in pbs and incubated for 1 h at 37 u00b0 c the animal serum samples were diluted rates 1 60 1 2000 in pbs containing 1 of bsa tween 20 0 01 w v incubated for 3 h at 37 u00b0 c the plates were washed with pbs several times and were put to react with anti igg antibodies from rat conjugated with horseradish peroxidase sigma biochemical usa after other washes the plates were incubated with a mixture containing ortophenilen diamine opd and h 2 o 2 substrate in citrate buffer the titers were expressed as the maximum dilution of samples that reaches values 2 times higher than the assay cutoff value the testosterone levels were determined using testo ct2 kit cisbio international france 25 u03bcl of each serum sample were added directly in pre coated tubes samples were incubated in duplicate finally the tubes were washed with distilled water and read using a gamma the results were expressed in nmol l the animals were anesthetized and slaughtered one month after the 5 th immunization 100 days according to good laboratory practices the significant statistic differences regarding the testicles and prostate size were evaluated using the duncan test stat graphic software likewise testosterone levels were analyzed using the same statistic procedure sas institute inc sas stat u2122 user 39 s guide release 6 03 edition cary n c sas institute inc 1988 1028 pp gnrh molecule as.