**4. Discussion**

To the best of our knowledge, there have been no studies of the serum protein electrophoretic fractions in healthy tigers. The total serum protein electrophoretic pattern obtained with agarose gel electrophoresis separated the protein into six fractions, albumin, α1, α2, β1, β2 and γ globulins as in other mammals [14], resulting in a typical electrophoretic pattern for the distribution of proteins. Mean values of albumin and globulin fractions, with the exception of α2 and β1 globulins mean concentration, in our healthy tiger population fell within the reference values for domestic cats [17]. The average values of α2 and β1 globulins were above the higher reference values indicated for protein serum electrophoresis performed with AGE in domestic cats [17]. In mammals, the α2 globulin fraction mainly consists of acute-phase proteins, such as α1- acid glycoprotein, and often these proteins increase as a result of activation of the inflammatory response [28] to regulate different stages of inflammation [29]. Complement is one of the main proteins present in the β globulin fraction, corresponding to the sum of β1 and β2 globulin fractions [14]. Both α2 and β1 globulins fractions may be elevated if there is increased production of some acute phase proteins which migrate into these regions [14]. Recently, acute phase proteins have also been proposed as useful stress biomarkers. In humans, cows and experimental animals, psychological and physical stress elevates plasma interleukin-6 and APP levels [22,30]. Acute phase proteins are synthesized predominantly in the liver, in response to secretion of pro-inflammatory cytokines. In response to stress signals, the hypothalamic–pituitary–adrenal (HPA) axis may trigger cytokine production resulting in an increase in hepatic APP synthesis and release into the bloodstream [22]. Although lacking specificity, the detection of an increase in α2 globulin could help in monitoring the stress status of tigers in captivity.

Although the sample size analyzed was limited, the data obtained in this study could sugges<sup>t</sup> that healthy tigers may have a higher concentration of acute phase proteins than domestic cats, or that the tiger population studied could have been in an inflammatory state. The interpretation of serum protein electrophoretic patterns depends on the variations among different groups of animals. Moreover, acute phase proteins are a variable group of serum proteins and concentrations vary widely between different animal species [31]. Domestic cats are in the same family and share a similar physiology to tigers and could be an acceptable alternative for comparison of normal values for many biochemical parameters; however, they are a different subfamily and extrapolation of all results is dangerous. Depauw et al. (2014) [13] reported results of captive cheetah (*Acinonyx jubatus*) serum protein electrophoretic fractions by capillary electrophoresis (CE). Although the AGE and CE are different techniques for protein fraction separation, the shape of the electrophoretogram of cheetah serum was comparable to that found in our healthy tiger sample [13].

The percentage of variation observed between protein fractions of two healthy tiger serum samples repeatedly submitted to agarose gel electrophoresis to evaluate inter-assay-accuracy were within the accepted ranges of within-subject biological variation for people [27]. Only the α1 globulin fraction exceeded the acceptable value of 20.83%. This result could be due to the low concentration of α1 globulins in the serum. In fact, the accuracy of analysis is usually better for protein fractions found in higher serum concentrations because low concentrations are more susceptible to small changes [32]. Cushing et al. (2019) [19] described a cases series of myeloma associated with hypergammaglobulinemia in five adult tigers. Diagnosis of myeloma is based on a variety of clinical signs often associated with monoclonal gammopathy found in serum. It is interesting to note that in this case series, the serum protein electrophoresis was done in the absence of reference values for the serum protein pattern typical of this species. This underlines the importance of acquiring a database of reference values even for the rarest wild carnivores.

There were a number of limitations with this study. Firstly, although each tiger was clinically examined, and screened for visible alterations and low body condition score, the history was sometimes incomplete or unavailable, which may have compromised the accurate categorization of animals according to health status and disease type. In addition, samples were from animals in different types of housing, so the diversity of habitat and diet could have affected the results. [25]. Furthermore, due to the small number of subjects, we are not able to define reference ranges. In fact, following the reference interval guidelines of the American Society for Veterinary Clinical Pathology, reference ranges should not be calculated when the sample size is <20 subjects. In these cases, mean or median and minimum and maximum values should be provided [33]. For the same reason, we did not evaluate the influence of gender or age on serum protein electrophoretic patterns.

In veterinary medicine, serum protein electrophoresis is recognized as a useful tool in the diagnosis, prognosis and monitoring of a number of diseases [15,17]. Alterations of blood biochemical values are interpreted by comparison with the same value in healthy subjects. This study provides useful data on the serum protein electrophoretic values in healthy captive tigers that increases our understanding of this endangered species. Furthermore, the paucity of reports of variations in serum protein electrophoretic patterns in free-ranging or captive tigers makes this preliminary study a useful aid for the evaluation of physiological and pathological alterations in both wild and captive tiger populations.
