Reference values of blood parameters in rams of the Bosnian Pramenka breed

Pramenka is a breed of sheep widely distributed on the Balkan Peninsula (over 30 strains). A number of factors affect hematological and biochemical parameters in sheep blood such as breed, age, sex, region, season and health. The aim of this paper is to determine reference ranges for hematological and selected biochemical parameters in the blood of Bosnian Pramenka rams. The experiment included 362 blood samples. The rams were healthy, and were raised under farm conditions in Banat (Serbia). The blood was collected by puncture of v. jugularis, in vacuum cleaners with a purple stopper (EDTA, for hematological analysis) and with a yellow stopper (for biochemical analysis). The reference intervals of the blood parameters in Bosnian Pramenka rams were: WBC 4.34–17.0 ×109/mL; RBC 4.34–17.0×1012/mL; HGB 72.73–119.8 g/L; HTC 21.97–36.77 %; MCV 26.55–34.23 fL; MCH 8.57–11.34 pg; MCHC 311.32–345.16 g/L; PLT 118–556.45 ×109


Introduction
Pramenka is an indigenous sheep breed, the most prevalent on the Balkan Peninsula. This is a very hardy breed raised under extensive conditions and it has no major requirements in the field of nutrition and care (Savić et al., 2007). The effect of different geographical areas has led to the development of more than 30 strains of sheep, including Sjenička  (Ivanov et al., 2005;Krajinović, 2006).
The determination of reference values in blood has multiple implications in science and the profession. According to international standards, each laboratory should determine its own reference ranges on a number of healthy, strictly controlled and uniform subjects, in order to compare the results with other laboratories and world literature, thereby also checking the quality of the laboratory (CLSI, 2008;Cincović, 2015, 2019). The reference values are important for differentiation between healthy and diseased individuals, and their comparisons establish similarities and differences in different species and strains of animals. For all these reasons, the Laboratory of Pathophysiology at the Department of Veterinary Medicine in Novi Sad examined reference ranges in different animal species Belić et al., 2011Nikolic et al., 2020).
In sheep, breed can have a significant effect on the value of blood parameters. In addition, a significant influence of age or gender on different blood parameters has been found (Oramari et al., 2014). In small ruminants, there is metabolic adaptation to the negative energy balance, and therefore knowledge of these parameters and their reference ranges is of great importance (Mohammadi et al., 2016;Milošević-Stanković et al., 2020). The values of blood parameters in sheep can be affected by different infections (Amarante et al., 2004;Lipecka et al., 2010) as well as by heat stress (Wojtas et al., 2014). It has been shown that there is a difference between manual counting of blood cells and counting with an automatic analyzer (Ibrhim, 2014).
The aim of this study is to determine reference values for hematological and selected biochemical parameters in the blood of rams of the Bosnian Pramenka breed.

Material and method
Samples -The sample included 362 blood samples of rams of the Bosnian Pramenka breed. The rams were healthy, and were reared under farm conditions in Banat. They were fed a diet based on hay and a balanced concentrate portion of the meal. Water was available ad libitum. Rams weighed 40-80 kg.
Laboratory tests -Blood was taken by puncture of v. jugularis after shearing, shaving and disinfection. Blood was drawn into vacuum cleaners with a purple plug (EDTA, for hematological analysis) and a yellow plug (for biochemical analysis). The blood was transported to the Laboratory for Pathological Physiology at the Department of Veterinary Medicine of the Agricultural Faculty in Novi Sad. Analyses were performed within 3-6 hours of sampling. Hematological analyses were performed using a Nihon Kohden veterinary hematology analyzer (Japan) equipped with software for sheep. The following parameters were determined: leukocytes (WBC), erythrocytes (RBC), hemoglobin (HGB), hematocrit (HTC), erythrocyte indices (MCV, MCH, MCHC) and platelets (PLT). Blood biochemical analyses were performed using a Rayto Chemray analyzer (China). The biochemical reagents Biosystems (Spain) were used. The following parameters were determined: total proteins (TPROT), albumin (ALB), globulins (GLOB), urea, glucose (GLU), cholesterol (CHOL), aspartate aminotransferase (AST), beta hydroxybutyrate (BHB) and lactate dehydrogenase (LDH).
Statistical analysis -Descriptive statistical analysis involved the determination of central tendency parameters and variation of examined parameters. A 95% confidence interval was also calculated for each parameter tested, for the lower and upper reference values separately. The shape of frequency distribution was determined using skewness and kurtosis parameters, and the normality of frequency distribution was determined using the Kolmogorov-Smirnov test and was presented graphically using Q-Q plots. Frequency distribution is presented in the form of histograms and polygons, and the polygon represents an ideal normal distribution. For the parameters for which a linear trend was determined between body weight (which depends directly on age) and biochemical parameters, a regression analysis was performed and the trend was presented graphically.

Results
The reference values of the blood parameters of Bosnian Pramenka rams were: WBC 4.34-17.0 ×10 9 /mL; RBC 4.34-17.0×10 12 /mL; HGB 72.73-119.  (Table  1). Normal frequency distribution was determined for RBC, HTC, PLT, ALB, GLOB, and CHOL, while other parameters did not have a normal frequency distribution. The form of frequency distribution is shown in Figures 1-18, and the deviation of the obtained values from the normal distribution is presented for each parameter in Figures 18-36. The values of individual parameters are linearly related to the body weight (age) of the rams. A positive correlation was found between body weight and RBC and HGB values, and a negative correlation between body weight and WBC and BHB values (Figures 37-40). The 95% CI for the population average has been within the known reference value so far, but because of the range of minimum and maximum population values, there was a need to establish a reference value for this population.

Discussion
Determining the form of frequency distribution helps in the appropriate statistical interpretation of the data obtained. If the distribution is not normal, logarithmic transformation may be used or nonparametric statistics may be used when using the median as a positional mean in the population estimate.
Shek Vugrovečki et al. (2017) in the Lika Pramenka sheep determined normal distributions for RBC, HGB, MCV, MCH, UREA and TPROT and log normal or nonnormal distributions for other hematology and biochemistry parameters. Our results show that a large number of parameters in the tested rams do not show a normal distribution. The use of smaller groups of sheep, in an appropriate statistical analysis, allows the determination of the reference interval without the transformation of raw data (Vojta et al., 2011), and the method was applied to the Dalmatian sheep.
The reference values obtained in our experiment can be compared with those obtained for other strains of Pramenka, but they cannot be compared with the results used in the world literature, which confirms the importance of determining reference ranges in our geographical area. The resulting reference values may be useful in interpreting the health and productive status of the Bosnian Pramenka population. Further studies should focus on ewes and lambs, as well as on the deviation of blood parameters in the case of various diseases.