one hundred twenty - one thoroughbred mares ( 222 years old ) in hokkaido , japan housed under natural conditions were used for measuring the levels of circulating 
 estradiol . for determining the correlation between the results for whole blood and serum , five pregnant mares , 819 years of age , were used .
whole blood samples were 
 collected weekly from the jugular vein into commercially supplied plastic tubes containing heparin sodium as an anticoagulant during day 233 of pregnancy and 
 the day of delivery . for serum sample collection , whole blood was drawn from the jugular vein of the same mares into plain blood collection tubes .
the concentrations of estradiol in all samples were measured by pathfast . for the experiments determining the correlation between the results of pathfast and time - resolved fia , two pregnant mares , 4 and 15 years old , were used .
serum 
 samples were collected monthly from the jugular vein into commercially supplied plastic tubes at the day of copulation and the day of delivery .
the 
 concentrations of estradiol in all samples were measured by pathfast and fia . for evaluating the pattern of circulating estradiol during gestation in mares , fifty - nine pregnant mares , ranging from 422 years of age , were used .
serum 
 samples were collected during gestation from the jugular vein into commercially supplied plastic tubes .
all procedures were carried out in accordance with the guidelines established by the tokyo university 
 of agriculture and technology for the care and use of horses in research .
pathfast : the concentrations of estradiol in whole blood and serum samples were determined using the pathfast analyzer with the pathfast reagent kit for 
 estradiol as described previously . in brief , estradiol measurement by pathfast
was performed using a single reagent cartridge containing 
 100 l of whole blood and serum samples without extraction . in the competitive assays ,
estradiol in the samples was inhibited from forming an immunocomplex by 
 an alkaline phosphatase - conjugated antigen .
following immunoreaction for 5 min , separation of bound and free hormones was performed using the magtration 
 technology .
after addition of the chemiluminescent substrate to the immune complex , the amount of chemiluminescence was measured .
the intra -assay coefficients of variance were 7.012.4% for whole blood , and 6.312.9% 
 for serum samples .
fia : serum estradiol concentrations were determined by a time - resolved fluoroimmunoassay using dissociation - enhanced fia systems ( perkinelmer , waltham , ma , 
 usa ) as described previously [ 19 , 20 ] . the intra- and inter - assay coefficients of 
 variation were 5.0% and 5.1% , respectively .
pearson s r was calculated to determine the correlation between variables using graphpad prism software version 5 for windows ( graph pad software , san diego , 
 ca , usa ) .
one hundred twenty - one thoroughbred mares ( 222 years old ) in hokkaido , japan housed under natural conditions were used for measuring the levels of circulating 
 estradiol .
for determining the correlation between the results for whole blood and serum , five pregnant mares , 819 years of age , were used .
whole blood samples were 
 collected weekly from the jugular vein into commercially supplied plastic tubes containing heparin sodium as an anticoagulant during day 233 of pregnancy and 
 the day of delivery . for serum sample collection , whole blood was drawn from the jugular vein of the same mares into plain blood collection tubes .
the concentrations of estradiol in all samples were measured by pathfast . for the experiments determining the correlation between the results of pathfast and time - resolved fia , two pregnant mares , 4 and 15 years old , were used .
serum 
 samples were collected monthly from the jugular vein into commercially supplied plastic tubes at the day of copulation and the day of delivery .
the 
 concentrations of estradiol in all samples were measured by pathfast and fia . for evaluating the pattern of circulating estradiol during gestation in mares , fifty - nine pregnant mares , ranging from 422 years of age , were used .
serum 
 samples were collected during gestation from the jugular vein into commercially supplied plastic tubes .
all procedures were carried out in accordance with the guidelines established by the tokyo university 
 of agriculture and technology for the care and use of horses in research .
pathfast : the concentrations of estradiol in whole blood and serum samples were determined using the pathfast analyzer with the pathfast reagent kit for 
 estradiol as described previously . in brief , estradiol measurement by pathfast was performed using a single reagent cartridge containing 
 100 l of whole blood and serum samples without extraction . in the competitive assays ,
estradiol in the samples was inhibited from forming an immunocomplex by 
 an alkaline phosphatase - conjugated antigen .
following immunoreaction for 5 min , separation of bound and free hormones was performed using the magtration 
 technology .
after addition of the chemiluminescent substrate to the immune complex , the amount of chemiluminescence was measured .
the assay range of pathfast was 202000 pg / ml . the intra -assay coefficients of variance were 7.012.4% for whole blood , and 6.312.9% 
 for serum samples .
fia : serum estradiol concentrations were determined by a time - resolved fluoroimmunoassay using dissociation - enhanced fia systems ( perkinelmer , waltham , ma , 
 usa ) as described previously [ 19 , 20 ] .
the intra- and inter - assay coefficients of 
 variation were 5.0% and 5.1% , respectively .
pearson s r was calculated to determine the correlation between variables using graphpad prism software version 5 for windows ( graph pad software , san diego , 
 ca , usa ) .
   a novel chemiluminescent enzyme immunoassay system , pathfast , for the measurement of estradiol in horses was evaluated . 
 the concentrations of estradiol in the 
 whole blood and serum of mares were measured using pathfast and the estradiol concentrations measured by pathfast were compared with those measured by a 
 time - resolved fluoro - immunoassay ( fia ) . to monitor physiological changes , serum estradiol concentrations in mares 
 were measured using pathfast throughout the 
 gestation period . 
 the serum estradiol concentrations correlated highly with those in whole blood samples . 
 the serum concentrations of estradiol measured by 
 pathfast also correlated well with fia . 
 circulating estradiol increased during mid - gestation and high levels of serum estradiol were maintained in late 
 gestation , followed by an abrupt decline to term . 
 these results demonstrate the utility of pathfast in equine clinics as an accurate diagnostic tool for the 
 rapid assay of estradiol within 26 min using unextracted whole blood . 