Animal Cytogenetics: some modifications in the peripheral blood lymphocyte culture technique

ABSTRACT

There is an increasing need for a close interaction between cytogenetics and veterinary practice, particularly in the detection of chromosomal abnormalities which may affect fertility and animal breeding programs in agriculture. This paper reports modifications of the standard lymphocyte culture technique frequently used for karyotyping. The main modifications included, a) the exclusion of antibiotics from the culture medium, b) the use of slow homogenization to avoid bubble formation, c) the use aluminium foil covered with self-adherent PVC film to seal the culture flasks and d) incubation at 39-40§C. Blood from cattle, buffaloes, dogs and monkeys was collected into heparinized syringes and inoculated into 199 medium, containing fetal calf serum and phytohemagglutinin. After incubation at 39-40§C for 72 h, the cells were exposed to colchicine and hypotonic solution followed by fixation on slides and staining with 3 per cent Giemsa stain. A total of 1000 nuclei and 13 good quality metaphases were counted and analyzed, respectively, for each animal. The mitotic index for the different species was 5.1ñ1.0 per cent for cattle, 5.5ñ0.9 per cent for buffaloes, 2.6ñ1.1 per cent for dogs and 6.0ñ0.7 per cent for monkeys. With the modifications described, marked cell growth, good metaphase quality, and better chromosome visualization were obtained.