Packed cell volume diagram

Packed cell volume diagram





Laboratory procedure
All animals were anesthetized using 5% sodium pentobarbital, in a 25 mg/kg dose. An incision on the abdominal skin was performed to expose the abdominal cavity. Blood was collected from the abdominal aorta artery by punction. After this procedure, animals were sacrificed by the sectioning of this artery.
For hematological analyses, 1 ml of blood was collected in a test tube with an anti-coagulant substance (EDTA). A COULTER T890 was used for white cell count, red cell count, hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (VCM), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (CHCM) and platelet count. In leukocyte differential count, blood smears, fixed and stained by the Romanowisk method were used. All these procedures were performed in the Seçمo de Hematologia pf the Instituto Adolfo Lutz (Sمo Paulo - SP).
For biochemical analyses, 5 ml of blood were collected, without anti-coagulant substances, in order to obtain serum samples. The following biochemical essays were performed: total protein, serum albumin, creatinin, alanin aminotransferase (GPT/ALT) and aspartate aminotransferase (GOT/AST), using WEINER LAB kits; alkaline phosphatase and urea, using LABTEST kits. Spectrophotometric reading was performed in a continuous flow system, in a BIO 2000 (BIOPLUS) analyzer, in the laboratory of Diagnَstico Toxicolَgico of the Departamento de Patologia of the Faculdade de Medicina Veterinلria e Zootecnia da Universidade de Sمo Paulo.
Statistical analysis
The variance analysis (ANOVA) was used to identify possible differences between hematological and biochemical values studied. P level was 0.05.

RESULTS
Values obtained for hemogram elements and for biochemical parameters from animals submitted to MEV were compared to those from the animals kept under GDV, which is the most common system used in animals facilities.
In Trial 1, significative differences were found for the following biochemical tests: albumin and urea in groups GDV and FV1 and ALT activity in the comparison between GDV and FV2 in male rats; for female rats, alkaline phosphatase values for FV1 and FV2 and urea, for the comparison GDV and FV1 and FV2 (Tab. 1).





Table 1

Values (mean ± standard deviation) for biochemical tests in male and female rats kept under Microenvironmental Ventilation System (MEV), using different levels of air speed (FV1 and FV2) and under General Diluting Ventilation system (GDV); n = 10. Sمo Paulo, 1998.



ALT = alanine aminotransferase; AST = aspartate aminotransferase; FV1: air speed level ranging from 0.03 to 0.26 m/s; FV2: air speed level ranging from 0.27 to 0.80 m/s. Different letters indicate that there are significative differences according to the ANOVA (p < 0.05)



In hemogram evaluation (Tab. 2) of male rats used in different groups in Trial 1, there were significative differences for the following components in groups FV1 and FV2: erythrocyte count, hemoglobin concentration, leukocyte count and absolute neutrophil count.






Table 2

Hematological values (mean ± standard deviation) obtained for male rats kept under Microenvironmental Ventilation system (MEV) using different levels of air speed (FV1 and FV2) and under General Diluting Ventilation system (GDV); n = 10. Sمo Paulo, 1998.



RBC = red blood cell; PCV = packed cell volume; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; WBC = white blood cell; FV1: air speed level ranging from 0.03 to 0.26 m/s; FV2: air speed level ranging from 0.27 to 0.80 m/s. Different letters indicate that there are significative differences according to the ANOVA (p < 0.05).



In the evaluation of the hemogram of female rats used in the different groups of Trial 1, there were significative differences between the number of erythrocytes of animals in groups GDV and FV2, and FV1 and FV2. In the analyses of hemoglobin values, there were differences between FV1 and FV2. In relation to hematocrit, significative differences occurred in groups GDV and FV2, and in groups FV1 and FV2. In CHCM, differences were found between GDV and FV1, and between FV1 and FV2. There was also a significative variation in the comparison between GDV and FV1 for the percentage of eosinophils (Tab. 3).



DISCUSSION
Reference values determined for biochemical tests and hemogram elements, as discussed and emphasized in specialized literature, may not represent precisely those of a certain population or animal species and should, therefore, be carefully interpreted4,13, once there is a wide range of physiological variation. Besides, these variations are influenced by environmental conditions, gender, age, origin, breeding system, feeding and lineage, which also may interfere with the results9,17,19 obtained in these tests. Therefore, the most adequate procedure would be to establish laboratory evaluation reference values for every animal facility.
Results obtained in biochemical analyses in Trial 1 male groups (Tab. 1) presented significative differences, in serum albumin, urea and ALT determinations. Among the MEV female groups, there were significative differences in relation to alkaline phosphatase and urea. Although these statistically significative differences have been noted, when these results are compared to the information found in specialized reports, they are considered to be inside the normal range of variation for the animal species and lineage4,13,16a.There is no biological reason for the variation to be clearly attributed to the management systems used in Trial 1. Alkaline phosphatase values observed in this trial were lower than the ones determined by Ringler; Dabich19. However, they were clearly higher for the male groups than for female ones, as stated by these authors.
When hematological values in Trial 1 are considered, there were significative differences between male FV1 and FV2 groups (Tab. 2), in relation to erythrocyte counts and hemoglobin concentration. When leukogram results are considered (Tab. 2), there were significative differences in the number of leukocytes and neutrophils, for both air speed levels in MEV, but not for the MEV and the GDV groups. In relation to female groups (Tab. 3), there were differences in the results for erythrocyte counts and hematocrit (FC2 X GDV; FV2 X FV1); hemoglobin values (FV1 X FV2) and CHCM (GDV X FV1; FV1 X FV2). There also was a significative difference for the relative number of eosinophils in animals of the GDV and FV1 (Tab. 3) groups. In spite of these evidence, values found were inside the range of physiological variation reported in the specialized literature9,12,20. No clear relation could be drawn from the differences found between the ventilation systems used (MEV and GDV).
When biochemical evaluation of the animals in Trial 2 is considered (Tab. 4), only alkaline phosphatase values for MEV groups IT7 and IT9 were significatively different from those of the GDV group. As already stated, these values were lower than the ones found by Ringler; Dabich19. However, this finding may be connected to physiological lineage variation, and environmental conditions, among other factors, for the values for alkaline phosphatase obtained were similar to those found by other authors4,13,16.
In relation to the variation of erythrogram elements (Tab. 5), in Trial 2 there were significative differences for the number of erythrocytes (IT3 X IT9), for HCM ((GDV X IT3; IT3 X IT9) and for platelet counts (GDV X IT9 and IT5 X IT9). However, values found in literature also place these results inside the normal range of variation5,19. Leukogram evaluation (Tab. 5) showed significative differences for the number of leukocytes and lymphocytes, when results for the GDV group and MEV groups with the smaller bed change interval (IT3 and IT5) were compared. These groups presented the lowest values for these counts. The similarity between the results for these counts in GDV animals and MEV groups with larger intervals for bed change (IT7 and IT9) may suggest a lower stress level in a more comfortable situation. It should be emphasized that IT3 and IT5 results were inside the normal range of variation for the species found in specialized reports4*.
Using the same animals of Trial 2, Carissimi6 studied ponderal development and feed intake of animals kept under MEV and GDV, and demonstrated that there was no significative difference for ponderal development in animals kept in both systems. In relation to feed intake, rats submitted to MEV ingested a statistically higher quantity than the ones submitted to GDV, no matter the bed change interval used. However, this fact did not produce any differences in erythrogram or total protein and albumin variations, in the comparison of the results for both groups. Therefore, intake observed may be related to higher energy consumption for body temperature maintenance in animals submitted to MEV.
It was noted that statistical differences observed occurred in a disperse way, and none of the systems studied was favored. Each of the items analyzed seem to point out for one of the ventilation systems, when they are analyzed individually. However, when all the items of each biochemical / hematological parameter are evaluated as a group, no correlation is found. It may be concluded that the differences observed are result of casual variability.

CONCLUSION
Results obtained in the present research trial demonstrate that the use of the MEV system does not produce significative alterations on biochemical or hematological parameters studied. This fact is significative and extremely important, for researchers should always be concerned with the standardization of laboratory animals, because they will influence one of the basic principles in research: result reproducibility. The need for determining reference values for different laboratory exams in every animal colony should be emphasized, because of the different and countless influence factors to which they are submitted in animal facilities. Reproductive, sanitary and bed change interval advantages of the MEV system, besides the data obtained in this trial and the possibility of higher control of microenvironmental variables suggest that this ventilation system should be used in the atmospheric control of animal facilities.