Various studies have reported differences in laboratory animal blood biochemistry parameters, as well as haematological parameters, related to species, strain, sex and age [11, 13–15]. Blood collection procedures related to duration of preceding fasting, time of sampling, time of samples to stand, hemolysis, use of plasma instead of serum, storage until time of measurement, method of analysis, are factors that can affect blood parameters measured [11, 13]. Although there is a multitude of reports on the lipidemic changes of heritable hyperlipidemic rabbits in chronic studies [16–19], there are relatively fewer reports on detailed changes in normal rabbits that are used as comparative controls [20, 21].
In the present study we sought to verify if during a short-term (3-month) study, normal young male NZW rabbits under a normal diet, which are often used as controls, have a stable biochemical profile, by examining their plasma values after a 12-hour fasting period. Particular attention was paid to the blood sampling procedures in order to exclude preanalytical variation. With the use of mild short-acting sedation, stress related to the blood-sampling procedure was avoided. Additionally, auricular vasodilation is prominent under sedation, which makes the sampling procedure simple and brief. The person in charge of the sampling had years of experience and training. The previously mentioned 12/12h lighting schedule of the animal house has been shown to elicit the fewest variations in blood biochemistry parameters  and the same sampling time was followed in order to avoid potential diurnal variations .
The baseline blood biochemical values of our study at 3 months were similar to those of other researchers in NZW rabbits [13, 23, 24]. In our study, plasma glucose levels of male NZW rabbits increased from the age of 3 months (135 mg/dL) throughout 6 months (177 mg/dL), even from the 4th month of age, with a statistically significant difference (p < 0.001 vs baseline, Table 1). Glucose appears to vary in studies with male NZW rabbits, with normal values at 112 mg/dL  or 187 mg/dL (10.42 mmol/L) . As glucose values are reported to present diurnal variation and affected when animals are frightened when handled or restrained without anesthesia , different values between studies may be due these reasons. In the present study, as previously mentioned, all samplings were carried out under sedation and at the same time of the morning.
Total cholesterol values similarly increased statistically significantly throughout the study, beginning at 51 and reaching 87 mg/dL (p < 0.001 vs baseline, Table 1, Figure 1). In contrast to our findings, Orlandi et al. found minimal differences in total cholesterol values of 4-month-old and 5-year-old NZW rabbits, which ranged between 28 and 34 mg/dL . Another study on younger (2-month-old) NZW rabbits reported 81 mg/dL (2.11 mmol/L) .
HDL-C values presented a small increase one month after our study start (p < 0.05 vs baseline), similar to other studies (23.5 mg/dL)  returning to baseline values at the end. LDL-C values however significantly increased at 6 months of age (p < 0.001 vs baseline). Triacylglycerol initial values were 56 mg/dL and reached 123 mg/dL at 6 months of age (p < 0.001 vs baseline).
Additionally, the activity of hepatic enzymes ALT and AST increased with age In the present study. This is in agreement with Matsuzawa et al., who studied age-related biochemical changes in a large number of other animals (monkeys, dogs and rats) . Their study supported that as a general trend, these hepatic enzymes increase with age, which however does not necessarily reflect a corresponding liver pathology, as their increase has been noted to occur due to fear of venepuncture. The authors also supported that liver enzymes are not elevated in heparinized blood. Therefore, the significant increase of values in the samples of the present study was not due to an anticoagulant effect, but most probably was age-related.
MDA also increased significantly towards the end of the study (p ≤ 0.001 vs baseline, Table 1). MDA has been the most studied product of polyunsaturated fatty acid peroxidation, indicating oxidative stress .
The aortas of the rabbits of the present study had normal both macro- and microscopical appearance, which is not compatible with the observed biochemical changes. Other studies with control groups of rabbits of 6 months of age had no pathologic findings in their aortas similarly to ours [3, 9].
As previously mentioned, the rabbits of this study consisted the control group of an experimental atherosclerosis study. It is acknowledged that their number (n = 8) may be considered small compared to other studies that have as main objective to establish reference values of blood parameters. However, their values' SD did not have a wide range and it is also ethically desirable to use as few animals as possible in experimental research . Similar numbers of rabbits per group to ours have also been used in other experimental atherosclerosis studies [1, 9, 21].
Diet composition is known to influence blood biochemical parameters. A change in lipid blood parameters in a short period of time could be due to a change of diet lipid composition. However, the rabbits of the present study were fed the same diet (2.5% fat content) in the breeding establishment and subsequently in our experimental establishment. Therefore, their lipid profile change cannot be attributed to a change of diet.
New Zealand White rabbits are the breed that has been and continues to be used in many studies of atherosclerosis research [1, 28, 29]. However, it has not been sufficiently taken into account that this breed has been also considered a spontaneous model of diabetes . It could be possible that with ad libitum feeding throughout time, diabetes could emerge. This is supported by the finding that plasma glucose levels were significantly increased compared to baseline already by the 4th month of age, and continued to rise. Additionally, this could be the beginning of a "metabolic syndrome", which perhaps did not have the time to manifest itself fully, because the animals had to be euthanized at 6 months of age. It is possible that with time, their metabolism undergoes an age-related change. It consists a limitation of the present study that it was not considered to monitor their blood pressure non-invasively throughout this period, as this would assist in accepting or rejecting the metabolic syndrome hypothesis. A longer observation period in a future study might also give answers. Aging has been proved to induce blood biochemistry changes in many animal species [10–12].
A possibility that the prenatal environment could have had a role in these biochemical alterations was also considered. The breeding establishment may have had some adverse effect on the pregnant does during our rabbits' prenatal life, which is practically impossible to investigate retrospectively. It is well known that maternal stress and nutritional imbalance during pregnancy affects fetal postnatal development adversely and can lead to several diseases in adult life, such as metabolic diseases, hypertension, renal insufficiency, etc. [31, 32], which consists the "fetal origins" hypothesis . This possibility cannot be ruled out.
Another factor that may have developed a stressful situation to our rabbits is their housing conditions. In the breeding establishment, they were housed in rows of cages in physical contact with each other. In their new housing condition in the experimental establishment, they were housed singly in stainless steel cages, with no visual or physical contact. It is well known that isolation stress is a potent stressor [34, 35], which very likely induced the biochemical changes. On the contrary, one blood sampling per month by experienced personnel and under the aforementioned precautions cannot be considered a stressing situation. Taking into account their overall experimental housing conditions, the isolated laboratory life of the rabbits appears to be a probable cause for these biochemical changes.