Reference physiological and biochemical values of adult albino rats

Introduction

The order Rodentia is the biggest group of mammals, consisting of more than 440 genera and over 2000 distinct species. The order consists of five primary families: Muridae, Sciuridae, Echimyidae, Heteromyidae, and Dipodidae. The Muridae account for approximately 66% of all rodent taxa (1). Rodents exhibit remarkable intelligence according to their physiological characteristics (2). Many rodent species are characterized by their small size, short gestation periods, early sexual maturity, secretive behavior, nocturnal activity, adaptability, and highly developed senses of touch, taste, and smell (3). Researchers noticed that sex and age substantially impacted hematological and biochemical markers in India's Wistar (Han) rats (4). Rodents, such as mice and rats, are the preferred preclinical animal models in pharmaceutical research due to their strong resemblance to humans and other mammals. They are also more suitable for comparison than larger and longer-lived species (5). Most human diseases can be replicated in these rats by altering corresponding genes or physicochemical stimuli. Additionally, rodents are crucial for conducting toxicity and safety studies while developing novel medications (6). Therefore, any variations in hematological, biochemical, or vital signs deemed significant are documented in reference studies. Based on prior research, the lymphocyte counts in rats typically vary according to age. Generally, as rats age, the number of lymphocytes falls while the percentage of neutrophils, eosinophils, and monocytes increases (7). However, a separate study indicates that age does not substantially influence the quantity of monocytes (8). Although the basophil numbers in rats fall within the usual range for most species, they make up less than 1% of blood leukocytes (9). Both factors, whether they positively affect biology, could potentially result in discovering new biomarkers or therapeutic targets for different diseases (10). Although the normal range for RBC count in rats is not mentioned, further research is needed to establish reference values (11). Understanding the typical range of hematocrit is essential for differentiating between normal and pathological conditions. In awake hamsters, reducing HCT levels led to a rise in mean arterial blood pressure and a consistent increase in cardiac output (12). Rats within the normal range of mean corpuscular volume (MCV) exhibit distinct physiological and behavioural responses compared to other strains of rats (13). The behavioural patterns commonly observed in rats within the MCH group are affected by various factors, including anxiety levels, which are internally related to measures of inquisitive and seeking activities (14). The abstracts presented do not directly specify the normal range of Red Blood Cell Distribution Width (RDW) in rats. However, they do include information about the importance and association of RDW with various diseases and disorders. RDW is a metric that quantifies the variability in size and volume of red blood cells (RBCs) and is correlated with anisocytosis. Elevated RDW levels are connected to unfavourable cardio-renal outcomes (15). RDW-SD, a metric for erythrocyte mean corpuscular volume (MCV) variability, exhibits variation among rat species. A study conducted by Chan et al. revealed notable variations in reported baseline RDW-SD values among different rat strains and detection methods (16). These findings indicate that variations in RDW-SD outcomes can be attributed to factors such as species and detection methods. The study suggests utilizing corresponding negative control models as a fundamental RDW-SD reference range for each investigation assessing RDW-SD levels in rats (17). Platelet indices, such as platelet count, plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW), undergo significant changes in rats with the duration of diabetes. Additionally, platelet count is a crucial test used in drug development studies to evaluate the well-being of rats (18). Platelet-large cell ratio (P-LCR) is a regularly computed measure in hemogram examinations. The literature does not provide a particular reference to the typical range of P-LCR in rats (19). The abstracts do not expressly state the specific range of triglyceride levels considered normal in rats. Nevertheless, the abstracts indicate that rats' triglyceride levels were within the range of normal values in multiple trials (20). In addition, the rate of cholesterol breakdown in rats decreases as they age, resulting in prolonged cholesterol retention in the body (21). The abstracts do not specify the typical range of blood urea values in rats (22). Investigating the disparities in ALT U/L metabolism between rats and humans holds promise for comprehending sickness and well-being (23). Understanding the variations in respiratory rate is valuable for studying disease and health. Knowing the normal values is necessary to differentiate between physical activity, exercise, disease, and healthy conditions. This knowledge is crucial for preventing metabolic diseases and promoting longevity (23,24).

Finally, the study aims to integrate all physiological, hematological, hormonal, and biochemical indicators affected by various causes in normal conditions. Consequently, we must remain current with these principles, especially in each country.

Materials and methods

Rats' management

This study utilized a sample of 50 male adult Wistar rats weighted at an average of 223.310-243.169 g raised in the animal facilities at Mosul University's Veterinary College, Iraq. Throughout the experiment, conducted in October, November, and December of 2023, the rats were raised in a controlled environment with a consistent daily cycle of twelve hours of light and darkness at a temperature of 25ºC±3 after two acclimations ten cages made of plastic with a lid made of steel. Before collecting any parameters, the rats were upraised from one month to mature (3-3.5 months) and provided unrestricted access to fresh water and a standard regular diet. The rats were handled and managed following the regulations of the Ethics Committee at the College of Veterinary Medicine, University of Mosul. The study was approved under the reference number UM.VET.2023.080. Each enclosure has a total of 50 rats.

Limitation procedures                                                                                                                                  

Handling and restraining exotic animals can be difficult because of their strong jaws and the requirement for pharmacological immobilization and sedation. In a recent study, sedative medications were omitted because they tend to affect regular readings. Consequently, physical constraint was employed as an alternative. We employed stainless steel buckles to manage and confine the creatures.

Vital signs

The individuals at the resting stage were gathered using a specialized instrument for veterinary purposes called the Veterinary Monitor Operation Manual (25), which is included in table 1.

Table 1: The table displays vital signs using a Veterinary Monitor

Hematological parameters

The initial portion of the blood samples was collected from the animals' eyes using an ocular puncture technique into a capillary tube containing heparin and subsequently transferred into a 0.5 mL tube containing EDTA. Following persistent agitation, the blood tube is inserted into the Veterinary auto-hematology analyzer (26-28). Table 2 display the parameters for full blood counts (CBC).

Table 2: The table displays complete blood counting (CBC) using Automatic Hematology Analyzer

Hormonal parameters

To determine hormone levels, the second blood sample was collected in a gel tube, subjected to centrifugation to obtain pure serum, and subsequently stored at a temperature of -26°C until analysis using a Microplate Reader (29,30), the following kit as shown in table 3 (Shanghai Ideal Medical Technology CO; LTD, Chani).

Table 3: The table displays hormonal assay using a Microplate Reader

Biochemical parameters

The biochemistry analyzer measures biochemical parameters using serum sample. Its components are listed in table 4.

Table 4: The table displays biochemical parameters using Automatic Biochemistry Analyzer

Statistically analyze

Analyzed data was subjected to descriptive and inferential statistical analysis using JMP Pro16.1 software (2021 SAS Institute Inc., North Carolina, USA). The descriptive statistics encompassed the measures of central tendency, such as the mean median, and the measures of dispersion, such as the range and quantiles. The impact of the treatments on the animals was assessed by analyzing variance (ANOVA) in the data. The treatment groups, chosen from a population with a normal distribution, were compared using the student's t-test and the Duncan multiple range test. The results were considered statistically significant at a significance level of P<0.05 (31).

Results

After assessing various health indicators, all rats appeared to be in good health (Table 5). These indicators encompass oxygen saturation, rectal temperature, heart rate, respiratory rate, and blood pressure (systolic, diastolic, and even plus) as a range (11.137-13.752, 8.012-9.547, 51.644-66.255, 81.882-88.197, 33.532-34.727, 60.489-66.990, 71.130±75.549) respectively. All of these parameters were taken in the morning and obtained by a patient monitor specialized in veterinary medicine. The average rat weight was 233.24 g, with a 223.310-243.169 g range.

The complete blood picture of adult male rats involves leukocytic count (14.217-16.675) and differential leukocyte count (DLC) (1.959-2.661, 9.305-11.360, 2.683-3.128, 0.078-0.096, 0.015-0.020) and their percentage (13.879-18.116, 63.814-69.525, 18.431-20.292, 0.538-0.681, 0.108-0.113), whereas the range of counting erythrocytes (11.434-13.010) and their distribution width (13.739-14.164 and 33.064-34.067) respectively. The hemoglobin concentration in grams per 100 milliliters ranges from 181.492 to 207.147, while the hematocrit percentage ranges from 67.283 to 78.320. On a median, the anemic index comprises MCV (fl), MCH (pg), and MCHC (g/100 l) (58.258-60.037, 15.616-16.079, 265.53-272.662). The mean platelet count (597.942-688.557) and percentage mean platelet crit are 0.445-0.519, respectively, along with the mean platelet volume (7.474-7.281) and platelet distribution width (10.367-10.792). Yet, the mean platelet-large cell ratio (4.824-5.751) and platelet-large cell count (28.621-34.138) are all parameters illustrated in table 6.

Table 7 displays the normal range of hormones for adult male rats, which includes leptin, TSH, insulin, T4, and cortisol (2.935-3.142, 3.097-3.445, 25.993-27.549, 81.207-87.307, and 45.979-52.888). Table 8 reveals the average value of biochemical analysis for control rats, encompassing glucose (117.39-104.56), total cholesterol (79.21-73.98), triglycerides (71.45-61.50), high-density lipoprotein cholesterol (37.796-33.923), blood urea nitrogen (43.303-39.816), serum creatinine (0.546-0.513), Alanine transaminase (64.562-60.117), aspartate transferase (235.20-220.173), and total protein (6.046-5.729).

Table 5: The table displays vital signs in male adult rats

Table 6 displays complete blood counting (CBC) in male adult rats

Table 7: The table displays hormonal assay in male adult rats

Table 8: The table displays biochemical parameters in male adult rats

Discussion

This discussion shows that the normal physiological, hematological, hormonal, and biochemical levels of people with albinism are significant compared with control groups from other research from different countries. These differences between values may come from environmental, nutritional, genetic, geographical, managemental, and other factors that the researchers consider.

Starting with the vital signs, Kyoto City, Japan recorded a slight difference in systolic and diastolic pressure but the pulse pressure values did not fall within the current reference range which was 51-66, and their result of 27.40 (32), while the albino rats in Zagazig, Egypt  showed no such difference in both higher and lower pressure (33), on the contrary in research established in Abha, Sudia Arabia noted a clear difference in systolic pressure 80.3 and diastolic was 49.3 (34), besides in 3 remote countries documented a diverse pulse pressure from this study like Nigeria was incredibly greater than this research otherwise in Berlin, Germany, and Jamaica was a little lesser than the current study (35-37) Vital signs are regarded as the first line for determining healthy status; thus, any variation within a group or even geographical area is recorded as a pathological cause; however, when the variables are in different countries, ambient temperature, ration type, ventilation, humidity, and even animal manipulation can cause deviations from the norms of the trail results (38,39).       

Research talks about a differential comparison of the oxygen saturation level in rats between 2 land levels starting in La Paz, Bolivia (3600 m above the sea level), documented 71% SPO2 at 90 mmHg and 95% SPO2 at 160 mmHg. In contrast, the sea level in Quebec City, Canada, was 89% SPO2 at 160 mmHg and 72% SPO2 at 90 mmHg. All these values differed from this experiment value, which showed approximately 81-88% SPO2. These distinctions could clarify the disparity in the country distribution spotted in wild rats and mice (40).

 A study done in the UAE in 2005 and in Bosnia and Herzegovina revealed the body temperature was different from the Mosul record by about 2.3ºC (41), while the heart rate in Banaras, India, was enormously altered from ours; nevertheless, the respiratory was close to the same rate (42). According to a famed international website about rat guide of physiological and other parameters, their temperature was 37.7 C, above the manuscript record of about 33-34 C, blood pressure was the same, and the HR and RR were exactly dissimilar from the Mosul rat's heart and respiratory rates (43). Indeed, the surrounding temperature and other circumstances, such as circadian variations, have all been attributed to set point temperature shifts of rats or any other animal considered a homeotherm; these recent papers aim to address this issue (44).

In research found in Egypt, the hematological significances of the control group of RBC count, Hb, HCT, MCV, MCH, MCHC, and WBC count exhibited differences with the experiment values, especially in HCT and MCHC (45); however, in Ibadan, Nigeria the values of MCV and MCH is higher than this study but RBC count, Hb, HCT, and MCHC were exposed the lower from the Mosul albinos (46). In Nsukka, Nigeria, the total WBC count with each type did not present much difference (47), but in Uttar Pradesh, India, the DLC indicated little deviation from the study rates (48). The platelet indices in Calabar, Nigeria, were almost equal to this experiment's (49). The book covers rodent normal ranges, the RBC count, and PCV. The trial reports did not fall in the standard range, and all forms of WBCs were in the same range, excluding monocytes, which were lower than our evidence, although thrombocytes were in virtually the same range (50). Since the blood criteria are regarded as a good indicator of animal health, variations in the feed component's type, amount, and state significantly affect the total blood picture (51).  

During this study, the rats were not exposed to any stress agent, and the cortisol rates did not match with the control group of another study in Shiraz, Iran (52), while in Mumbai, India, they did not fall within the acceptable range of the current study (53). Likewise, in rodents, both hormones cortisol and corticosterone might change in ratio depending on the demands and physiological accordance to regulating metabolites (54).

The insulin evaluation in 2 studies in Cairo, Egypt, was lower than the Mosul study (55), and the 2nd one was higher than this trial (56). A study in Kurdistan, Iraq, measured the leptin level and was lower than the study evaluation (57); however, in Ankara, Turkey, it was the same (58), while the Medical Faculty of the LMU Munich evaluated some metabolic hormones using various methods and found a leptin value in the serum by ELISA close to and within the same range as current study (59). In these investigations in Harlan Labs, Indianapolis, Faculty of Georgia, male Sprague-Dawley rats (SD rats) were employed in the experiment to obtain results regarding serum insulin and leptin levels in line with the trial data (60). In general, leptin monitors calorie intake and spending; thus, its average level is determined by requests for energy, and variations in the common value do not differ at the physiological level. Also, the noted impacts of leptin on the production of thyroid hormone are contested, with research focusing primarily on energy imbalance (61). In 2 trials at Menoufiya University, Egypt, and Jorden, the hormone T4 of the control rats was lower than ours, but TSH was somehow like the manuscript levels (62,63).

In Giza, Egypt, a group of researchers calculated biochemical standards of ALT, AST, total protein, urea, creatinine, and glucose were roughly similar except AST was lower (64), while in Colombo, Sri Lanka the total cholesterol, total triglycerol were lower. However, the ALT was higher (65). Twenty-one days later, Wistar rats in Buea, Cameroon, noted a lipid picture of total cholesterol, triglycerol, and HDLC much higher than the experiment picture (66). In line with vet books about rats, the biochemical ranges of total protein, glucose, creatinine, TG, and cholesterol were roughly the same range; nonetheless, the urea nitrogen data was lower than ours even though the liver enzymes range was higher than the present experiment (67,68). Variations in various biochemical tests may solely be ascribed to individual variances, potentially linked to hereditary factors stemming from inbreeding and outbreeding (65). Others affirm that the solitary housing of rats induces alterations in specific biomarker markers (69).

Conclusion

The intervals are computed based on data gathered from a collection of healthy animals who have undergone statistical methods and are in comparable circumstances. Reference intervals are the primary instrument used in healthcare decision-making. They serve as the foundation for laboratory investigations and are crucial to the interpretation of findings, enhancing the accuracy of the results. After a months-long study, standards and levels were collected for 4 basic parameters ​​covered by most research in Iraqi veterinary colleges for the sake of one goal: accuracy and scientific honesty in examining these standards. To prove this, these results were compared with those of other countries worldwide and from biochemistry and physiology books, indicating a noteworthy variance in vital, hormonal, and biochemical values ​​and a slight variation in blood pictures.

Acknowledgments

We are grateful to the College of Veterinary Medicine, the University of Mosul, and the University of Alnoor for their financial support of this work.

Conflict of interest

The authors declare that they have no conflict of interest