Clinical hematological and biochemical parameters in Swiss, BALB/c, C57BL/6 and B6D2F1 Mus musculus

Abstract Background Animal models are widely used in scientific research in order to obtain information from a whole organism under a specific set of experimental conditions. Various lineages of mice have been used to investigate diseases and new therapeutic strategies, and, consequently, hematological and biochemical tests in these laboratory animals are essential to validate scientific studies. Our study seeks to establish reference values for hematological and biochemical parameters of four lineages of mice. Methods We evaluated the hematological and biochemical profiles of 20 males and 20 females from the lineages Swiss (heterogeneous), BALB/c and C57BL/6 (isogenic), and B6D2F1 (hybrid), totaling 160 mice. Analysis were standardized using the systems pocH‐100iV Diff™ for 19 hematological parameters and VITROS® 350 for 12 biochemical parameters. Results Results are shown as means and standard deviation, grouped by lineage and genre. Comparing the values obtained in this study with the values from previous studies, some variations were detected, which could be explained by differences in methodologies or individual variability. Conclusion Thus our study shows that knowledge and disclosure of the values of physiological parameters of laboratory animals is necessary, and emphasises the importance of considering variations influenced by gender, lineage and genotype in the choice of the best experimental model.


| INTRODUC TI ON
Laboratory mice are the most commonly used animal model for biological studies of human health leading to the establishment of new diagnostic and therapeutic strategies. 1,2 The demand for rat models has also increased in pharmacological, oncological and toxicological research, as well as in studies on drug efficacy, 2 due to their easy creation, short generation time and the availability of inbred lineages (at least 20 generations of brother-sister mating). 3,4 The extensive mapping of the mice's genome and the detailed understanding of its immunological properties 5 have improved the standardization of experimental models, increasing the reproducibility of studies and the comparison of results by researchers worldwide, thereby minimizing the need to repeat experiments. 2 Advances in transgenic research and genetic targeting in models that use laboratory animals have led to a deeper understanding of the mechanisms of many diseases, and revealed new possibilities for treatment in human and veterinary medicine. 1,5 Swiss heterogeneous mice (non-consanguineous, heterogamous, outbred) present 99% heterozygosity between allele genes and therefore they have been used as a source of consanguineous animals representing natural populations and to obtain hybrid and transgenic descendants. 6 Their aggressive behavior makes them good models in studies related to causes and/or mechanisms of aggression. 7 This lineage is widely used for scientific purposes such as biomedical research on metabolic and autoimmune diseases, complement fixation, and mammary gland or lung tumors, as well as in pharmacology as stock in security testing for drugs. 8 Due to their superior maternal abilities, Swiss females are used as ideal pseudogestants in the transfer of transgenic embryos and embryos of other lineages of mice. 9 Both BALB/c and C57BL/6 lineages are isogenic (consanguineous, isogamous, inbred), obtained by crossing brothers and sisters for 20 consecutive generations, which allows the production of genetically homogeneous populations and, consequently, the need for fewer samples per experimental group. 6 BALB/c mice have 99% homozygosity among allele genes, and are used for the production of monoclonal antibodies from plasmocytes. 6 They have a low incidence of mammary tumors; however, they may develop other types of cancer, such as reticular neoplasias, and primary tumors in the lung and kidneys over their lifetime. This lineage has been frequently used in studies of infection with Listeria monocytogenes due to their great susceptibility: the mean time to death for females is three days. 10 The C57BL/6 lineage has 6.5% of its genome originating from the Mus spretus species, 6 and is widely used for conducting spontaneous and induced mutations, since it can totally express such mutations. Its main characteristics are low susceptibility to tumors and hearing loss induced by noise, and it is commonly used in studies on cardiovascular biology, development and genetics, and immunological, neurological and sensory stimuli. More specific characteristics are expressed in sublineages such as C57BL/6J, which is widely used in the production of transgenic mice. C57BL/sublineages are genetically prone to develop obesity induced by diet, diabetes type 2, atherosclerosis, high incidence of microphthalmia, reduced bone density and hereditary hydrocephalus. 11 Unlike the other lineages, the B6D2F1 lineage is a hybrid (originally from crossing C57BL/6 [B6] females with DBA/2 [D2] males), and is heterozygous for the B6 and D2 alleles at all locations in the genome (genetic and phenotypic uniformity). The females have been used as embryo donors for microinjection in the creation of transgenic/knockout and deletion mutations, and in behavioral research, radiation and nutrient bioassays, drug and hormone research, and transplant (tumor, ovaries and skin) research using other lineages. 12 Variations in the hematological and biochemical parameters of mice may be related to lineage, genotype, and genre, and are influenced by age, diet, environment, place of collection, and other factors. 3,13,14 Therefore, knowledge of the physiological parameters and appropriate interpretation of hematological and biochemical serum rates are relevant to an evaluation of homeostasis and the alterations induced by pathological processes in different organs, 15,16 since they provide information about the animal's clinical conditions, nutritional balance, iron deficiency in hemoglobin and presence of infections, and are useful in monitoring the efficacy and the prognosis of treatments. 17 Our study sought to establish values to serve as reference values in various hematological and biochemical of serum parameters in mice of the Swiss (heterogeneous), BALB/c and C57BL/6 (isogenic) and B6D2F1 (hybrid) lineages, to guide researchers in the best choice of an experimental model. This study used 20 male and 20 female mice from each of the Swiss (heterogeneous), BALB/c and C57BL/6 (isogenic) and B6D2F1 (hybrid) lineages, totaling 160 individuals; with 60 days of life mice were 60 days old (age). The mice were examined and showed no lesions or inflammation of the skin and no ectoparasites at the time of collection. The animals were kept in the bioterium of the School of Medical Sciences of the UERJ, in rigorous sanitary conditions with quarter-hourly monitoring. They were free of specific pathogens (SPF), and were housed in racks of mini isolators (5 mice/cage; cage dimensions 45 × 31 × 19 cm) lined with sterile pine wood shavings of (ASPEN -LIGNOCEL ® ), which were periodically renewed. The mice were kept in filtered air at positive pressure, with a 12 hours light/ dark cycle, starting at 6:30 am, temperature ~21 to 22°C (±2°C) and humidity ~50 to 55%. All animals received sterile water (autoclaved) and appropriate rations (Nuvilab ® CR-1) ad libitum, meeting the nutritional requirements for the species. The animals were not exposed to chemical or drug treatments that could change their natural physiological state.

| Blood sampling method and sample handling
All biological material was collected by the same qualified professional. Material was collected after an 8-hour fast, in the morning, to avoid variations in the parameters. Initially, the absolute weights of the animals were measured using a precision balance (Marte -AD 2000, maximum load capacity 210 g; sensitivity of 0.01 g).
Subsequently, they were sedated by intraperitoneal injection (lower right quadrant) of 16 mg/kg of xylazine hydrochloride ® as a muscle relaxant and 120 mg/kg of ketamine hydrochloride ® for deep sedation, using BD Ultra-Fine TM syringes (needle 25 × 5 mm). After Aliquots of 300 µL of blood were collected in tubes (10 × 45 mm, maximum volume 500 µL -VACUPLAST) containing ethylenediamine tetra-acetic acid (EDTA-K2) and carefully mixed by inversion in a homogenizer (Electra -Homolaby 22T) for a complete blood count (CBC). For biochemical analysis of serum, aliquots of blood were deposited in tubes (10 × 45 mm, maximum volume 500 µL -VACUPLAST) containing separator gel and coagulation activators, to the maximum volume indicated by the manufacturer, waiting 30 minutes to blood clot retraction. The aliquots were then centrifuged for 5 minutes at 2500 rpm (Eppendorf ® Minispin ® model SPIN 1.000, Hamburg, Germany) to separate the serum. The biological samples were then sent to the Institute of Science and Technology in Biomodels, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

| Clinical biochemistry parameters
The blood serum was processed in an automated spectrophotom-   Santa, Minas Gerais, Brazil), and were used according to protocols established by manufacturer.

| Statistical analysis
The variables of interest monitored were absolute body weights and the values obtained by hemogram and serum biochemistry in all four lineages of mice (Swiss, BALB/c, C57BL/6 and B6D2F1) of both genders (male and female). The data obtained for analysis were reported as mean values and standard deviation (SD), with 95% confidence intervals (CI); P > .05 was considered statistically significant. The statistical analysis was conducted using software GraphPad Prism version 6.01.  (Table 3). Immature forms of neutrophils were not observed. Anucleate and slightly flattened platelets were observed, as well as few platelet aggregates (Figures 1 and 2).

| RE SULTS
The blood serum was limpid and semi-transparent in aspect.
The analyses of biochemical parameters demonstrated higher ALB and UR levels in Swiss males; higher CHL levels in BALB/c males, and higher AP and ALT in females; higher TP and UA levels in C57BL/6males, and higher AST and GLB in females; and finally, higher TG levels in B6D2F1 males and higher GLC levels in females (Table 4).

| D ISCUSS I ON
The   Currently, many automated hematological counters provide different parameters for platelet analysis. Despite still being little understood, they are used in medical and laboratory practice, particularly because of the difficulty in standardization. In analogy to the erythrogram and leukogram, the parameters that analyze the platelets composed of PLT, PDW, MPV and P-LCR came to be called plateletogram. 27 The platelets are anucleate fragmented cells derived from megakaryocytes. They have hemostatic (buffer) function, and maintain endothelium integrity by releasing pro-angiogenic cytokines. 28 These cells can be activated spontaneously or in response to stimuli, depending on lineage activation stage. 21 In mice, platelet count is ~900 to 1600 × 10 3 /mm 3  Understanding immunological characteristics is essential to evaluating inherited or acquired disorders. 30 Leukocytes participate in immune and inflammatory processes, being responsible for mediating the innate and adaptive immune response. In mice, the total WBC count is ~2 to 10 × 10 3 /mm 3  rates. We could not find any comparable studies using automated assessments of small, medium and large white blood cells.
LYNs are mostly mediators of adaptive immunity, 17 and comprise ~ 70% to 80% of leukocyte counts in mice, which can increase to above 80% in young animals. 21,31 MON comprise ~0% to 2% of the total leukocyte count in mice 21,31 and, along with free macrophages and those fixed in the tissues, are responsible for initiating the immune response, and phagocytozing and destroying microorganisms such as bacteria. 32 Our automated and microscopic eval- The assessment of hepatic function in response to anatomical or biochemical alterations 42 is commonly subjected to dosage testing of ALT, AST, AP and TP. 43 The origin of ALT is predominantly plasmatic; it is found abundantly in the liver, in moderate amounts in the kidneys and in small quantities in the heart and skeletal musculature. 43,44 AST is an intracellular enzyme present in the cytoplasm and mitochondria 43 in various organs and tissues, including liver, kidneys, heart, brain, skeletal muscle and erythrocytes. 45  Changes to the ALT, AST, AP and TP can be symptomatic of some diseases. The rapid elevation of ALT indicates hepatic lesion. 43,44 When associated with an increase in AST concentration, it indicates a profound damage from hepatocytes. 43 In small animals, increased AP is observed in inflammatory and degenerative disorders of the skeletal musculature, hepatocellular dysfunction, and heart diseases (eg ischemia, congestion, necrosis, neoplasia, trauma). 45,50 ALB and GLB are important in maintaining osmotic pressure, to avoid blood extravasation, and are commonly evaluated in conjunction with TP as determinants of metabolism homeostasis. 47 The plasma reduction of both may have similar causes to a decrease in TP. 45,51 ALB is synthesized in the liver and comprises 50% of total protein in the serum. It is responsible for 80% of colloidal osmotic pressure and acid-base equilibrium (in metabolic acidosis). 52  We could not find comparable studies mentioning parameters for this index using this lineage.
GLC present in the serum is obtained mainly through the diet, and to a lesser extent from hepatic glycogen. Its main function is to generate energy (adenosine triphosphate -ATP) for biological functions. 55

| CON CLUS ION
The determination of values for hematological and biochemical parameters in Swiss, BALB/c, C57BL/6 and B6D2F1 mice is important in the choice of experimental model and study design because of the significant differences in these parameters between lineages, genders and routes of blood collection. Automated analyses should always be confirmed by microscopy evaluation and compared with the data from the literature for optimal interpretation. Each bioterium should establish its own reference values, since a wide range of variations in physiological parameters can be due to the conditions to which the animals are subjected, and this can affect the results of research. It is important to consider the many variables that directly interfere with metabolism and, consequently, hematological and biochemical values: species, age, genetic variation and the environmental conditions to which the animals are subjected, namely temperature, relative humidity, ventilation, lighting, noise, manipulation, feeding, water, microbiota, presence of pathogens and contact with other animals.

CO N FLI C T O F I NTE R E S T S
The authors declare that there are no conflicts of interests.