Proceedings of The Physiological Society

Puerto de la Cruz, Tenerife (2003) J Physiol 548P, P170

Poster Communications

The effects of acute stress on leukocyte activation

I. Montes*†, G.W. McLaren†, D. Macdonald† and R. Mian*

*School of Science and The Environment, Coventry University, Cox Street, Coventry CV1 5FB, UK and †Wildlife Conservation Research Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK


Acute stressors can activate circulating leukocytes and change their composition and number (Ellard et al. 2001; Mian et al. 2002). Here we report that transport stress causes leukocyte activation in wild badgers (Meles meles) that were trapped and transported as part of an ongoing long-term ecological study (Macdonald & Newman 2002). We also show that rest after transport results in a decrease in the number of activated circulating leukocytes.

The animals were caught during the night in cage traps baited with peanuts. Traps were checked at 06.30 h and trapped animals then experienced one of three handling regimes: anaesthetised (0.2 ml kg-1 of 100 mg ml-1 ketamine hydrochloride I.M.) at site of capture (T), n = 5; transported for less than 10 min to a field laboratory and anaesthetised (TR), n = 9; and as TR, but with a period of rest after transport of at least thirty min (TRR), n = 8. A sample of blood was taken from each animal (< 5ml), and all animals were then marked and examined as part of the ecological study, and released back into the wild after recovering from anaesthesia. Levels of leukocyte activation were determined using a nitroblue tetrazolium (NBT) staining test.

Nitroblue tetrazolium (Sigma 840-10) was diluted in phosphate buffered saline (PBS 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride pH 7.4) at a concentration of 1 mg of NBT in 1 ml of PBS buffer. This solution was then stored in the dark at 4°C when not directly in use. Immediately after the blood sample was collected from the animal a sample of 50 ml was transferred into an Eppendorf tube from the EDTA vial. This tube was incubated at 37°C for 45 min. After the incubation period, 50 ml of NBT was added to the tube, which was then incubated for a further 10 min at 37°C. After incubation, 15 ml was taken from the tube and smeared onto a microscope slide and left to dry.

The dry slides were flooded with 1 ml of Accustain Wright stain modified (0.3 % w/v buffered at pH 6.9 in methanol; batch no. 096 H4372) for 45 s. The slides where then washed with 1 ml of distilled water, allowed to stand for 1 min, washed with excess distilled water, and allowed to dry. Using the X 100 oil immersion objective, four areas of each slide were examined. The total number of activated and non-activated leukocytes were counted. Leukocytes were classified as active if they appeared irregular in shape and diffusely granular with intracytoplasmic formazan deposits. At least 100 neutrophils were counted in each slide; the count was repeated several times and the mean average taken. The number of NBT-positive cells from these counts is considered to be the percentage neutrophil activation.

Transport regime had a significant effect on leukocyte activation (F2,19 = 12.4, P < 0.001). Activation was highest immediately after transport when a mean (S.E.M.) of 62.6 % (2.6 %) of leukocytes were active. Non transported animals had significantly lower activation levels than transported (Tukey test, P < 0.001) and transported and rested (Tukey test, P < 0.05) animals. However, activation did not differ significantly between rested and non-transported animals (Tukey test, P > 0.05) where recorded mean (S.E.M.) activation levels were 51.8 % (2.4 %) and 42.4 % (3.6 %), respectively.

Thus leukocytes respond rapidly to transport stress by increasing their levels of activation, but this is a short-term response that is reversed by resting for 30 min. These results can help improve the welfare of wild animals handled for ecological and other research purposes.

Where applicable, experiments conform with Society ethical requirements