Comparison of serum protein electrophoretic pattern in cows and small ruminants

Determination of the physiological electrophoretic patterns in animals is very useful for clinicians in diagnosing healthy and sick animals. The objective of this study was to investigate the serum protein electrophoretic pattern in cows, sheep, and goats in order to evaluate the differences in the size and number of protein fractions between the evaluated ruminant species. Ten adult multiparous high-yielding dairy cows, 10 adult female sheep and 10 adult female goats were included in this study. All the evaluated animals were clinically healthy. Serum was analyzed for total serum protein concentrations, and for the relative and absolute values of protein fractions with calculation of albumin/globulin ratios. Serum protein fractions were separated by zone electrophoresis on buffered agarose gel. Serum protein electrophoresis identified 6 distinct bands, comprising albumin, alpha1(α1), alpha2(α2), beta1(β1), beta2(β2), and gamma(γ) globulins in cows. In sheep, serum proteins exhibited 6 fractions: albumin, α1-, α2-, β-, γ1and γ2-globulins. In goats, serum proteins were separated into 5 fractions: albumin, α1-, α2-, βand γ-globulins. Significant differences in the relative as well as absolute means were found for the albumin/globulin ratio and most of the protein fractions, except γ-globulins. No significant differences were found in the concentration of total proteins. These results describe the marked species differences in most of serum protein fractions between the evaluated groups of animals, and contribute to the current knowledge about the physiological electrophoretic pattern of serum proteins in ruminants, which can be used for diagnostic purposes. Cattle, sheep, goat, total proteins, protein fractions, gel electrophoresis Hundreds of different proteins with a wide variety of functions circulate in blood and concentrations of total proteins and several specific proteins are of clinical value (Joliff 1992). When tissues responsible for the synthesis or excretion of proteins are altered by disease, the resulting serum produces distinctive patterns of proteins that could be helpful in diagnosis (Joliff 1991). Modifications of total serum protein concentrations, the fractional distribution of blood proteins and albumin/globulin ratio are usually the first signs of protein abnormality (Dede et al. 2014). Electrophoresis is a suitable separation technique for fractination and quantification of serum proteins, and for the monitoring of changes in serum protein fractions. It is based on the movement of charged particles through a solution when subjected to an electrical field (Azim et al. 2004). Serum proteins have a negative charge, so they migrate in an electrical field and are separated from each other in different bands (Esmaeilnejad et al. 2014). Following electrophoresis, serum proteins can be separated into four fractions including albumin, and alpha (α)-, beta (β)and gamma (γ)-globulin fractions (Bossuyt 2006). Each band is made up of a group of individual proteins, each of which is characterized by independent metabolic properties. Although protein electrophoresis has been extensively used as an effective diseasescreening tool in human medicine for many years, this technique is not commonly used in veterinary medicine (Wijnen and Van Dieijen-Visser 1996; O’Connell et al. 2005). Although this test has low specificity in the diagnosis, determination of the physiological electrophoretic patterns in all domestic animals and correct interpretation of their results is very useful for clinicians in diagnosing healthy and infected animals (Lutz et al. 2009). ACTA VET. BRNO 2015, 84: 187–195; doi:10.2754/avb201584020187 Address for correspondence: Doc. MVDr. Oskar Nagy, PhD. University of Veterinary Medicine and Pharmacy, Clinic for Ruminants Komenského 73, 041 81 Košice, Slovak Republic Phone: +421 915 986 695 E-mail: [email protected] http://actavet.vfu.cz/ The distribution and concentration of blood proteins is affected by many diseases. Identifying and quantifying protein fractions enable the identification of animals with altered serum protein pattern, which may reflect responses to changes in homeostasis or disease (Alberghina et al. 2011). Many times, abnormalities found in the proteinogram are not related to illness but to physiological and individual conditions (França et al. 2011). The interpretation of biochemical constituents depends on the knowledge of variation that exists not only among different species of animals, but also among different groups of animals. Some researchers noted that serum protein fractions display important differences among all domestic animals (Keay and Doxey 1981; Alberghina et al. 2010). As the number of protein fractions varies with the species, determination of the normal electrophoretic pattern of each species appears to be essential (Ahmadi-Hamedani et al. 2014). However, only a few studies have described the differences in the electrophoretic mobility of serum proteins between ruminant species. Therefore, the aim of this study was to determine the physiological serum protein electrophoretic pattern for selected ruminant species, and to describe the possible differences in protein fractions between the evaluated groups of animals. Materials and Methods To obtain the differences in the serum protein electrophoretic pattern between ruminants, 10 adult multiparous high-yielding dairy cows at the peak of lactation, 10 adult female sheep and 10 adult female goats from conventional farms were included in this study. The evaluated cows were of the Slovak Pied breed and its crossbreeds at the age of 3–5 years, with a mean body weight of 453 ± 32 kg and daily milk yield of 28 ± 5 litres. The cows were housed in free-stalls, and fed twice a day diets for lactating cows with free access to water in automatic drinking troughs. They were milked twice a day. The evaluated sheep were of the merino breed at the age of 2–4 years, with a mean body weight of 48 ± 6 kg. The goats were of the white shorthaired breed, 2–4 years old and with a mean body weight of 35 ± 5 kg. All the evaluated sheep and goats had free access to water and alfalfa hay. The concentrate was provided once daily. Before blood sample collection, the animals used in the study were examined clinically using standard clinical examination procedures. All the animals were clinically healthy and in good general health condition without any obvious clinical signs of disease. Blood samples were collected through jugular venipuncture using plastic serum gel separator tubes without anticoagulant (Meus, Piove di Sacco, Italy). Blood samples were allowed to clot at room temperature, and then centrifuged at 3000 × g for 30 min to separate serum. Harvested blood serum was dispensed into plastic tubes, and stored at -20 °C until analysed. Serum was analysed for the concentrations of total proteins (TP, g/l) and serum protein fractions (albumin, a-, β-, and γ-globulins). Total protein concentrations were assessed on an automated biochemical analyser Alizé (Lisabio, France) by the biuret method using commercial diagnostic kits (Randox Laboratories, United Kingdom). Serum protein fractions were separated by zone electrophoresis on buffered agarose gel at pH 8.8 on an automated electrophoresis system (Hydrasys, Sebia Corporate, Evry-Paris, France) using commercial diagnostic kits (Hydragel 7 Protein, Sebia Corporate, Evry-Paris, France) according to the procedure described by the manufacturer. Electrophoretic gels were scanned, and the serum protein fractions were visualized and displayed on densitometry system (Epson Perfection V700, Epson America Inc., Long Beach, California, USA) by light transmission and automatic convertion into an optical density curve presentation. Protein fractions were identified and quantified by computer software (Phoresis version 5.50, Sebia Corporate, Evry-Paris, France), and if necessary, corrected by visual inspection of the electrophoretogram. The relative concentrations (%) of the protein fractions were determined as the percentage of optical absorbance, and the absolute concentrations (g/l) were calculated from the total serum protein concentrations. Albumin:globulin ratios (A/G) were computed from the electrophoretic scan. Arithmetic means (x) and standard deviations (SD) for each evaluated variable and group of animals were calculated using descriptive statistical procedures. One-way analysis of variance (ANOVA) with a Tukey’s multiple comparison test were applied to evaluate the significance of differences in means between the groups of animals. All statistical analyses were done using the programme GraphPad Prism V5.02 (GraphPad Software