**2. Blood collection and investigation**

The collection of blood for laboratory investigations is comparatively difficult in both free range and captive tigers and only possible when animal is sedated or restrained properly in squeeze cage.

Withstanding facts, chemical capture is comparatively safe, if accomplished by trained and experienced wildlife veterinarians. There are several drugs available for sedation. Each drug works in a different manner and is more suited to some species only. The time required for a drug to have an effect depends upon the factors such as route of administration, absorption rate, concentration and physiological status of the animal while it is difficult to generalize the choice of drug and doses (WII, 1985). It depends upon circumstances like species of the animal, age, sex, weight, location, temperature regimes in season, time of the day and emotional state. Shrivastav et.al.(2011) have used Xylazine hydrochloride + Ketamine hydrochloride as sedative drugs with the help of Tel-inject projectile syringe to immobilize the tigers of free range while Yohimbine hydrochloride was used as reversal drug.

Prior to collection of blood from immobilized animal, it is an essential protocol to obtain normal values only through free flow of the collected blood drawn from the animal either at rest or under conditions of least excitement to minimize the physiological variations in cell count (Jain, 1986). Normally the cephalic saphenous, femoral and jugular veins are used for collection of blood from dog, cat and non human primates while in tigers these sites are not convenient because the blood collector remained in front of face of the tiger. The caudal vein is convenient and safer site for blood collection (Shrivastav et.al.2011).

From sedated free range tigers, 2-5 ml of blood is drawn by venipuncture of the caudal vein through 18 no gauge disposal syringe in a tube containing Ethylenediaminetetraacetate (EDTA at 2 mg/ml of blood) as the anticoagulant (Shrivastav et.al. 2011). The blood samples should be processed as soon as possible after collection. If a delay is anticipated, it should be refrigerated at 4oC (Jain, 1986).The blood sample should be mixed several times before a portion is removed for test procedure (Shrivastav and Sharma, 2000). Automatic devices providing a continuous rocking or circular motion have been found satisfactory, but prolonged mixing should be avoided, particularly on a device with circular motion, to prevent a mechanical trauma to various blood cells, especially erythrocyte. In any event, blood smear must be made immediately after blood collection, either directly from fresh blood or after anticoagulation. Blood films should be dried quickly and protected from dust and flies till stained (Shrivastav and Sharma, 2005). Blood films can be made on glass slides and on cover slips.The haematological analysis needs precautionary measures and blood smear is stained with Romanowsky stains and at least 200 white cells should be examined for the differential leukocytes count. Simultaneously, the blood smears must be screened for parasitic blood protozoa, flagellates and rickettsial infections.
