with a steady decline in leukocyte counts (9-11) while the absolute and relative leucocyte counts, especially neutro- phils and lymphocytes, vary considerably (11) to reach a neutrophil to lymphocyte (N/L) ratio of 2:1 in older horses (2). Minor differences in leukocyte counts have been found between the different breeds of horses, with warm-blood- ed horses having higher WBC counts than to cold-blooded horses (6, 12). The horse population investigated in this study was the autochthonous Posavje Horse breed, originating from the Lower Sava River flatlands of the southeastern part of Slovenia (especially in the districts of Krsko and Brezice) and in Croatia, this breed resulted from the crossing of local warmblood mares with Norik stallions. In addition, Ardene stallions were used to improve the Posavje horses’ abilities for heavy draft work (13). The Posavje horse is the smallest cold-blooded breed in Europe, characterised by a good-na- tured temperament and a pronounced sexual dimorphism. It was mainly selected for heavy draft work, especially in steep forest areas, but it has also been used for meat pro- duction (13). In 1993, a Slovenian breeding and conserva- tion programme was established for this horse breed. Since then, the breed has been bred according to the principles of conservation genetics: narrow relation of breeding stallions (sires) to mares with balanced breeding using different sires and moderate selection (13). Currently, reference values of haematological parameters are widely available for horses in general and for the most common and popular breeds (2, 5, 14). However, literature data for endemic breeds are sparse and only a few reports address haematology in au- tochthonous draft horse breeds (15-17). The aim of this study was therefore to investigate the characteristics of the haematological parameters in the Posavje Horse breed and to test the hypothesis that age, sex and reproductive status cause some haematological changes in horses. In addition to variations in haematological parameters in the Slovene Posavje Horse breed, the study was particularly focused on the characteristics of differential leucocyte counts and their variations with age gain. The measured haematological val- ues and their variations could serve as guideline values for further haematological investigations and as a basis for the development of an approach to determine haematological reference values for the Posavje breed (1,2, 6, 18). Materials and methods The study was conducted as a part of the routine annual breeding and registration procedures of the Slovenian breeding and conservation programme for the autoch- thonous Posavje horse breed at different locations in the region of south-eastern Slovenia during July and August. The stallions were located as sires in breeding stations and separated from the mares and foals kept on the farms of local breeders. Regardless of category and location, the horses were kept on pasture in natural environmental con- ditions during the day and stabled in individual boxes dur- ing the night. While stabled, they were fed hay, considering the needs of each category, and had free access to water. They were dewormed regularly, clinically sound on the day of sampling and did not receive medication in the last 3 months before blood sampling. The mares included in the study were not pregnant. As the horses were familiar with humans and accustomed to different handling procedures, no restraint was required during sampling. The study included 39 stallions aged 3 to 22 years (aver- age 11.3 years), 94 mares aged 4 to 22 years (average 11.1 years) and 30 foals aged 30-180 days (average 102 days). The grouping of horses by age and sex is presented in Table 1. Blood samples were collected from the jugular vein with double-ended needles and evacuated tubes contain- ing K2EDTA as an anticoagulant (Vaccuette; Greiner Labortechnik GmbH, Kreimsmünster, Austria) and stored at 4 °C for haematological analyses (6, 18), which were per- formed within the next 6 hours at the Laboratory for Clinical Pathology of the Clinic for Reproduction and Large Animals at Veterinary Faculty, University of Ljubljana. Routine hae- matological analyses included the following: Red blood cell tests (red blood cell count (RBC), haematocrit (HCT), haemoglobin concentration (HGB), red blood cell indices (mean cell haemoglobin concentration (MCHC), mean cell Table 1: Arrangement of horses to age groups Age group Age Sex Total (n)Mares (n) Stallions (n) Foals 30–180 days / / 30 Adults 3–22 years 94 39 134 Group A 3–6 years 21 8 29 Group B 7–9 years 22 9 31 Group C 10–13 years 20 8 28 Group D 14–15 years 10 6 16 Group E 16 and more 21 8 29 SloVetRes_NOTRANJOST ver2.indd 16 09/05/2023 10:14 Slov Vet Res | Vol 60 No 1 | 17 volume (MCV), mean cell haemoglobin (MCH)) and red blood cell distribution width (RDW)), white blood cell tests (total white blood cell count (WBC), absolute differential leu- kocyte count), platelet count (PLT) and mean platelet vol- ume (MPV) were performed using an automated veterinary haematology analyser (Scil Vet abc Plus+, Horiba, Japan), validated for equine samples and following original instruc- tions for use. The relative differential leukocyte count (neu- trophils - NEU, eosinophils - EOS, basophils - BAS, mono- cytes - MON, lymphocytes - LYM) was measured under the microscope using blood smears stained with the commer- cial staining kit Hemacolor (Merck Cat. No. 1.11661, Merck KGaA, Darmstadt, Germany). The neutrophil/lymphocyte ratio (N/L ratio) was calculated by dividing the neutrophil proportion by the lymphocyte proportion. Statistical calculations were performed using the Statistical Package for Social Sciences (SPSS for Windows, release 8.0.0). The normality of the data distribution was assessed using a Shapiro–Wilk test and significance was determined using all pairwise multiple comparisons (Tukey’s test). Differences between values calculated for horses grouped by age or sex were statistically analysed by one-way analy- sis ANOVA. When significant differences were found, a posthoc analysis was performed (Bonferroni–Holm test) to clarify the groups between which these differences existed. Differences were considered significant at P≤0.05. The val- ues measured are presented as the mean ± standard devia- tion in the text (x ± SD) and as mean ± error of the mean (x ± SE) in the figures. Results Red blood cell tests The mean values of haematological parameters for all ex- amined foals, stallions and mares of Posavje horses are shown in Table 2. Statistically significant differences be- tween mares and stallions were found for RBC, HCT and haemoglobin concentrations (P < 0.001). In foals, RBC lev- els were significantly higher (P < 0.001) than in stallions and mares, but HCT, MCV and MCH levels were significantly lower (P < 0.001). The mean HGB concentration was sig- nificantly lower in foals than in stallions (P < 0.001) and higher than in mares (P < 0.01). The differences between foals, mares and stallions were not significant for MCHC and RDW (Table 2). Age-dependent variations in RBC and indices in mares and stallions of the Posavje breed are presented in Fig. 1. The RBC values (Fig.1A) were highest in foals and signifi- cantly decreased thereafter in both sexes with age gain (P < 0.001). With the exception from foals and 3 to 4-year-olds, the RBC values in mares were lower than those in stallions (P < 0.001). The lowest MCV (Fig.1 B) and MCH (Fig.1 D) were measured in foals and increased significantly with age in both sexes (P < 0.001 for both parameters). MCV was significantly lower in mares aged 3 to 6 years than in older animals (P < 0.001). The differences between stallions and mares of all ages were insignificant for MCV and MCH. HCT (Fig. 1C) and HGB (Fig. 1E) values in mares decreased with age (P < 0.001), reaching the lowest values in the 14- to 15-year-old Table 2: Haematological parameters in Posavje horse foals, stallions and mares in total (x ± SD) Variable (unit) Foals (n=30) Stallions (n=39) Mares (n=94) RBC (× 1012/L) 9.84 ± 1.26a,b 8.79 ± 0.99c 7.45 ± 1.01 HGB (g/L) 124.27 ± 12.82 a,b 138.41 ± 14.93c 117.08 ± 12.82 HCT (L/L) 0.35 ± 0.04a,b 0.40 ± 0.05c 0.33 ± 0.04 MCV (fL) 36 ± 1.86a,b 45.18 ± 2.76 44.79 ± 3.35 MCH (pg) 12.68 ± 0.66a,b 15.78 ± 1.01 15.83 ± 1.25 MCHC (g/L) 351.93 ± 12.49 349.54 ± 8.26 353.52 ± 15.16 PLT (× 109/L) 350.93 ± 101.08a,b 229.54 ± 67.12 242.65 ± 67.62 MPV (fL) 7.48 ± 0.79a,b 6.36 ± 0.38c 6.61 ± 0.37 RDW (%) 17.86 ± 1.11 17.94 ± 0.74 17.99 ± 0.71 Legend: RBC - red blood cell count; HGB - haemoglobin concentration; HCT - haematocrit; MCV - mean cell volume; MCH - mean cell haemoglobin; MCHC - mean cell haemoglobin concentration; PLT – platelet count; MPV – mean platelet volume; RDW – red cell distribution width. Values in a row with the same superscript show significant differences (adifferences between foals and mares; bdifferences between foals and stallions; cdifferences between mares and stallions; P < 0.05) SloVetRes_NOTRANJOST ver2.indd 17 09/05/2023 10:14 18 | Slov Vet Res | Vol 60 No 1 5 6 7 8 9 10 11 12 RB C [× 10 12 /L ] Age [years] Mares Stal lions 30 35 40 45 50 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CV [f L] Age [years] Mares Stal lions 0,25 0,30 0,35 0,40 0,45 0,50 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < PC V [1 /1 ] Age [years] Mares Stal lions 12 13 14 15 16 17 18 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CH [p g/ L] Age [years] Mares Stal lions 100 110 120 130 140 150 160 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < HG B [g /L ] Age [years] Mares Stal lions 345 350 355 360 365 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < M CH C [g /L ] Age [years] Mares Stal lions Figure 1: Age-dependent changes of red blood cell number (RBC; Panel A), mean cell volume (MCV; Panel B), Haematocrit (HCT; Panel C), mean cell haemoglobin (MCH; Panel D), haemoglobin concentration (HGB; Panel E) and mean cell haemoglobin concentration (MCHC; Panel F) in Posavje mares and stallions (mean ± SE) group, while in stallions, a decrease was observed with peak values for HCT and HGB in the 7- to 9-year-old age group, followed by a gradual decrease in both values with age. Stallions aged 7 to 9 years or more had significantly higher HCT and HGB values than mares in the same age group (P < 0.001, respectively). The MCHC value (Fig. 1F) in stallions decreased slightly and reached the lowest val- ues at the age of 7 to 9 years and increased thereafter. In mares, MCHC increased with age, peaking at 10 to 13 years of age and decreasing thereafter; the differences in MCHC between mares and stallions of all ages were not signifi- cant (P > 0.05). RDW values remained stable with age in both mares and stallions and differences between sexes were not signifi- cant (hence, the changes are not shown graphically). The PLT count and MPV were significantly higher in foals than in stallions and mares (P < 0.001). The mean platelet volume (MPV) was higher in mares than in stallions (P < 0.001). SloVetRes_NOTRANJOST ver2.indd 18 09/05/2023 10:10 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < C D A B E F Slov Vet Res | Vol 60 No 1 | 19 White blood cell tests The mean total WBC and the relative and absolute differen- tial leucocyte counts in foals, stallions and mares are shown in Table 3. The mean total WBC and LYM counts (Table 3) were significantly (P < 0.001) higher in foals than in mares and stallions, while the NEU, MON, EOS and BAS counts were significantly lower in foals than in adults (P < 0.05). The highest WBC value (Fig. 2A) was found in foals of both sexes and then gradually decreased in mares to reach the lowest value in the age group of 16 years and over (P < 0.001). A significant decrease in WBCs was observed in stallions at 3 to 6 years of age (P < 0.01), followed by an increase in WBCs at 10 to 13 years of age and a gradual decrease thereafter. WBC counts were also significantly lower in stallions aged 3 to 6 years (P < 0.01) than in mares of the same age. In mares, WBC counts were significantly higher in the 3- to 6-year-old group than in the older group (P < 0.001). The NEU count increased and the LYM count decreased significantly (P < 0.001 and P < 0.001, respec- tively) with increasing age in stallions and mares (Fig. 2B). Mares aged 3 to 6 years had a significantly higher NEU count (4.61) compared to stallions of the same age (3.36, P < 0.05; Fig.2B). The N/L ratio was significantly lower in the foals than in Groups B (7 to 9 years), D (14 to 15 years) and E (16 and more years) (P < 0.001) (Table 4). The interactions between age and sex of NEU and LYM are shown in Fig. 2. MON did not significantly differ between Posavje foals, stal- lions and mares (Table 3). The BAS counts in foals were statistically lower than those in mares and stallions (P < 0.001 and P < 0.05, respectively). Discussion Red blood cell tests The mean RBC of Posavje stallions and mares was at the lower end of the normal range for warm-blooded horses (19) but at the upper end of the normal range for draft hors- es (16, 17) and slightly lower than in warmbloods (9, 20). In general, erythrogram values in this study were mostly com- parable to those for warmblooded horses (5, 14, 21), which is surprising considering that the Posavje horse is a cold- blooded breed, although some warmblood characteristics are still present. In Posavje foals, the mean RBC was higher than in adult horses, consistent with the literature (2, 9). However, MCV and MCH were lower than in adult horses, as in warm-blooded foals (19). The erytrogram values determined in our study were also related to the age of horses as described recently (14, 5, 22), with the RBC decreasing with age, followed by a Table 3: Total WBC count and relative/absolute differential leukocyte count (x ± SD) in foals, stallions and mares Variable Unit Foals(n=30) Stallions (n=39) Mares (n=94) WBC ×109/L 11.20 ± 2.03a,b 8.76 ± 1.81 9.14 ± 2.03 NEU % 39 ± 11.42 56.26 ± 12.93 51.9 ± 11.65 ×109/L 4.55 ± 1.57 4.99 ± 1.84 4.71 ± 1.45 LYM % 52.67 ± 10.39a,b 37.26 ± 12.38 39.2 ± 11.62 ×109/L 5.78 ± 1.66a,b 3.20 ± 1.11 3.63 ± 1.54 MON % 0.93 ± 1.27 1.23 ± 1.37 0.85 ± 0.97 ×109/L 0.12 ± 0.15 0.11 ± 0.13 0.08 ± 0.10 EOS % 3.52 ± 3.20 4.10 ± 3.14 5.66 ± 3.62 ×109/L 0.37 ± 0.25 0.36 ± 0.29 0.50 ± 0.32 BASO % 0.11 ± 0.42a 0.38 ± 0.63 0.79 ± 1.07 ×109/L 0.01 ± 0.04a 0.03 ± 0.06 0.07 ± 0.09 Ratio N/L 1/1 0.88 ± 0.54a,b 1.86 ± 1.23 1.55 ± 0.84 Legend: WBC – white blood cell count; NEU – neutrophil; LYM – lymphocyte, MON –monocyte; EOS – eosinophil; BAS – basophil; N/L –neutrophil/ lymphocyte ratio. Values in a row with the same superscript indicate significant differences (adifferences between foals and mares; bdifferences between foals and stallions; cdifferences between mares and stallions; P < 0.05) SloVetRes_NOTRANJOST ver2.indd 19 09/05/2023 10:14 20 | Slov Vet Res | Vol 60 No 1 compensatory increase of MCV and MCH in both sexes (5, 14, 18, 21, 22, 23, 25, 26). A gradual increase in MCV appears to be a common finding associated with equine ageing (22-24) causing changes in the dynamics of eryth- rocyte maturation (24). The MCV values of the Posavje horses studied were lower than those in the Przewalski and Kathiawari horse breeds (27, 28) but higher than those in the Zemaitukai horses (21) of comparable ages, while in foals they were consistent with those reported in many other breeds (29, 30). HGB concentrations differed between sex and age groups of Posavje horses in the present study and were consis- tent with (15, 31) or higher (32) literature data. In contrast to Lahora working horses (33) and Lipizzans (5), a sig- nificant increase in HCT and HGB was observed only in Posavje stallions up to the age of 7 to 9 years, followed by a gradual decrease in older groups, while the values in mares decreased with age. In general, the mean total RBC, HCT and HGB levels were significantly higher in Posavje stal- lions than in mares, as common for horses (15, 33, 34, 35). This trend is most likely due to the effect of testosterone, which is also known to increase circulating HGB, HCT and RBC in humans (36) stimulating haematopoietic tissue and erythropoiesis in men more than in women (37). The role of testosterone in haematopoiesis was also supported by the study in castrated goats (38). The mean RDWs of Posavje horses did not differ between age groups and sexes and were lower than (7, 39, 40) or sim- ilar to reported values (17, 19, 41, 42, 43, 44). A significant de- crease in RDW was previously observed in stallions after ex- ercise (7), but this decrease was not observed in our study. Platelet count in Posavje horses decreased with age, which is consistent with literature data (21, 22, 23), although no dif- ferences between age groups were reported (24). MPV was higher in Posavje mares than in stallions; in both sexes, the values were higher than in Shetland ponies (41) and lower than in Holstein horses (7, 44). Elevated MPV has been pro- posed as an indicator of platelet activation in humans, but the lack of defined limits to distinguish between activated and nonactivated platelets and the failure of platelet aggre- gation inhibitors to reverse a high MPV limit its utility as a platelet activation marker in human medicine (45). White blood cell test In the present study of horses of the Posavje breed, the highest mean WBC value was found in foals, followed by mares, and the lowest in stallions, with the later exhibiting the lowest value (14, 21, 25). In contrast, the WBC levels of Thoroughbred (9) and Lipizzan (46) stallions were higher in than in mares, and some studies failed to find signifi- cant differences between the sexes (47). In all age groups of Posavje horses WBC values in mares were higher than those in stallions, although this difference was significant 6,00 7,00 8,00 9,00 10,00 11,00 12,00 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < W BC [* 10 9 / L] Age [years] Mares Stal lions 0 1 2 3 4 5 6 7 8 foals 3 to 6 7 to 9 10 to 13 14 to 15 16 < N EU a nd L YM [* 10 9 / L] Age [years] Figure 2: Changes of total white blood cell number (x ± SE) (A), and neutrophil (NEU) and lymphocyte (LYM) counts (B) in Posavje mares and stallions with age Table 4: Neutrophil/lymphocyte ratio (N/L) (x ± SD) in mares and stallions of various age groups (A: 3–6, B: 7–9, C: 10–13, D: 14–15, E: 16 and more years old) Sex Age group Foals Group A Group B Group C Group D Group E Mares 0.84 ± 0.54 0.94 ±0.35 1.14 ±0.53 1.78 ±1.08 1.91 ±0.55 2.20 ±0.82 Stallions 0.75 ± 0.55 0.96 ±0.57 1.43 ±0.47 2.50 ±1.47 2.84 ±1.82 1.86 ± 0.81 SloVetRes_NOTRANJOST ver2.indd 20 09/05/2023 10:10 A B NEU mares NEU stallions LYM mares LYM stallions Slov Vet Res | Vol 60 No 1 | 21 only at the age of 3 to 6 years. The in WBC count of Posavje horses decreased with age gain (11, 24, 44) which could be attributed to the gradual decline in immunocompetence and cannot be considered as leukopenia (24, 44). In Posavje horses, slightly higher NEU and lower LYM counts were measured in stallions than in mares (48). A significant age-related decrease in LYM count, proportional to the decrease in WBC, was observed in both sexes of the Posavje horse, as also reported in other horse breeds (2, 15, 16, 46). This decrease may be the reason for the de- creased immunocompetence in older horses (10). The ab- solute NEU count has been reported to be higher in foals than in adult horses and remains stable with age gain (9, 25, 46), whereas it increased significantly in Posavje horses of both sexes. This increase was also the reason for a steady increase in N/L ratio with age, ranging from 0.84 to 2.01 in mares and from 0.75 to 1.68 in stallions. Similar changes in the N/L ratio in Andalusian horses (11) indicate a natural state reflecting a decreased bone marrow response. The predominance of NEU in the Posavje horses studied re- flects the cold-blooded origin of this breeds (12). In stallions aged 3 to 6 years the NEU count decreased sig- nificantly in parallel to the WBC count; however, it remained within physiological limits. In all other age groups, NEU counts were higher in stallions than in mares, as also re- ported for other horse breeds (32, 48, 49, 50); this difference could be attributed to increased testosterone production in reproductively active stallions. The plasma levels of tes- tosterone are an important regulator of NEU function and the associated inflammatory response in humans (51, 52) which represents the first line of defence against invading pathogens and tissue injury (53, 54). Therefore, the physi- ological increase in NEU in the blood of the stallions studied could be an evolutionary adaptation to prevent infections caused by injuries of stallions, fighting for mares within a harem. Surprisingly, that the described changes in the NEU counts of stallions have thus far gone unnoticed. Modest increases in NEU counts within the normal range may have been ignored and the stallions in studies that addressed this issue (14, 21, 34, 46) were not sufficiently old or were reproductively inactive. Another reason for leucocytosis in horses with increased NEU and decreased LYM numbers could be increased plasma cortisol levels under stress (53). We can exclude this cause in the Posavje stallions, as all age groups of the examined stallions were housed under similar environmental conditions and treated in the same manner. Neither age nor sex affected the EOS and MON, confirm- ing the results of previous studies in horses (46, 49). Higher BAS values in older Posavje stallions were likely the result of altered immunological load (14). Conclusions In conclusion, our study indicates breed-related differenc- es in haematological parameters of horses, and we have shown that haematological parameters vary with age and sex in the Posavje breed. The haematological traits iden- tified in our study represent interesting breed-, age- and sex-specific adaptations/responses but are of limited di- agnostic value. In general, the values of the haematologi- cal parameters in our study most closely matched those of warm-blooded horses, although the Posavje horse is a cold-blooded breed. The leucogram values and the N/L ratio determined in our study corresponded to those of cold-blooded horses. Furthermore, the results confirm and extend previous reports on age- and sex-related changes in haematological variables. 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J Equine Sci. 2021; 32(1): 21–5. 49. Romić S. Krvna slika noniusa. Zagreb : Poljoprivredni nakladni zavod, 1973: 289–98. (Poljoprivredna znanstvena smotra; 16/2) 50. Nidl C, Merino JO, López EA, et al. Effect of age, gender, and season on hematological pa-rameters in Quarter horses. J Vet Sci Med Diagn 2017; 6(2). doi: 10.4172/2325-9590.1000224 51. Marin DP, Bolin AP, dos Santos Rde C, Curi R, Otton R. Testosterone suppresses oxidative stress in human neutrophils. Cell Biochem Funct 2010; 28(5): 394–402. 52. Gagliano-Jucá T, Pencina KM, Guo W, et al. Differential effects of testosterone on circulating neutrophils, monocytes, and platelets in men: findings from two trials. Andrology 2020; 8(5): 1324–31. 53. Davis AK, Maney DL, Maerz JC. The use of leukocyte profiles to mea- sure stress in vertebrates: a review for ecologists. Funct Ecol 2008; 22: 760–72. 54. Hines MT, Schott HC, Bayly WM, Leroux AJ. Exercise and immunity: a review with emphasis on the horse. J Vet Intern Med 1996; 10: 280–9. 55. Krumrych W. Variability of clinical and haematological indices in the course of training exercise in jumping horses. Bull Vet Inst Pulawy 2006; 50: 391–6. Karakterizacija hematološkega profila pri posavskem konju M. Mesarič, M. Nemec, N. Čebulj Kadunc Izvleček: Cilj raziskave je bil proučiti vpliv spola in starosti na hematološke parametre pri pasmi posavski konj. V razis- kavo je bilo vključenih 163 konj posavske pasme (30 žrebet, 94 kobil in 39 žrebcev), pri katerih smo v vzorcih krvi določali hematološke parametre s hematološkim analizatorjem. Diferencialna bela krvna slika in razmerje med nevtrofilci in lim- fociti (N/L) je bilo določeno na krvnih razmazih. Konje smo razdelili v pet starostnih skupin: žrebeta (od 1 do 6 mesecev, n = 30), 3 do 6 let (n = 8 žrebcev/21 kobil), 7 do 9 let (n = 9 žrebcev/22 kobil), 10 do 13 let (n = 9 žrebcev/20 kobil), 14 do 15 let (n = 6 žrebcev/10 kobil) ter 16 in več let (n = 8 žrebcev/21 kobil). Rezultati naše raziskave kažejo vpliv spola na preis- kovane hematološke parametre; pri žrebcih so število rdečih krvnih celic (RBC), hematokrit (HCT) in koncentracija hemo- globina (HGB) značilno višji (P < 0,001), pri kobilah pa je višje število belih krvnih celic (WBC). Med starostnimi skupinami posavskih konj smo ugotovili zmanjšanje RBC in HGB in posledično kompenzacijo s povečanjem povprečnega volumna in hemoglobina eritrocitov, zmanjšanjem števila levkocitov, trombocitov (PLT) in limfocitov ter povečanjem razmerja med nevtrofilci in limfociti (N/L) s starostjo (P < 0,001). Posavski konj po zunanjosti spada med hladnokrvne konje, v raziskavi ugotovljeni hematološki profil pa kaže značilnosti, ki so skupne toplokrvnim pasmam konj, z izjemo razmerja N/L. Ena od pomembnejših ugotovitev te študije je večje število nevtrofilcev pri aktivnih plemenskih žrebcih. Višje vrednosti RBC, HGB, HCT in števila nevtrofilcev pri posavskih žrebcih kažejo učinek androgenov (testosterona), kar bi lahko bil učinkovit mehanizem za preprečevanje okužb, ki lahko vplivajo na preživetje žrebcev in s tem na evolucijo vrste. Ključne besede: avtohtone pasme; posavski konj; hematologija; starost; spol SloVetRes_NOTRANJOST ver2.indd 23 09/05/2023 10:14