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Journal of EnvironmentalScience and Health .Part A: EnvironmentalScience and Engineeringand Toxicology: Toxic/Hazardous Substances andEnvironmental EngineeringPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lesa19

Factors affectingthe concentration oftestosterone in poultrylitterLaurence S. Shore a , Elian Harel‐Markowitz a

, Michael Gurevich a & Mordechai Shemesh aa Department of Hormone Research , KimronVeterinary Institute , Bet Dagan, P.O.B. 12,IsraelPublished online: 15 Dec 2008.

To cite this article: Laurence S. Shore , Elian Harel‐Markowitz , MichaelGurevich & Mordechai Shemesh (1993) Factors affecting the concentrationof testosterone in poultry litter, Journal of Environmental Science andHealth . Part A: Environmental Science and Engineering and Toxicology: Toxic/Hazardous Substances and Environmental Engineering, 28:8, 1737-1749

To link to this article: http://dx.doi.org/10.1080/10934529309375974

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J. ENVIRON. SCI. HEALTH, A28(8), 1737-1749 (1993)

FACTORS AFFECTING THE CONCENTRATION OF TESTOSTERONE IN

POULTRY LITTER

Key words: Testosterone, estrogen, poultry litter, antibiotics

Laurence S. Shore Elian Harel-MarkowitzMichael Gurevich Mordechai Shemesh

Department of Hormone Research, Kimron Veterinary InstituteBet Dagan, P.O.B. 12, Israel

Abstract

Poultry litter used as cattle feed can contain high levels oftestosterone (>400 ng/g) and estrogen (>300 ng/g) which caninterfere with reproduction. However such high levels oftestosterone are not usually found in broiler chicken litter(< 6% of samples). We tested the following parameters to determinewhat factors could be responsible for highly elevated concentra-tions of testosterone: sex, age, fermentation. It was found that(1) there is no difference in testosterone content in poultrylitter obtained from male (133±12 ng/g) or female broilers(133±13 ng/g); (2) poultry litter from adult roosters haveunacceptably high levels of testosterone (670±95 ng/g) and layershave high levels of estrogens (533±50 ng/g); (3) althoughfermentation of poultry manure generally lowers steroidal hormonecontent, if antibiotics are added to the incubation, there was a afive-fold increase in radioimmunoassayable testosterone content(controls 94±6 ng/g vrs treated 578±80 ng/g).

INTRODUCTION

Chicken manure litter has been widely used in Israel for over

10 years (and more recently in the US) as an important source of

protein for beef cattle and non-lactating calves as well as means

1737

Copyright © 1993 by Marcel Dekker, Inc.

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1738 SHORE ET AL.

of disposing of animal waste. More than 25.000 tons are supplied

from commercial sources alone each year. However, chickens (as do

most birds) produce ugs/day of testosterone (T) regardless of sex

[1]. We have previously reported that the average content of T in

7-8 wk old broiler chicken litter was 268±14 ng/g [2], but

occasionally Ct/69) very high levels concentration of T (0.5 to

1 ug/g) were found. The presence of such high concentrations of T

in the litter could be problematic as implants of both T and its

analogue trenbolene acetate have adverse effects on fertility in

cattle. These compounds have been reported to cause masculiniza-

tion [3], delay puberty [i(-6] and temporarily interfere with the

estrous cycle [7]. Field evidence [8] suggests that feeding

chicken litter containing T levels greater than UOO ng/g T can

delay puberty and enlarge the clittoris. However, disruption of

the estrous cycle (such as seen with T implants) can occur when

heifers are fed chicken litter containing only 200 ng/g [8]. In

addition, we have reported that estrogen (E) present in poultry

litter (>300 ng/g) can cause premature udder development in

heifers [9].

We have previously reported that conditions of silaging, type

of processing or time of year are not major factors in the level

of T in the litter [2]. In the present paper we examined three

other possibilities for high T content in chicken litter. 1) Use

of litter from adult hens or roosters; 2) sex of broilers (coops

are separated by sex) and 3) fermentation on the floor of the

coop. The latter possibility was considered as Calvert et al. [10]

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TESTOSTERONE IN POULTRY LITTER 1739

reported that digestion of adult poultry litter by fly larvae

could increase T content by 4-fold (as determined in bioassay) but

decreases the E content.

EXPERIMENTAL

Extraction and Determination of Testosterone and Estrogen in

Poultry Litter

Extraction and determination of T and E in poultry litter were

done as described in detail in Shore and Shemesh [2]. Briefly 0.5

g samples were extracted with in 802 methanol and the extracts

eluted on C-18 maxi-extraction columns (Alltech, Deerfield, IL).

The elutants were evaporated to dryness under air and the residue

redisolved in the buffer used for steroid analysis. The concen-

trations of T and E were then determined by radioimmunassay. The

antibody for T was specific for T and dihydrotestosterone and the

antibody for E recognized both estrone and estradiol but had

negligable cross-reaction with other steroids.

Incubations

One half gram samples of poultry litter obtained from the

floor of the coop were incubated at 30°C for up to 1 week in 10 ml

of one of the following solutions: 1 N HC1 (pH 1); 0.1 M sodium

acetate (pH 5); 0.1 M phosphate buffer solution (pH 7); 2 N sodium

bicarbonate (pH 12). To some of the incubations 50 U penicilin and

1 ug streptomycin were added. Addition of antibiotics to control

samples prior to extraction had no effect on hormone values.

Samples were neutralized and extracted at the termination of the

incubation. The experiments were done in 3 trials using duplicate

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1740 SHORE ET AL.

TABLE 1.Concentration of Estrogen and Testosterone in Poultry Litter from7 Wk Old Broilers, 5 Mo. Old Layers and 5 Mo. Old Roosters.

Source

ImmatureBroilers

FemalesMales

Laying Hens

Roosters

Testosterone(ng/g D.W.)

133±13133±12

25i)±22

670±95

Estrogen(ng/g D.W.)

65± 7lk± ft

533±ttO

93±13

N

1010

17

10

incubations. Treatments were compared with controls by Student's t

test and P>0.05 was considered significant.

RESULTS

Determination of Hormones in Chicken Litter from Various Sources.

The concentration of T and E from adult and immature male and

female poultry litter are summarized in Table 1.

There was no difference in the T content of 7 wk old male or

female broiler litter. However there was five times (P<0.001) more

E in female broiler litter than in male broiler litter. In litter

from laying hens, the estrogen concentration was ten times higher

than that found in immature female broiler litter and adult

rooster litter had five times the amount of testosterone than

immature male broiler litter.

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TESTOSTERONE IN POULTRY LITTER 1741

TESTOSTERONE males

600

1400

200

without antibiotics 0with antibiotics •anaerobicaerobic

DAYS OF INCUBATION

FIG. 1. Effect of incubation of 7 wk old male broiler litter, in thepresence or absence of antibiotics, on radioimmunassayable testoster-one. 0.5 g of litter was incubated in 10 ml of PBS, pH 7.0 at 30°C for0, 2, h or 7 days in sealed 20 ml vials in the presence or absence of50 U pennicilin and 1 ug streptomycin. After i» days, half of the vialswere opened and plugged with sterile gauze. Each point represents themean of at least two determinations.

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1742 SHORE ET AL.

Effect of Time and Aerobic/Anaerobic Conditions on Hormonal

Content of Chicken Litter.

The effect of incubating 0.5 g of male broiler litter with 10

ml PBS in the absence or presence of antibiotics is shown in

Figure 1. Initial incubations were done at pH 7 as the pH of

chicken litter was between pH 6.5 and 7. It was found that:

a. T concentration increase linearly with the length of

incubation; b. more T was produced in anaerobic than aerobic

conditions and c. the presence of antibiotics dramatically

increase the concentration of T. All subsequent incubations were

therefore done for 1 wk in sealed vials.

Effect of Incubation at Various pH's on the Estrogen and

Testosterone Content of Chicken Litter from Female Coops.

The effect of incubating 0.5 g samples of 7 wk old female

broiler litter on concentration of E is shown in Figure 2 and the

effect on T is shown in Figure 3. In the absence of antibiotics,

there was significant decrease in the amount of E at pH 5 and pH 7

and in the amount of T at pH 5. However, in the presence of

antibiotics the E or T concentration was not different than that

found in controls.

Effect of Incubation at Various pH's on the Estrogen and

Testosterone Content of Chicken Litter from Female Coops.

The effect of incubating 0.5 g samples of 7wk old female

broiler litter on concentration of E is shown in Figure 2 and the

effect on T is shown in Figure 3. In the absence of antibiotics,

there was significant decrease in the amount of E at pH 5 and pH 7

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TESTOSTERONE IN POULTRY LITTER

TESTOSTERONE

1743

pll

Female

80I

1 »I

1 1/////////////////////

1

1* 12

FIG. 2. Effect of incubating for 1 wk at various pH's on the testo-sterone content of female broiler litter. Data represents three trialsof duplicate samples of 0.5 g of litter /10 ml media in sealed vials.Legends: C= unincubated control; 7+= incubation with antibiotics.Asterisk indicates significantly different from control (P<0.05).

and in the amount of T at pH 5. However, in the presence of

antibiotics the E or T concentration was not different than that

found in controls.

Effect of Incubation of Hale Broiler Litter.

As found with female broiler litter, E concentrations of the

incubated male broiler litter was significantly below (P<0.05)

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1744 SHORE ET AL.

ESTROGEN Female

pH

FIG. 3. Effect of incubating for 1 wk at various pH's on the estrogencontent of female broiler litter. Legends same as in FIG. 2.

than that of control values of 13.2±0.3 ng/g (n=6) at pH 5 and pH7

(9.3±0.5 and 9.9±0.8 ng/g respectively). However, in presence of

antibiotics the E content was not significantly different from

controls.

The effect of incubation on T values is shown in Fig. k. In

the presence of antibiotics the amount of radioimmunoassayable T

at pH 7 (678±80 ng/g) in the male broiler litter was significantly

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TESTOSTERONE IN POULTRY LITTER

TESTOSTERONE"g /s

1745

200

males

C 1 5 7 7+ 12

FIG. 4. Effect of incubating for 1 wk at various pH's on the testo-sterone content of male broiler litter. Asterisks indicatessignificantly different from control (* = P<0.05; *** = P<0.001).Other legends same as FIG. 2.

(P<0.001) elevated above control values (94.5±5.7 ng/g). In the

absence of antibiotics, there was no significant difference

between controls and incubations with the exception of pH 1.

DISCUSSION

Chicken manure litter is widely used in Israel as a feed for

non-lactating calves and beef cattle. It is an excellent solution

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1746 SHORE ET AL.

for a major environmental problem and a good source of cheap

protein. However, hormones present in the litter are a limitation

in its use. We have previous reported that levels of T above 300

ng/g can interfere with the reproductive cycle in the cow and

cause delay in puberty. However, in an extensive survey chicken

litter with levels above 300 ng/g were rarely detected Ct/69). We

therefore in the present paper investigated three possibilities to

explain the occasionally high T concentrations: age, sex and

conditions of the litter on the coop floor. As expected, adult

chicken litter has much higher hormone concentrations than

immature broiler litter but this is probably not important

except in cases where litter from laying hens is misrepresented as

broiler litter (which is in contradiction to regulations). Female

broiler litter had five times higher concentrations of E than male

broiler litter and this estrogen (50 ug/kg) may contribute to

hyperestrogenism especially if other sources of estrogenic

activity are consumed, e.g. phytoestrogens in alfalfa, soya or

clover. In contrast, there was no difference in T concentration

between male and female broiler litter.

The conditions of fermentation are a factor in hormone

content. E concentrations decreased after incubation in both male

and female broiler litter. The effect was not apparent when

antibiotics were added nor was it seen at extreme pH's

indicating that microbial agents are responsible for the decrease.

In contrast, when antibiotics were added to male broiler litter,

there was a significant increase in T concentration. This

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TESTOSTERONE IN POULTRY LITTER 1747

indicated that under certain conditions, changes in the microflora

can dramatically affect hormone concentrations.

The observation that the microflora can decreases E content

and increase T content is in general agreement with the obser-

vations of Clavert et al. [10] who measured the effect of

digestion with fly larvae on hormone content as determined by

bioassay. However, Clavert used litter from layers while we report

that T production by female broiler litter does not increase with

incubation.

There remains the problem of what is the origin of the

additional T following incubation of the male broiler litter.

Clavert et al [10] suggested that some microorganism may be

producing T from progesterone. The present data suggest that the

source may be degradation of conjugated T. The basis for this

hypothesis is that in the same manner that female broilers produce

five times more E than male broilers, it would be expected that

the males (which are about 300 g larger than the females at the

same age) would produce more T than the females. This additional T

however is apparently excreted as sulfate or glucorinide

conjugates which under certain conditions are returned to the

free, radioimmunoassayable form on the floor of the coop.

CONCLUSIONS

If high levels of T or E are found in broiler chicken litter

two possiblities should be considered:

1. Misrepresentation: The litter is from layers or adult roosters.

This should be readily apparent from tests for protein (at least

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1748 SHORE ET AL.

20/0 and ash (>20/0 which should be done before using any poultry

litter.

2. Use of antibiotics: The broilers received antibiotics (probably

in contradiction to regulations) which changed the microflora of

the coop. Anaerobic conditions in poorly ventilated coops with

excessive humidity could also be a factor.

REFERENCES

1. Bendheim U., Shore LS., Harel-Markovitz E., Weismann Y.,Yagil R., Shemesh M. Hormonal sexing versus surgical sexingin birds. In: Proc. 1st Conf. Europ. Committee of the Assoc.Avian Veterinarians, Rubel A., Baumgartner R., eds. TheNetherlands: European Chapter of the Assoc. of AvianVeterinarians 1991; 115-117.

2. Shore LS., Shemesh M. Analysis of testosterone levels inchicken manure. Isr. J. Vet. Med., 1993; 48:xx (In press).

3. Greene WA., Mogul LG., Lein DH., McCauley AD., Foote RH.Growth and reproductive development in freemartins hormonallytreated from 1 to 79 weeks of age. Cornell Vet. 1979; 69:248.

4. Gassner FX., Reifenstein Jr. EC., Algeo, JW., Mattox WE.Effects of hormones on growth, fattening and meat productionpotential of livestock. Recent Prog. Horm. Res. 1958; 14:183.

5. Hargrove DA. Use of growth promotants in replacement heifers.In: (Fields MJ., Sands RS., eds), Factors Affecting CalfCrop. Boca Raton, Florida: CRC Press 1993: 91-103.

6. Moran C., Prendville DJ, Quirke JF., Roche, JF. Effect ofoestradiol, zeranol or trenbolene acetate implants onpuberty, reproduction and fertility in heifers. J. Reprod.Fert. 1990; 89: 517.

7. Reynolds IP., Harrison LP., Mallinson CB., Harwood DJ.,Heitzman RJ. The effect of trenbolene acetate on the bovineoestrous cycle. Ani. Reprod. Sci. 1981; 4: 107.

8. Shemesh M., Shore LS. Effects of hormones in the environmenton reproduction in cattle. In: Fields MJ., Sands RS., eds.Factors Affecting Calf Crop. Boca Raton, Florida: CRC Press1993: 289-299.

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TESTOSTERONE IN POULTRY LITTER 1749

9. Shore LS., Shemesh M., Cohen R. The role of oestradiol andoestrone in chicken manure silage in hyper-oestrogenism incattle. Aust. Vet. J. 1988; 65: 68.

10. Clavert CC., Smith LW., Wrenn TR. Hormonal activity inpoultry excreta processed for livestock feed. Poultry Sci.1978; 57: 265.

Date Received: March 31, 1993Date Accepted: May 10, 1993

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