APPLICATION OF ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR DETERMINING TESTOSTERONE LEVELS IN THE FOLLICULAR-FLUID OF PREPUBERTAL AND PUBERTAL HEIFERS

An enzyme-linked immunosorbent assay (ELISA) was used to measure folli cular fluid testosterone concentrations in small size antral follicles (less than 5 mm in diameter) obtained from the ovaries of PrePubertal (4 months old) and pubertal (1-2 years old) heifers. The homologous E LISA was developed by raising specific antibodies against testosterone in New Zealand White rabbits, using testosterone-3 carboxy methyl oxi me (CMO): bovine serum albumin (BSA) as immunogen, and by labelling te stosterone-3 CMO with horseradish peroxidase. The standard dose-respon se curve covered the range between 0 and 1 ng per well with follicular fluid analysed without previous extraction. Sample interferences were minimised by dilution. The intra- and inter-assay coefficients of var iation were less than 7%. Recovery of added testosterone averaged 96.9 +/- 1.2% and the minimum limit was 0.4 pg per well with sensitivity o f 50% binding at 81.7 pg per well. A comparison with radioimmunoassay had a correlation of 0.98. Mean +/- SE follicular fluid testosterone c oncentrations in samples from Prepubertal heifers were 54.81 +/- 5.85 ng ml-1 and in pubertal heifers 56.94 +/- 5.49 ng ml-1. These concentr ations are typical for testosterone-rich follicles. This ELISA is simp le and direct and can be used for determining follicular fluid testost erone concentrations in cattle.