USGS PWRC Draft Report, March 28, 2012



### Draft Preliminary Summary Report: Effects of 17 β Trenbolone exposure on multiple

generations of Japanese quail – bi ochemical endpoints in adults

Natalie Karouna-Renier and Paula F.P. Henry, USGS Patuxent Wildlife research Center,

Beltsville, MD

We investigated the effects of continuous exposure to 17 β trenbolone (17 β T), a synthetic

anabolic steroid, on three successive generations of Japanese quail ( Coturnix japonica ). For the

F0 generation, 6 week-old Japanese quail were randomly assigned to one of six doses of 17ßtrenbolone: 0, 1, 5, 10, 20 and 40 mg/kg feed (n=16 pa irs per dose). Eggs were collected from F0

and F1over weeks 7-8 to generate pairs for the ne xt generation. F1 birds were assigned to the

same dose as their parents and received dosed fe ed from day 1 of hatch. The F2 birds were

exposed solely in ovo and were provided only untreate d feed. The 40 ppm dose was dropped

after F0 since the pairs exposed to this dose were unable to produce sufficient numbers of eggs to

populate the F1 generation. F0, F1 and F2 parents we re euthanized at 16 w eeks of age to harvest

tissues for genomic and biochemical analysis.

Methods

Plasma, liver samples, and thyroid glands were collected at necropsy. Thyroid glands were

excised, weighed and frozen at -20 o 

### C until analysis. Liver tissue was excised and immediately

placed in RNALater and then frozen at -20 o 

### C until analysis. Plasma was frozen at -20 o 

### C until

analysis. Fecal samples were collected from F1 adults at 11, 13, and 15 weeks of age.

Levels of testosterone, thyroxine (T4) and estr adiol were analyzed using ELISA. Testosterone

levels in plasma and feces were analyzed using the DRG® Testoste rone ELISA (EIA-1559)

(DRG International, Mountainside, NJ) followi ng the manufacturer’s protocol with slight

modification for plasma. Male samples were analy zed using 25 ul of plasma and female samples

used 75 ul of plasma. Estradiol levels we re determined using the Estradiol Salivary

Immunoassay Kit (1-3702) from Sa limetrics (State College, PA). A 10 ul aliquot of plasma was

analyzed from each bird following the manufacturer ’s instructions. Fecal samples were extracted

in 1.5 ml methanol followed by hydrolysis in 300 μ L of 1:200 diluted

Glucuronidase/Arylsulfatase in aceta te butter (0.2 M, pH = 4.8) at 37  ̊ C overnight.

Levels of T4 were analyzed in plasma and thyroid glands using th e AccuwellTM T4 EIA Kit

(Neo-genesis, Clackamas OR). Thyroid glands we re digested in 350 ul of digestion medium

containing 25 mg of PRONASE® Protease (Calbiochem), Trizma base (Sigma), L-Glutathione

reduced (Sigma), 6-Propyl-2-Thiouracil (Sigma) and Triton X-100 (Sigma) at 37 o 

### C for 24 h.

Following the digestion, 1.0 ml of absolute etha nol was added to each tube, vortexed, incubated

for 24 h at 20 o 

### C and centrifuged at 14,000 x g for 8 min. The supernatant was removed and

stored at 20 o 

### C until analysis. Diluted (as necessary) thyroid gland extracts (15 ul) and plasma

samples (15 ul) were analyzed using an ove rnight incubation following the manufacturer’s

instructions.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013



### All assays were validated in the laboratory for parallelism, recovery, inter- and intra-assay

variability. All samples were analyzed in duplic ate and positive controls were included on each

plate. Samples for which duplicate CV exceed ed 15% were re-analyzed or excluded from

analysis.

Expression of Vitellogenin II (V TG) mRNA was analyzed by qRTP CR. Total RNA was isolated

from liver using RNAzol (Molecular Research Cent er, Inc., Cincinnati, OH) in combination with

the RNeasy Mini Kit (Qiagen, Valencia, CA). Samples were reverse transcribed into cDNA

using the SuperScript III First-Strand Synthesi s SuperMix system (Invitrogen). Expression of

VTG and two reference genes (albumin [ALB] a nd eukaryotic translatio n elongation factor 1

alpha 1[EEF1A1]) was analyzed by qPCR usi ng the DyNAmo Flash SYBR Green qPCR Kit

(Thermo Fisher Scientific, Cincinnati, OH). A ll reactions were performed in duplicate and

included interpolate calibrators a nd blanks. The threshold for determ ination of Cq was set in the

exponential phase at 0.1 normali zed fluorescence units. We us ed Genex Version 5.3.2 (MultiD

Analyses AB, Germany) for preprocessing of data (efficiency correction, reference gene

normalization, and calculation of relative quant ities). We calculated the reaction-specific

efficiencies for each sample and primer pair. Re lative quantities were calculated using the mean

Cq for the control samples as the calibrator.

Statistical analyses were performed using R (ver 2.15.3). p<0.05 was cons idered statistically

significant, and p<0.07 was considered marginally si gnificant. Data were an alyzed for violations

of assumptions for parametric analysis (heteroscedacity and normality of residuals). If the

absolute value of the standardized residual for a sample was greater th an 3, the observation was

classified as an outlier and removed from th e analysis. Data were log transformed where

appropriate. Analysis of variance (ANOVA) followed by post-hoc multiple comparisons was

used to determine differences in biomarke rs between dosed samples and controls.

Results

Expression of steroid-res ponsive genes in adults

Liver VTG mRNA levels decrease d significantly with dose in F0 females (Figure 1) and were

significantly lower in females exposed to 5, 10, 20 and 40 ppm 17 β T than in control birds

(P<0.05). In F1 females, liver VTG mRNA levels at 5, 10, and 20 ppm were significantly higher

than those at 0 ppm (p<0.05). In F0 males, VTG mRNa levels were significantly higher at 10

ppm than in controls. No signifi cant changes in expression of VT G were observed in livers from

F1 and F2 males or F2 females (Fig. 1 and 2).

Steroid hormone levels in adults

Testosterone levels in males and F1 females we re not significantly affected by exposure to 17 β T

(Fig. 3 and 4). F0 females showed a dose-depe ndent decrease in testosterone levels and

significantly lower levels at 10, 20 and 40 ppm than at 0 ppm (p< 0.05; Fig. 4). Testosterone in

F2 females was significantly higher at 20 ppm than in controls (p<0.05) (Fig. 4).USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013



### Plasma estradiol levels in F0 and F1 males incr eased significantly with dose (Fig. 5). Estradiol

concentrations in males exposed to 5 ppm trenbol one and higher were significantly greater than

those in controls for both generations (p <0.05). In females (Fig. 6), ANOVA identified

significant differences in estradiol levels between doses in F0 and F1 females, with significantly

higher levels at 40 ppm in F0 and 10 ppm in F1 bi rds (p<0.05). Estradiol le vels in F2 males and

females did not differ si gnificantly between doses.

Fecal levels of testosterone in F1 males did not differ significantly between treatments (Figure

7). In females (Figure 8), during the first samplin g period (week 11), fecal testosterone levels in

1 ppm birds were significantly higher than in controls (p<0.05). Ho wever, no significant

differences in testosterone levels were obser ved during the remaining two week so collection

(weeks 13 and 15).

Fecal levels of estradiol in F1 males differed significantly between treatments for all sampling

weeks (Figure 9). Birds receiving feed treated with all doses (1, 5, 10, and 20 ppm) exhibited

significantly higher levels of estradiol than in co ntrol males. In females, fecal estradiol levels

differed significantly between treatments for we eks 11 and 15 (Figure 10). Estradiol at 20 ppm

was significantly higher than in controls birds (p<0.05).

Thyroid Hormone Levels

No significant changes were obser ved in plasma (Fig. 11 and 12) or glandular (Fig. 13 and 14)

T4 in any generation.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 2. Relative expression of VTG mRNA in adult female Japanese qua il livers. Error bars =SE. F0

females exposed to 5 ppm trenbolone and higher exhibite d statistically significant lower expression of VTG

mRNA than controls (p < 0.05), and F1 females exposed to 5 ppm trenbolone and higher exhibited

significantly higher levels of expression than controls. Figure 1. Relative expression of VTG mRNA in adul t male Japanese quail livers . Error bars =SE. F0

males exposed to 10 ppm trenbolone exhibited statis tically higher expression th an controls (p < 0.05).USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 3. Plasma testosterone levels in ma le adult Japanese quail (±SE). No significan t

differences in testosterone leve ls were observed in males.

Figure 4. Plasma testosterone concentrations in female adult Japanese quail (±SE). A

significant decrease in testosterone was observ ed in the F0 females (p<0.05). At 20 ppm, F2

females showed a significant increa se in testosterone (p<0.05).USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 5. Effects of trenbo lone exposure on estradiol levels in adult male Japanese quail (±SE). F0

and F1 male quail exhibited increased levels of es tradiol with increasing t renbolone dose. In both

generations, exposure to 5 ppm trenbolone and higher doses resulted in significantly higher levels

of plasma estradiol than obs erved in controls (p<0.05).

Figure 6. Effects of trenbo lone exposure on estradiol levels in a dult female Japanese quail (±SE). In

contrast to males, female quail did not show a cl ear pattern of effects on plasma estradiol levels.



## * =significantly different fr om controls (p<0.05).USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 7. Mean fecal testosterone levels in F1 male adults ±SE. No significant differences

were observed between treatments.

Figure 8. Mean fecal testosterone levels in fe male adult birds (±SE). *p<0.05 vs. controls.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 9. Mean fecal estradiol levels in adult males ± SE. *= p<0.05 vs. controls

Figure 10. Mean fecal estradiol levels in adul ts females (±SE). * = p<0.05 vs. controls.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 11. Plasma T4 levels in adult male Japa nese quail exposed to trenbolone (±SE). No si g nificant

changes in plasma T4 levels w ere observed in any generation.

Figure 12. Plasma T4 levels in adult female Japanese quail exposed to trenbolone (±SE). No

significant changes in pl asma T4 levels were observed in any generation.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

Figure 14. Glandular T4 levels in adult female Japanese quail exposed to trenbolone (±SE). No

significant changes in pl asma T4 levels were observed in any generation.

Figure 13. Glandular T4 levels in adult male Ja panese quail exposed to trenbolone (±SE). No

significant changes in pl asma T4 levels were observed in any generation.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0 0.2 0.4 0.6 0.8

1

interval 1 interval 2 interval 3 interval 4 interval 5 interval 6% 

### F0 generation on Japanese quail exposed to trenbolone:

set weekly % egg laying

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0 0.2 0.4 0.6 0.8

1 1.2

interval 1 interval 2 interval 3 interval 4 interval 5% 

### F1 generation on Japanese quail exposed to trenbolone:

set weekly % egg laying

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0 0.2 0.4 0.6 0.8

1 1.2

interval 1 interval 2 interval 3% 

### F2 generation on Japanese quail exposed to trenbolone:

set weekly % egg laying

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

interval 1 interval 2 interval 3 interval 4 interval 5 interval 6% 

### F0 generation on Japanese quail exposed to trenbolone:

set weekly

‐ total eggs laid per day

0 ppm

1 ppm

5 ppm

10 ppm

40 ppm

Series6

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

interval 1 interval 2 interval 3 interval 4 interval 5% 

### F1 generation on Japanese quail exposed to trenbolone:

set weekly

‐ total eggs laid per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

interval 1 interval 2 interval 3% 

### F2 generation on Japanese quail exposed to trenbolone:

set weekly

‐ total eggs laid per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

interval 1 interval 2 interval 3 interval 4 interval 5 interval 6% 

### F0 generation on Japanese quail exposed to trenbolone:

set weekly

‐ eggs laid per treatment group per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 0.20 0.40 0.60 0.80 1.00 1.20

interval 1 interval 2 interval 3 interval 4 interval 5% 

### F1 generation on Japanese quail exposed to trenbolone:

set weekly

‐ eggs laid per treatment group per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

interval 1 interval 2 interval 3% 

### F2 generation on Japanese quail exposed to trenbolone:

set weekly

‐ eggs laid per treatment group per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0 0.2 0.4 0.6 0.8

1

interval 1% 

### F0 gener a

0.00 0.20 0.40 0.60 0.80 1.00 1.20

interval 1 interval 2 interval 3 interval 4 interval 5% 

### F1 generation on Japanese quail exposed to trenbolone:

set weekly

‐ eggs laid per hen per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

interval 1 interval 2 interval 3% 

### F2 generation on Japanese quail exposed to trenbolone:

set weekly

‐ eggs laid per hen per day

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm Male behavioral trials – These 3-day, 3 minute trials are conducted on a subset of males, exposed to the

same diets and handling as the study quail but are kept separated from females once transferred from

brooders at 5 weeks. Two males are placed in the breed er pens to acclimate to the pens and water system.

On the day of the trials, one of the resident males is removed and an untreated female is introduced into the

pen. Observations on the male-female interactions are recorded for 3 min. The female is removed and the

males are switched. After 30+ minutes, an untreated fema le is introduced into the pen and observations on

the 2 nd male-female interactions are recorded for 3 min. The female is then removed, and the 1 st male

returned to the pen. The trials are repeated on da y 2 and day 3. The males are then removed from the

studyUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

F1 Behavioral trial:

Start time: 8 am. 6/30-7/1/10

Sample size:

0 ppm – 12 10 ppm - 12

1 ppm – 12 20 ppm - 11

5 ppm - 11 40 ppm – 10

Observations: latency time decreased or

stayed the same for all but the 1 ppm males.

Completed cloacal contacts decreased or

stayed the same for all but the 1 ppm males.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013



### F2 Behavioral trial:

Start time: 10 am. 10/18-

10/20/10

Sample size: 6 males for

each treatment.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

24 hr 2 week 6 week 16 weekmass(g) 

### Body weight of female Japanese quail exposed to

trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

24 hr 2 week 6 week 16 weekmass(g) 

### Body weight of female Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

24 hr 2 week 4.7 weeks 13 weeksmass(g) 

### Body weight of female Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

25.0 26.0 27.0 28.0 29.0 30.0 31.0 32.0 33.0 34.0 35.0

2 weeks 6 weeksmm 

### Tarsus length of female Japanese quail exposed to

trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

25.0 27.0 29.0 31.0 33.0 35.0 37.0

2 week 6 weekmm 

### Tarsus length of female Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

25.0 27.0 29.0 31.0 33.0 35.0 37.0

2 week 4.7 weeksmm 

### Tarsus lengh of female Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 20.0 40.0 60.0 80.0 100.0 120.0

24 hr 2 week 6 week 16 weekmass(g) 

### Body weight of male Japanese quail exposed to

trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.0 20.0 40.0 60.0 80.0 100.0 120.0

24 hr 2 week 6 week 16 weekmass(g) 

### Body weight of male Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.0 20.0 40.0 60.0 80.0 100.0 120.0

24 hr 2 week 4.7 week 13 weekmass(g) 

### Body weight of male Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

25.0 26.0 27.0 28.0 29.0 30.0 31.0 32.0 33.0 34.0

2 week 6 weekmm 

### Tarsus length of male Japanese quail exposed to

trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

25.0 26.0 27.0 28.0 29.0 30.0 31.0 32.0 33.0 34.0 35.0

2 week 6 weekmm 

### Tarsus length of male Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

25.0 26.0 27.0 28.0 29.0 30.0 31.0 32.0 33.0 34.0 35.0

2 week 4.7 weekmm 

### Tarsus length of male Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmeantissueweight,g+SE 

### Liver weights in female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 0.10 0.20 0.30 0.40 0.50 0.60

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmeantissueweight,g+SE 

### Ovary weights in female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmeantissueweight,g+SE 

### Adrenal weights in female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 1.00 2.00 3.00 4.00 5.00 6.00

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksCounts 

### Numbers of yolk follicles present in adult female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmm 

### Diameter of largest yolk follicle present in adult

female Japanese quail exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksg+SE 

### Oviduct mass in adult

female Japanese quail exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Left Thyroid Right Thyroidmeantissueweight,mg+SE 

### Left and right thyroid weights in adult female Japanese quail

exposed to trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Left Thyroid Right Thyroidmeantissueweight,mg+SE 

### Left and right thyroid weights in adult female Japanese quail

exposedd to trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Left Thyroid Right ThyroidMeantissueweight,mg+SE 

### Left and right thyroid weights in adult female Japanes quail

exposed to trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Left Thyroid Right Thyroidmeantissueweight,mg+SE 

### Left and right thyroid weights in adult male Japanese quail exposed

to trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Left Thyroid Right Thyroidmeantissueweight,mg+SE 

### Left and right thyroid weights in adult male Japanese quail exposed

to trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Left Thyroid Right Thyroidmeantissueweight,mg+SE 

### Left and right thyroid weights in adult male Japanese quail exposed

to trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmeantissueweight,mg+SE 

### Adrenal weights in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 0.5 1.0 1.5 2.0 2.5

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksg+SE 

### Liver in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.00 0.50 1.00 1.50 2.00 2.50

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmeantissueweight,mg+SE 

### Cloacal weights in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013

0.0 50.0 100.0 150.0 200.0 250.0 300.0

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksmm2+SE 

### Cloacal gland area (L*W) in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

F0:16

‐ 17 wks F1: 16 wks F2: 13 wksg+SE 

### Testes weight in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppmUSGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013



### o 55kg and 110 kg batches of trenbolone mixed feed were picked up from Wildlife International Ltd in

Easton MD and stored in walk –in refrigerated units at USGS PWRC, Laurel MD. Smaller working

buckets were color coded by treatment and used to store approximately 1 week of feed, to be used to fill

pen feeder cups or troughs daily or as needed. Filled bu cket weights were recorded at the start of the week.

Once a week, any left over feed from feeder cups was weighed, and the weight was added to the feed left in

buckets. Weekly total feed consum ption for each treatment was estimated by subtraction divided by the

number of pens/treatment. This was then adjusted by number of birds tallied daily to account for any

mortality. Unless otherwise noted, estimated feed consumption is in feed consumed per individual per day.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013



### Trials were run to track feed

consumption in group brooder pens.

However, birds were transferred

within treatment to additional pens

during their initial rapid growth

phase to minimize crowding; systems

to effectively minimize spillage from

troughs were ineffective or

obstructive; and feed spillage was

significant. Therefore estimated feed

consumption was based on weight of

feed before and after feeding to track

any major discrepancies in feed

consumption between treated groups.

No attempts were made to estimate

feed / chick per day.USGS PWRC Draft Report: 17 β Trenbolone , March 28, 2013