USGS PWRC Draft Report, March 28, 2012 

Draft Preliminary Summary Report: Effects of 17βTrenbolone exposure on multiple 

generations of Japanese quail – biochemical 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 17β trenbolone (17βT), 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 pairs per dose). Eggs were collected from F0 

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

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

exposed solely in ovo and were provided only untreated 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 were euthanized at 16 weeks 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 -20o

C until analysis. Liver tissue was excised and immediately 

placed in RNALater and then frozen at -20o

C until analysis. Plasma was frozen at -20oC until 

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

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

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

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

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

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

Immunoassay Kit (1-3702) from Salimetrics (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 μL of 1:200 diluted 

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

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

(Neo-genesis, Clackamas OR). Thyroid glands were 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 37oC for 24 h. 

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

for 24 h at 20o

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

stored at 20oC until analysis. Diluted (as necessary) thyroid gland extracts (15 ul) and plasma 

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

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

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

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

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

analysis. 

Expression of Vitellogenin II (VTG) mRNA was analyzed by qRTPCR. Total RNA was isolated 

from liver using RNAzol (Molecular Research Center, 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 Synthesis SuperMix system (Invitrogen). Expression of 

VTG and two reference genes (albumin [ALB] and eukaryotic translation elongation factor 1 

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

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

included interpolate calibrators and blanks. The threshold for determination of Cq was set in the 

exponential phase at 0.1 normalized fluorescence units. We used Genex Version 5.3.2 (MultiD 

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

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

efficiencies for each sample and primer pair. Relative 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 considered statistically 

significant, and p<0.07 was considered marginally significant. Data were analyzed 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 than 3, the observation was 

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

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

used to determine differences in biomarkers between dosed samples and controls. 

Results 

Expression of steroid-responsive genes in adults 

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

significantly lower in females exposed to 5, 10, 20 and 40 ppm 17βT 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 significant changes in expression of VTG 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 were not significantly affected by exposure to 17βT 

(Fig. 3 and 4). F0 females showed a dose-dependent 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: 17βTrenbolone , March 28, 2013 

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

concentrations in males exposed to 5 ppm trenbolone 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 birds (p<0.05). Estradiol levels in F2 males and 

females did not differ significantly between doses. 

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

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

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

differences in testosterone levels were observed 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 control males. In females, fecal estradiol levels 

differed significantly between treatments for weeks 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 observed in plasma (Fig. 11 and 12) or glandular (Fig. 13 and 14) 

T4 in any generation. 

 
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

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

females exposed to 5 ppm trenbolone and higher exhibited 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 adult male Japanese quail livers. Error bars =SE. F0 

males exposed to 10 ppm trenbolone exhibited statistically higher expression than controls (p < 0.05). 
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

Figure 3. Plasma testosterone levels in male adult Japanese quail (±SE). No significant

differences in testosterone levels were observed in males. 

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

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

females showed a significant increase in testosterone (p<0.05). 
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

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

and F1 male quail exhibited increased levels of estradiol with increasing trenbolone dose. In both 

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

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

Figure 6. Effects of trenbolone exposure on estradiol levels in adult female Japanese quail (±SE). In 

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

*=significantly different from controls (p<0.05). 
USGS PWRC Draft Report: 17βTrenbolone , 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 female adult birds (±SE). *p<0.05 vs. controls. 
USGS PWRC Draft Report: 17βTrenbolone , 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 adults females (±SE). * = p<0.05 vs. controls.
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

Figure 11. Plasma T4 levels in adult male Japanese quail exposed to trenbolone (±SE). No significant 

changes in plasma T4 levels were observed in any generation. 

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

significant changes in plasma T4 levels were observed in any generation. 
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

 

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

significant changes in plasma T4 levels were observed in any generation. 

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

significant changes in plasma T4 levels were observed in any generation. 
USGS PWRC Draft Report: 17βTrenbolone , 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0

0.2

0.4

0.6

0.8

1

interval 1

%

F0 genera

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 breeder 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 female is introduced into the pen and observations on 

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

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

study
USGS PWRC Draft Report: 17βTrenbolone , 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: 17βTrenbolone , March 28, 2013 
USGS PWRC Draft Report: 17βTrenbolone , 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: 17βTrenbolone , March 28, 2013 

 
USGS PWRC Draft Report: 17βTrenbolone , 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 week

mass (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 week

mass (g)

Body weight of female Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 weeks

mass (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 weeks

mm

Tarsus length of female Japanese quail exposed to

trenbolone: F0 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

25.0

27.0

29.0

31.0

33.0

35.0

37.0

2 week 6 week

mm

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 weeks

mm

Tarsus lengh of female Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0.0

20.0

40.0

60.0

80.0

100.0

120.0

24 hr 2 week 6 week 16 week

mass (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 week

mass (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 week

mass (g)

Body weight of male Japanese quail exposed to

trenbolone: F2 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 week

mm

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 week

mm

Tarsus length of male Japanese quail exposed to

trenbolone: F1 generation

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 week

mm

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 wks

mean tissue weight , g + SE

Liver weights in female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 wks

mean tissue weight , 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 wks

mean tissue weight , g + SE

Adrenal weights in female Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 wks

Counts

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 wks

mm

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 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 wks

g + 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 Thyroid

mean tissue weight , 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Left Thyroid Right Thyroid

mean tissue weight, 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 Thyroid

Mean tissue weight, 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Left Thyroid Right Thyroid

mean tissue weight , 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 Thyroid

mean tissue weight , 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 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Left Thyroid Right Thyroid

mean tissue weight , 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 wks

mean tissue weight , mg + SE

Adrenal weights in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , March 28, 2013 

0.0

0.5

1.0

1.5

2.0

2.5

F0:16‐17 wks F1: 16 wks F2: 13 wks

g+ 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 wks

mean tissue weight ,mg + SE

Cloacal weights in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 wks

mm2 + 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 wks

g+ SE

Testes weight in male Japanese quail

exposed to trenbolone

0 ppm

1 ppm

5 ppm

10 ppm

20 ppm

40 ppm
USGS PWRC Draft Report: 17βTrenbolone , 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 bucket 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 consumption 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: 17βTrenbolone , 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: 17βTrenbolone , March 28, 2013