Amniotic cell culture during different ages of gestation for karyotype analysis in bovine

Abstract

Bovine karyotyping has become an important diagnostic tool in animal breeding. In the prenatal period it can diagnose several chromosomal abnormalities such as Robertsonian translocations, testicle feminization syndrome, gonadal dysgenesis and Klinefelters syndrome. An important cell source for karyotype analysis is the amniotic fluid. It has been extensively used in humans but in bovine, however, this is not the case despite its diagnostic value. Since a small percentage of cells is viable, cells and their growth conditions as well as the handling of the material should be optimal to insure a successful analysis. For this, we have compared the growth efficiency for bovine amniocytes in two media, employing cells from 10 to 14 weeks of gestation. Amniocytes were cultured in the Amniomax (Gibco-BRL/ Life Technologies, Rockville, MD USA) medium during eleven days and in the RPMI 1640 (Gibco-BRL) medium during sixteen days at 37ºC and 5% CO2, then fixed and GTG banded. All the cultures with RPMI showed a poor cell growth, regardless the gestational age. Out of the samples cultured in Amniomax one presented 100% of cellular confluence at day 11 (10 weeks of gestation) and the others resulted in an increased proliferation compared with those that were cultured in RPMI. To ensure a successful karyotyping, amniotic fluid from cows with gestational ages of 10-12 weeks should be used and care should be taken for critical steps in preparation of spread metaphases - hypotonic and trypsin treatments.