study area and experiment animals : this study was conducted on 5 pregnant 
 asian elephants which were domesticated in a semi - captive condition in the ayuttaya elephant 
 palace camp in pranakornsri - ayuttaya province , thailand . all elephants in this study were 
 chosen randomly from a mixed group . they varied in age between 21 and 37 years , had no 
 reproductive problems and had not calved for at least 3 years previously . the animals were 
 habituated to humans and used for tourist treks outside of the camp in the early morning or 
 late afternoon . they were brought back to the camp for forage and supplementary feeding each 
 evening . during their working period , they were given a variety of foods , such as banana , 
 sugar cane and hay from tourists . in addition , they had access during this time to water 
 ad libitum and natural grass . in camp , the elephants were divided into 
 different groups according to age ( calves , sub - adult , adult and old adult ) . the cows were 
 separated from the bulls except during the breeding period . during this time , the bulls were 
 walked to detect estrus for 30 to 60 min , 3 to 5 times per week in the early morning . when 
 the cows were found to be in standing heat , they were allowed to mate until , they refused 
 mounting . pregnancy diagnosis was determined monthly by transrectal ( linear transducer ; 5.0 
 to 12.0 mhz ) and transabdominal ( convex transducer ; 2.0 to 8.0 mhz ) ultrasonography scanning 
 ( model medison portable ultrasound machine ; version sonoace r3 , samsung medison co. , ltd . , california , u.s.a . ) between the 2nd and the 4th month after the last mating . under their 
 general management schedule , the elephants were dewormed and looked after by a veterinarian 
 from the elephant camp . these experiments were approved by the animal care and use 
 committee , faculty of veterinary medicine , mahanakorn university of technology , nong - chok , 
 bangkok , thailand ( project : vet - mut008/2553 ) . sample collection : blood and feces samples were collected monthly during 
 the pregnancy period ( beginning 30 days after the last mating ) for routine progestagen 
 ( progesterone hormone and its metabolite ) and glucocorticoid hormones and their metabolites 
 analysis . the samples were collected twice a day ( around 7.00 to 9.00 am and 16.00 to 18.00 
 pm ) , one day per month from each of the five pregnant elephants . blood samples of around 10 
 ml were collected from the ear vein , while the cows were controlled by a 
 mahout . blood samples were kept for 8 to 16 hr at 4c in a sealed box with ice packs , while 
 the blood was transported to a laboratory . in the laboratory , the blood samples were 
 centrifuged for 5 min at 1,500 g. the serum was collected and stored at 20c until 
 analysis . the fecal samples were collected from each elephant on the same day as the blood 
 samples , 1 hr after blood sample collection . the fecal samples were taken from the rectum or 
 chosen from the middle of a bolus of freshly dropped feces and were approximately 50 g. 
 fecal samples were packed in resealable plastic bags and kept for 8 to 16 hr at 4c in a 
 sealed box during transport and then stored at 20c until analysis . serum and fecal hormone analysis : serum and fecal progestagens , cortisol , 
 corticosterone hormones and their metabolites were analyzed by enzyme immunoassay ( eia ) at 
 conservation research and animal health , khao - kheow open zoo , cholburi province , thailand . serum , feces extraction and eia analysis were conducted following the standardized protocol 
 used by brown et al . . the 
 concentrations of progestagen were determined using monoclonal antibody ( dilution 1:10,800 ; 
 quidel clone no . 425 ; supplied by coralie munro ( cm ) , university of california ( uc ) davis , 
 ca , u.s.a . ) against 100% progesterone hormone and cross - reacted with various progesterone 
 metabolites . horseradish peroxidase ( hrp ) 
 conjugated antibody was used on already treated and visualized progestagens ( dilution 
 1:60,000 ; supplied by cm , uc davis ) which were examined with a elisa plate reader 
 ( tecan sunrise absorbance reader ; tecan austria gmbh , grdig , austria ) for 
 quantitative progesterone detection by comparison with progesterone standard levels . the 
 sensitivity of this assay was 0.02 ng / ml , and the 
 inter - assay coefficients of variation ( cv ) for high and low concentration controls were 
 9.39% and 10.22% , respectively . the intra - assay cv for high and low concentration controls 
 were 5.12% and 4.57% , respectively . cortisol hormone and its metabolite assays used 
 polyclonal cortisol antiserum ( dilution 1:8,500 ; quidel clone no . r4866 ; supplied by cm , uc 
 davis ) , against cortisol-3-carboxymethyloxime , linked to bovine serum albumin and 
 cross - reacted with cortisol ( 100% ) , prednisolone ( 9.9% ) , prednisone ( 6.3% ) , cortisone ( 5% ) 
 and < 0.1% with androstenedione , androsterone , corticosterone , desoxycorticosterone , 
 11-desoxycortisol , 21-desoxycortisone and testosterone . the horseradish peroxidase ( hrp ) conjugated antibody was labeled cortisol 
 ( dilution 1:20,000 ; supplied by cm , uc davis ) , and the use of the elisa plate reader was for 
 quantitative cortisol detection by comparison with cortisol standard levels . the sensitivity 
 of this assay was 0.08 ng / ml . the inter - assay cv for high 
 and low concentration controls and the intra - assay cv for high and low concentration 
 controls were 8.23 , 8.28 , 4.75 and 4.93% , respectively . corticosterone hormone and its 
 metabolite assays were conducted by using polyclonal corticosterone antiserum ( dilution 
 1:30,000 ; quidel clone no . cjm06 ; supplied by cm , uc davis ) , 100% binding with 
 corticosterone hormone and cross - reacted with 14.25% desoxycorticosterone and 0.9% 
 tetrahydrocorticosterone ( cm , uc davis ) . the horseradish peroxidase - conjugated antibody was 
 labeled corticosterone ( dilution 1:40,000 ; supplied by cm , uc davis ) and used with the elisa 
 plate reader for quantitative corticosterone detection . the sensitivity of this assay was 
 0.08 ng / ml . the inter- and intra - assay cv for high and low 
 concentration controls were 8.56 , 9.82 , 5.81 and 6.08% , respectively . statistical analysis : correlations between serum and fecal progestagens , 
 cortisol and their metabolites and corticosterone and their metabolites were analyzed using 
 spearman s correlation test , using spss ( spss 10.0 for windows , spss inc . two of the pregnant elephants calved at about 21.5 months , and the other 3 pregnant 
 elephants gave birth after approximately 22 months within 2 to 7 days after the last blood 
 and feces samples collection ( last sample collections were at day 21 of the 22nd month ) . the average serum progestagen concentration profile during gestation in all pregnant 
 elephants was 12.08  3.87 ng / ml . overall , mean serum 
 progestagen gradually rose from the 1st month to the 4th month and then slightly decreased 
 in the 5th month of pregnancy ( fig . 1fig . 1.the mean (  sd ) of serum and fecal progestagens concentration level profiles in asian 
 elephants during pregnancy ( 22 months ) . ) . after this , it increased rapidly from over 12 
 ng / ml in the 6th month to a peak concentration of 18.44 
  2.28 ng / ml in the 11th month ( mid - gestation ) . it 
 remained > 12 ng / ml until the 18th month and then began 
 to decline to its lowest level ( 2.11  0.60 ng / ml ) in the 
 22nd month shortly before parturition ( 2 to 7 days ) . fecal progestagen concentration varied 
 from 1.18  0.54 to 3.35  0.45 g / g with an average of 2.50  0.71 
 g / g during pregnancy until the prepartum period and was highest during 
 the 12th month ( mid - gestation ) of pregnancy ( fig . 
 1 ) . fecal progestagen levels showed a significant positive correlation with serum 
 progestagen in all five asian pregnant elephants ( r=0.88 , 
 p<0.001 , table 
 1table 1.correlations between serum and fecal progestagens , cortisol and their metabolites 
 and corticosterone and their metabolites analyzed using spearman s correlation 
 testcorrelation between hormone sortscorrelation coefficient ( r)p - valueserum and fecal progestagens0.880<0.001serum corisol and fecal cortisol metabolite0.1790.425serum corticosterone and fecal corticosterone 
 metabolite0.4870.021serum progestagen and serum cortisol0.719<0.001serum progestagen and serum corticosterone0.2060.357serum cortisol and serum corticosterone0.4380.042fecal progestagen and fecal cortisol metabolite0.0910.687fecal progestagen and fecal corticosterone 
 metabolite0.0570.801fecal cortisol metabolite and fecal corticosterone 
 metabolite0.6050.003 ) . in a pattern similar to serum progestagen , fecal progestagen rose slowly 
 from the 1st month to the 4th month and then decreased a little in the 5th month of 
 pregnancy . fecal progestagen remained higher than 3 g / g from the 7th until 
 the 15th month and then declined rapidly to reach its lowest level in the 22nd month of 
 pregnancy ( on average 2 to 7 days before calving ) . the mean (  sd ) of serum and fecal progestagens concentration level profiles in asian 
 elephants during pregnancy ( 22 months ) . the serum cortisol concentration profile during gestation in all five pregnant elephants 
 was 6.05  2.48 to 18.54  5.73 ng / ml(averaging 9.10  
 2.72 ng / ml ) . overall , serum cortisol concentrations 
 remained relatively stable almost the whole period from the 1st month to the 20th month of 
 pregnancy and then rapidly increased until parturition ( fig . 2.the mean (  sd ) of serum cortisol and fecal cortisol metabolite concentration level 
 profiles in asian elephants during pregnancy ( 22 months ) . ) . the fecal cortisol metabolite concentration level ranged from 10.3  2.74 to 20.68 
  3.09 g / g in general , 
 fecal cortisol metabolite concentration levels fluctuated from the 1st to the 21st months of 
 pregnancy and then rose to reach a peak in the 22nd month of pregnancy ( fig . serum cortisol concentrations showed no significant 
 correlation with fecal cortisol metabolite levels in all five asian pregnant elephants 
 ( table 1 ) . serum corticosterone concentration 
 levels during gestation in all 5 pregnant elephants ranged from 1.42  0.63 to 12.35  2.21 
 ng / ml ( averaging 4.77  2.67 
 ng / ml ) , and fecal corticosterone metabolite 
 concentrations varied from 64.81  26.45 to 127.07  29.04 g / g ( averaging 
 95.44  15.76 g / g ) . generally , both serum corticosterone and fecal 
 corticosterone metabolite concentrations fluctuated almost the whole period from the 1st 
 month to the 20th month of pregnancy and then reached a peak in the last month of the 
 gestation period ( fig . 3fig . 3.the mean (  sd ) of serum corticosterone and fecal corticosterone metabolite 
 concentration level profiles in asian elephants during pregnancy ( 22 months ) . ) . serum corticosterone and fecal corticosterone metabolites concentration levels 
 showed a significant positive correlation ( table 
 1 ) . the mean (  sd ) of serum cortisol and fecal cortisol metabolite concentration level 
 profiles in asian elephants during pregnancy ( 22 months ) . the mean (  sd ) of serum corticosterone and fecal corticosterone metabolite 
 concentration level profiles in asian elephants during pregnancy ( 22 months ) . the present study obviously demonstrated that serum glucocorticoid and fecal glucocorticoid 
 metabolite concentrations in the last month of pregnancy ( the 22nd month ) archived their 
 peak levels , while progestagens were in their lowest levels . these results could be 
 specifically explained by the fact that serum and feces collections in the last period of 
 pregnancy were done shortly ( 2 to 7 days ) prior to parturition . our findings were similar to a previous 
 report , in that the dramatically increased serum 
 cortisol and the sharply decreased progestagens levels around 5 days before calving and the 
 highest serum cortisol and the lowest serum progestagens levels were observed on the day of 
 parturition . brown and lehnhardt   reported a 
 gradual decline of serum progesterone and an increase of cortisol concentrations during the 
 last month of gestation in asian elephant . thereafter , 2 days before parturition , the serum 
 progesterone was obviously decreased to non - detectable levels , while the serum cortisol 
 concentrations increased to reach the peak on the day of parturition . for fecal 
 glucocorticoid metabolites and progestagens , our results did not agree with the other study 
 by foley et al .   who presented 
 that the fecal corticoid metabolites pursued fecal progestagens during periparturition until 
 postpartum if a dietary effect of seasonal changes was responded in free - ranging african 
 elephants . in our study , the elephants were domesticated in semi - captive condition which 
 they were fed with optimal diets . in addition , the high glucocorticoid concentrations could be due to 
 a calving stress response resulting in increased concentration levels in serum and feces 
 shortly before parturition . this means that measuring serum or fecal glucocorticoid 
 concentration levels as an indicator of stress could be used to monitor the progestagen 
 profile indirectly in the short period of periparturition . however , the prediction of 
 parturition required more frequent sample collections during the last month of gestation 
 period . although the number of elephants ( 5 pregnant elephants ) and the frequency of sampling 
 collections ( 1 day per month ) were limited , the present study could demonstrate a gestation 
 length of 21.5 to 22 months , similar to that found by other studies [ 2 , 16 , 17 ] . there would be only a minimal disruption of the pregnant elephant s 
 habitat and working routines . the monthly samplings , however , were carried out twice a day 
 in order to increase the sample number and reduce the possibility of error in the hormone 
 concentration levels readings . the serum progestagen concentration profile in our study was similar to the serum 
 concentration profiles of progesterone , 5-p-3oh and 5-dhp ( progesterone 
 metabolites ) in pregnant african elephants that were reported previously [ 15 , 16 , 24 , 25 ] . moreover , 
 the serum progestagen concentration profile in our research could indicate that luteal 
 activity was at its highest level in mid - gestation ( in order to maintain pregnancy ) and then 
 declined to its lowest level from 7 to 2 days prior to calving , which is supported by the 
 serum progesterone concentration profiles of pregnant elephants found in other studies 
 [ 5 , 6 , 16 , 17 , 19,20,21 , 24 , 25 ] . the average fecal progestagen concentration in this 
 study was lower than the 5-p-3oh ( major progesterone metabolite in elephant feces ) 
 concentration that was found previously . that 
 study used a 100% specific antibody , while our study used a 64% specific antibody with 
 5-p-3oh ( monoclonal antibody quidel clone no . 425 ) . however , the fecal progestagen 
 concentration profile in this study was quite similar to the fecal progesterone , 5-p-3oh 
 and 5-dhp concentration profiles found in a previous study of pregnant 
 african elephants . a significant positive 
 correlation with serum progestagen concentrations could mean that high concentration levels 
 of progesterone in serum during pregnancy are excreted mainly in the form of progesterone 
 metabolites in feces . if so , assays of fecal progesterone metabolites could be useful as an 
 indicator of luteal activity for assessing the pregnancy status of asian elephants according 
 to other studies [ 8 , 10 , 11 , 15 ] . glucocorticoid hormones ( cortisol and corticosterone ) were measured to evaluate stress 
 responsiveness in the elephants in this study . it was found that the serum cortisol 
 concentration profile was relatively similar to a previous study [ 17 , 20 ] . fecal cortisol metabolite 
 concentration levels during pregnancy in our study , however , were lower than the levels in 
 the pregnant african elephants reported previously . normally , serum glucocorticoid ( cortisol and corticosterone ) hormone is rapidly 
 and extensively changed into the form of metabolites before excretion in feces . therefore , the correlation between serum cortisol and 
 fecal cortisol metabolites should be positive . but , serum cortisol and fecal cortisol 
 metabolite concentration patterns in this study showed no significant correlation ( table 1 ) . this could be because the polyclonal 
 antiserum used in this study ( r4866 ) had a limited cross - reactivity with < 10% fecal 
 cortisol metabolites . this could cause a reduction in the detection of fecal cortisol 
 metabolites and help account for the lack of correlation between these concentration levels 
 and those of serum cortisol . in this study , however , there were significant positive 
 correlations between serum corticosterone and fecal corticosterone metabolites and serum 
 cortisol and corticosterone and fecal cortisol metabolites and corticosterone metabolites 
 ( table 1 ) . the average fecal corticosterone 
 metabolite concentration was about 7 times higher than the fecal cortisol metabolite 
 concentration during gestation in all five pregnant elephants . an increase of corticosterone 
 metabolites concentrations in feces during pregnancy was also reported previously . our research found that serum progestagen was 
 negatively correlated with serum cortisol and an inclination ( non significant ) with serum 
 corticosterone according to meyer et al . . furthermore , in our study , fecal progestagen did not correlate with fecal 
 glucocorticoid that it was different from a previous study which showed a positive 
 correlation in african elephants . one explanation 
 could be that our study had a relatively low sampling frequency ( twice a day and one day a 
 month ) . a second explanation could be that glucocorticoid metabolite concentrations are 
 significantly correlated with season . they are highest in the dry season , but our research did not control for dry season 
 influences . moreover , excited or stressful behavior of elephants during pregnancy could lead 
 to an increase in glucocorticoid levels which , in turn , could cause abortion or parturition 
 according to dobson and smith . in conclusion of our study , it was found that during pregnancy , the progestagen level 
 increased and only returned to low levels in the short period before calving . the 
 glucocorticoid level , in contrast , fluctuated from low to medium throughout the pregnancy up 
 until the last month shortly before calving when it increased dramatically . these findings 
 may help for evaluating and managing the pregnancy status and stress condition in order to 
 decrease the rate of fetal loss and predicting parturition in asian elephants .
abstractthe study was to find patterns of progestagen ( progesterone and its metabolite ) 
 and glucocorticoid and their metabolite concentrations in serum and feces of pregnant 
 asian elephants ( elephas maximus ) . 
 the 5 female asian domestic elephants 
 were naturally mated until pregnancy . 
 after that , blood and feces samples were collected 
 monthly during pregnancy for progestagen , glucocorticoid and their metabolites analysis by 
 enzyme immunoassay ( eia ) . 
 the results showed the serum progestagen concentration during 
 gestation was 2.11  0.60 to 18.44  2.28 ng / ml . 
 overall , serum progestagen concentration rose from the 1st month to reach peak in the 11th 
 month , after which it declined to its lowest level in the 22nd month of pregnancy . 
 fecal 
 progestagen concentration varied from 1.18  0.54 to 3.35  0.45 g / g 
 during pregnancy . 
 in general , fecal progestagen concentration increased from the 1st month 
 to its highest level in the 12th month . 
 after this , it declined reaching its lowest point 
 in the 22nd month of pregnancy . 
 glucocorticoid hormones and their metabolite 
 concentrations both in serum and feces fluctuated from low to medium throughout almost the 
 entire pregnancy period and then rapidly increased around the last week before calving . 
 our study suggests that this profile of progestagen and glucocorticoid hormones and their 
 metabolite concentration levels in serum and feces can be used to assess the pregnancy 
 status of asian elephants . 
 if serum and fecal progestagen concentrations were found in 
 very low levels and glucocorticoid and their metabolite concentrations were found in very 
 high levels , it was indicated that the cow elephant would calve within 7 days .