Method discussion

5.1.1. Animals

It was decided to use Hermann’s tortoises for this study, because these tortoises are one of the most commonly kept reptile species in Germany. From this concludes that they are one of the most frequently presented reptile species in veterinary surgery. Among the 456 chelonians presented at the Klinik f¨ur V¨ogel, Reptilien, Amphibien und Zierfische of the LMU Munich within one year (July 2014-June 2015) were 275 Hermann’s tortoises, which equates to 60,3 %.

The blood samples used in this study were obtained in the context of the propaedeutics classes for students of veterinary medicine at the Klinik f¨ur V¨ogel, Reptilien, Amphibien und Zierfische of the LMU Munich. For this study, the required amount of blood was taken out of the samples before they were discarded. The survey was restricted to male animals, as they usually have a longer tail than females, allowing easier access of the Vena coccygealis dorsalis for blood collection. Blood specimens of 0,33 ml were necessary and a minimum body weight of at least 200 g was assigned to absolutely ensure that the blood volume was sufficient for blood sampling without harming the animal, also bearing in mind the repeated samplings after one month. The actual body weights of the tortoises ranged from 445-1085 g, so that the smallest animal had the double minimum weight.

Since the emphasis of this study was on how the storage time and temperature influence the results, as opposed to the indiviual animals and other influences, it was attempted to standardise the procedure as much as possible for a better comparability. To achieve this, it was considered necessary to use animals of the same sex and to use the same individuals repeatedly, as this would rule out a variation due to different individuals.

In veterinary practice it is recommended to tortoise owners to have a blood test run at

CHAPTER 5. DISCUSSION

the veterinarian to establish individual reference intervals over time, which facilitates the early recognition of diseases in the individual animal. Repeated blood examinations are also necessary for the veterinarian to monitor the success of therapy in a sick animal, as has been recommended by Campbell [2006], who underlined the utility of repeated blood examinations in a single animal. In this study an interval of at least three weeks in between the blood samplings was set, which was considered a safe time for blood renewal and recovery from overall strain and also allowed the study to be carried out within one summer activity period of the tortoises.

Since no signs of illnesses were found in the clinical examinations and the X-rays, the animals used were regarded as healthy despite the lowered LDH values, as this was not considered to require treatment.

5.1.2. Evaluation of blood smears

The evaluation of all blood smears was conducted by the same person and comprised of several steps. First, the macroscopic quality was rated. Subsequently, the microscopic evaluation followed, beginning with a differential blood count of 100 leucocytes. During the performance of the differential blood count, the microscopic quality, the bacterial growth and the cytoplasmic vacuoles in the erythrocytes were also observed and docu-mented with graded assessments.

It was decided to evaluate the smears by a single person taking advantage of the fact that then the same criteria were used consistently. A problem using this strategy can arise because of a certain subjective bias of the evaluating person, which is difficult to rule out completely. In the presented study it was tried to restrict this bias by using predefined criteria for the macroscopic and microscopic quality and a staged assessment for the amount of bacterial growth and the cytoplasmic vacuoles in erythrocytes. Nonetheless, criteria evaluated by humans always show a more or less distinct bias due to slightly different assessments of the same information by different persons. To integrate this bias adequately during the clinical assessment of a differential blood count result, it would

CHAPTER 5. DISCUSSION

be necessary to have further studies comparing leucocyte differential counts obtained by several persons with varying stages of experience. This would allow to assess the influence of the examiner’s experience on the reliability of the results.

Corrons et al. [2004] listed vacuoles in human blood cells as a quality criteria and during preliminary tests it became apparent that especially the erythrocytes would begin to show cytoplasmic vacuoles when the blood sample was stored for longer periods. The preliminary assessments also showed an increasing bacterial growth in the longer-stored samples, therefore these two criteria were included in the final evaluations.

5.1.3. Statistical evaluations

When examining the obtained leucocyte values, it became apparent that the basophil, monocyte and plasma cell values represented only a small percentage of the differential blood count and were not influenced strongly by the various storage conditions. Therefore it was decided to pool these data as the group “rest” for the statistical evaluation of the leucocyte values. This group “rest” was weighted with 1, while the heterophil, eosinophil and lymphocyte values were weighted with 2 to emphasise the differences resulting from the storage conditions. The different weighting of the cell types was established for a better visualisation of the relevant changes in distribution within the differential blood count.

Hereby the cell types with the largest percentages were considered more representative for the influence of storage conditions of blood samples on the differential blood count.

Basophils, monocytes and plasma cells appeared in much lower percentages (basophils represented ≤5 % in 97.14 % of the smears, monocytes≤ 5 % in 99.04 % of the smears).

These numbers were considered too low to allow for a representative comparison of an influence of storage conditions on the percentage of these cells.

CHAPTER 5. DISCUSSION