Control of the illegal administration of natural steroid hormones in urine and tissues of veal calves and in plasma of bulls

57

Analytxa Chmuca Acta, 275 (1993) 57-74 Elsener Science Pubhsbers B V , Amsterdam

Control of the illegal administration of natural steroid hormones in urine and tissues of veal calves and in plasma of bulls M L Scrppo, P Gaspar, G Degand, F Brose and G Maghum-Rogtster Facubi de Mhiecme V&huwe,

Laboratove d%atyse des Dmkes Almwttarres d’Ongme Anunak?, Utuuersrte’de L&e, Sart Timan, B&t B42, B-4ooo age &lguun)

P Delahaut Laboratoue d’Homwnolog~?Ammak, Centre d’Economce Rumle de Marhe, B-6900 Marche @elgtum)

J P Wrllemart hvuwn

Agro-Vh%nan,

Rcwsel-U&J

163 A~wnue Gambetta, F-75020 Pans (France)

(Recewed 10th June 1992, revised manuscnpt received 20th October 1992)

Abstract

In the context of the control of the Illegal admmlstratlon of natural steroid hormones m cattle husbandxy, declslon levels of sex steroid hormones were estabhsshed, takmg mto account the effect of the treatment, for the cases of the urme of male and female veal calves treated with estradlol and/or testosterone-contammg implants, and the plasma of bulls treated by an me&on of an estradlol-testosterone cucktad At each d-Ion level, a score was assigned, that represents the percentage of treated ammals detected when the declslon hnut IS apphed Concenung the veal calves, a maxunum declslon level 1s proposed for the 17fi-estra&ol m m-me of 1 ng ml-’ m both male and female veal calves, gwmg a score of 95% A muumum declslon level was set at 2 ng ml -’ for testosterone m urme of male veal calves, anth a score of 95% (95% of the implanted male veal calves display a urmary testosterone level lower than 2 ng ml-‘) For female veal calves, a maxunum declslon level was set at 0 45 ng ml-’ for the unnary testosterone concentration (score of 90%) For bulls, the 17&estrad~ol concentrahon m plasma IS a good cntenon for detectmg mected bulls a maxunum decslon level was set at 40 pg ml-‘, dlsplaymg a score rangmg from 100 to 45%, 2 and 7 days after the mjection, respectwely Keyword9 Immunoassay, Radlochenucal True samples, Urme, Veal calves

methods, Anabohc

In the European Economrc Commumty (EEC), Duectrve 88/146/EEC prohrbrts the use of anabohcs as growth promoters for meat productron Correspondence to G Maghum-Rowter, Laboratolre d’Analystdes Den&s Ahmenkures d’Ongme Ammale, Faculti de Mdecme V&&maue, Umverslti de LG?ge, Sart Tdman, Bat B42, B-4UOl.lL&ge, (Belgmm)

steroids, Bovme samples, Bulls, Hormones,

Plasma,

As non-endogenous synthetrc hormones, such as zeranol, trenbolone and diethylstllbestrol (DES), can easily be detected m urme, plasma or trssue samples [ll, anabohc treatments wrth natural sterords are mcreasmgly applied [2] The problem 1s then to k-ate between physrologrcal concentratrons and elevated hormone levels due to the admlmstratron of natural anabohcs Thrs 1s

OOO3-2670/93/W 00 0 1993 - Elsewer Science Pubhshers B V All nghts reserved

58

ML Sctppo et al /Ad

not an easy task owmg to the large variabthty described m the hterature for the concentratrons of the various sterords m different matrrces In very well controlled expenments, mvolvmg annnals of the same age, fed and housed m the same way, Arts and co-workers [3,41 estabhshed reference values for 17/I-estradrol, 17a-estradrol, testosterone and eprtestosterone concentrations 111plasma and urme of male and female veal calves From these reference values, they determined decrston hnuts on veal calves treated by mJectron of an estradiol-testosterone cocktail They concluded that the best crrterion to detect treated animals 1s the urmary or plasma level of 17&estradtol (which mcreases after the treatment) Our approach 1s modelled on that of Arts and co-workers but is somewhat different The aim of the control IS to determme whether a sample ongmates from a treated annnal or not, wrthout knowmg rts age or Its housmg and feeding condttrons In order to achieve to this am, a statrstrcal study was performed on data from several dtfferent expernnents These data are concentrations of natural steroid hormones [determmed by radromunoassay (RIA)] m urme and tissues of male and female veal calves untreated or treated with sex sterord-contammg implants The need for reference values also extsts for adult ammals We therefore measured plasma levels of varrous sex steroids hormones and metabohtes m untreated bulls and m bulls treated by a single lnJectron of a testosterone-estradiol cocktail Decision lmuts were then determmed takmg mto account the treatment effect

EXPERIMENTAL

Samples Vhf calves Male and female veal calves were

treated m several drfferent expernnents (Table 1) wnh estradrol and/or testosterone-contammg 1l11plants for various periods of ume (15-120 days) Samples of urme were collected durmg the treatment per& and tissue samples were recovered at slaughter After hydrolyses wrth Heluc pomatza JUKX, diethyl ether extraction and purifi-

Chm. Acta 275 (1993) 57-74

cation on Bond Elut [51, the samples were analysed usmg specrfic RIAs for 17@estradtol, 17aestradlol, estrone and testosterone according to procedures descrrbed previously [5,6] Bush Eleven bulls, aged 9-11 months and weighting 310-470 kg, were treated by a smgle rmectron of a co&tad contammg 20 mg of 17/3estradrol benxoate (Intervet) and 200 mg of several esters of testosterone (Durateston, Intervet) Control plasma samples were collected from these bulls 4, 2 and 1 day before the treatment and plasma samples from treated ammals were collected 2, 3, 5 and 7 days after the steroid mJection After dtethyl ether extraction of the plasma samples, non-conjugated forms of the sex steroid hormones and metabohtes (17)3-estradrol, 170~ estradrol, estrone, epltestosterone and testosterone) were measured usmg specific RJAs The concentration of comugated forms of the same steroids were determmed after mcubatron of the aqueous phases, resulting from the first drethyl ether extraction step, wrth H&x pomatw Juice (20 ~1 ml-’ plasma) for 24 h at 37”C, and subsequent drethyl ether extraction

Radwtmnuuwassay

Antisera agamst 17&estradtol, estrone and testosterone were ratsed m rabbits (Laboratoue d’Hormonologie Ammale, Centre d’Econorme Rurale, Marlowe, Belgmm) and antr-l’ku-estradlol and anti-epitestosterone antisera were from BIOgenesis (Boumemouth, UK) Trrtiated 17&estradiol, estrone and testosterone were obtained from Amersham (Amersham, UK) Trmated l’la-estradrol and eprtestosterone were obtained by conversron of tnttated estrone and androstenedione, respectively, by mcubation with bovme erythrocytes at 37°C 171 Cross-reactwnres of these antisera are given m Table 2 The characteristtcs of each RIA m urme, ussue and plasma samples are described m Table 3 Control samples contammg a low, a medran or a high amount of each hormone were introduced mto the assay For each RIA, the accuracy and the reproduciblhty were checked by performmg

ML

Scpp~ et aL /Ad

59

Chum Acta 275 (1993) 57-74

repeated analyses of spdced samples As an example, the data for RIA m urme samples are presented m Table 4 In all mstances, the mean recovenes ranged from 88 to 124% of the amount of hormone added In the same expernnent, the mtra-assay relative standard devratlon (R S D 1 did not exceed 30% m any case The recovery after the dlethyl ether extraction step was calculated by addmg a known amount of tntlated hormone to each sample The mean recovenes are gwen m Table 3

StatWcal analyst and dmswn level detemuaatwn As m most mstances a non-Gaussmn dlstnbutlon of the hormone concentrations was found, a non-parametnc test, the Wdcoxon test, was apphed (usmg Statworks 13 software, Cncket Software, Phtladelptia, PA) m order to determme If the hormone levels measured m control and treated ammals are stgmficantly Merent (with p < 0 05) If this 1s the case for a hormone concentration, a declston lnmt 1s defined by the h@-

TABLE 1 Descnptlon of the different expenments concernmg the veal calves (the ammals were treated wth Implants) each experunent mvolved control anrmals, from which samples were collected at the same tune as those collected III treated veal calves Ammats

Treatment

!kilDkS

Hormones measured

Male veal calves (2-3 weeks of age at day 0)

Day 3 Imph BM (Roussel-Uclaf, France) (20 mg of 17gcstradlo1 + 200 mg of progesterone) Day50 Revalor (Roussel-Uclaf) (20 mg of 17j3-estradiol + 140 mg of trenbolone acetate)

Samples of urme collected on days 89,96,103,108 and 109

17B-Estradlol, 17a-estradlo1, testosterone

Samples of tissues (muscle, hver, ludney and fat) collected at slaughter on days 15, 30,50,65,80,100 and 120

17&Estra&ol, 17a-estradlol (m hver). testosterone

Day 50 Revalor

Samples of urme collected on days 89, 96,103,108 and 109

17/3-Ektra&ol, 17a-estradlol

Samples of tiues (muscle, hver, hdney and fat) collected at slaughter on days 65,80,100 and 120

17/3-Estrad~ol, 17 a-estradlol (m hver)

Male and female veal calves (23 weeks of age at day 0)

Male and female veal calves

Day 28 Crestar F (Intervet, France) (20 mg of 17@estradlol+ 200 mg of testosterone) Day 56 Crestar F

Samples of tissues (muscle, liver, ludney and fat)

Testosterone,

17&estra&ol, estrone

Male and female veal calves

Day 56 Crestar F

Samples of tissues (muscle, hver, bdney and fat)

Testosterone,

17&estra&ol

Male and female veal calves

Day 56 Crestar M (20 mg of 17/j-estradlol + 200 mg of progesterone)

Samples of tissues (muscle, hver, ktdney and fat)

Testosterone,

17@cstradlol

Male and female veal calves

Day 42 Revalor

Samples of unne collected ondays26,66and86

17@Zstrad~ol, 17a-estradlol

60

ML Sctppo et al. /AML Chm. Acta 275 (1993) 57-74

TABLE 2 SpedMy

(o/o)of the antisera rawd against testosterone, epitestosterone,

Crossreactwdy wth

Testosterone 17B-Estradlol 17a-Estradlol E&one Dlhydrotestosterone Androstenedlone Eplstestosterone Cholesterol Pregnelonone Dlhydroepumdrosterone CoWsone 17a-Hydroxyprogesterne Estrlo1

progesterone, 17@-estradtol, 17a-estradtol and estrone

Anhserum agamst Testosterone

17$t?Estradlol

17aEstradlol

Estrone

Epltestosterone

100
<007 100 047 14 ND ND ND <007 <007 ND ND ND 027

NDB 0 71 100 125 ND ND ND ND ND ND ND ND 01

0 015 054 039 100 ND 007 ND ND ND 0 15 <0005
14 ND <03 ND <04 37 ND ND ND ND ND ND ND

a ND = not determmed

est hormone concentration found m the control ammals, If the treatment effect 1s an elevation of that concentration, or the lowest hormone concentration found m the control animals, rf the treatment effect IS a dunmutlon of that concentration The percentage of posltlves detected among treated ammals IS an mdlcatlon of the score of efficacy of the proposed decision levels

Concemmg the veal calves, each experiment displayed m Table 1 mvolved control ammals As they were treated by Implants, which contmuously release hormones, the data obtamed from the SP expemnents (from control or treated amma19 were taken together, for each category of sample (urme, muscle, liver, ludney or fat) With the exception of testosterone concentration, no dlstmctlon was made between male and female

TABLE 3 Charactertstzs

of the radonnmunoassays

(after log@log transformation) used m this study for urme, plasma and tissue samples ’ Slope

Intercept

ED, (pg per tube)

15 70 15 25

-082 -102 -082 -077

237 397 237 2 33

18 45 18 22

34 49 48 55 44

55 55 17 13 65

-091 -100 -099 -095 -124

275 286 223 291 344

21 17 10 21 16

70 58 53 52

43 63 21 19

-116 -106 -095 -097

290 4 45 256 330

12 68 15 38

Steroid hormone

Mean recovery (%)

b/T (o/o)

:j

Urine

17&Estradlol 17a-Estradtol Testosterone Estrone

85 90 85 70

54 46 54 53

Plasma

17/3-Estradlol 17a-Estra&ol Testosterone Epltestosterone Estrone

70 89 85 85 50

Tissue

17/3-Estradlol 17a-Estradlol Testosterone Estrone

71 61 74 51

Sample

a B,/T = percentage of specific fixation, NS = non-specfic,

ED,, = dose at 50% of fixation

ML Scppo et al /Anal

61

Chm Acta 275 (1993) 57-74

TABLE 4

TABLE 7

Mean recovenes obtamed from sp&ed urme samples

17a-E!&radlol m urme and bver of male and female veal calves treated or not wtb natural sterord-contammg unplants

Steroid hormone

n

17@atradlol

3 3 3 3 3 3 3 3 3 2 2 2

17a-Estradlol

Testosterone

Es&one

Added (pg 1-l)

Measured (cg 1-V (meanfSD)

Recovery (%6)

05 10 20 05 10 20 05 10 20 05 10 20

05 *002 124&O 10 203kO 1 044*004 094*008 194*008 052*002 096+003 201*008 051*011 101*002 203f006

100 124 102 88 94 97 104 96 100 102 101 102

Controls Muumum Maxnnum Treated Mmlmum Maximum U test (p) Decision level Score (o/o)

17a-Estra&ol m urme (ng ml-‘)

17aXstradlol m bver (pg g-‘1

n = 51

n = 13 560 761 n = 56 294 4158
04 23 n = 103 3 242 <00005 <23ngml-’ 58

TABLE 5 17@stradlol

m urme and twsue of male and female veal calves treated or not wth natural sterold-contammg 17/?-Estradlol m unne (llg ml-‘)

Controls Mmlmum Maxunum Treated Mmmwm Maxunum u test (PI Decwon level Score of the decwon level (%)

n=40 02 1 n = 122 06 17 6 <00005 < 1 ng ml-’ 95

unplants

178~E-stradlol m twsues (pg g-l) Muscle

Lwer

I(ldney

Fat

I; 33 2 n = 80 11 280 <00005 <34pgg-’

n-24 53 53 n = 111 5 1650 <00005 <53pgg-’

n=23 79 69 n=lOO 64 589 <00005 <69pgg-’

n =21 56 50 n = 103 93 358
81

80

75

80

34

TABLE 6 17/3-Estradlol m plasma of bulls before (controls) and after treatment by an lqlectlon of a testosterone-estradol

Controls (n = 33) Mmunum Maxunum

Treated (n = 11) M1nlmum Maxmmm U test(p) Decwon level Score (%)

Uncoqlugated 17&estradlol m plasmaofbulls(pgml-‘1

Coqugated 17&estradlol m plasma of bulls (pg ml - ‘1



cocktad

Day2 b

Day3 b

Day5 b

Day7 b

Day2 b

Day3 b

Day5 b

Day7 b

205 1162
65 421 <0005



29 75 <0005



64

45

37 116 <0005 <32pgml-’ 100

90

18

ND

BW=l~tofdetetion=20pgml-1

100

bAfterIqlechon

62

ML Scrpp~et al /Ad

Chm Acta 275 (1993) 57-74

TABLE 8 17a-Estradml m plasma of bulls before (controls) and after treatment by an Iq)echon of a testosterone-estradlol co&ml *

Controls (n = 33) Mmlmum Maximum

Treated (II = 11) Mmlmum Maxmmm LItest (p) Decmlon level Score (%)

Uncoqlugated 17u-estra&ol m plasma of bulls (pg ml-‘)

ConJugated 17a-estrad~ol m plasma of bulls (pg ml- ‘1

14 70

12 l&33

Day2 b

Day3b

Day5 b

Day7 b

Day2 b

Day3 b

Day5 b

Day7 b

11 109 0043 <7opgml-’ 36

23 92 -Co005

30 51 0034

25 95 0 012

171 601 -Co005

61 480 -Co005

13 370 0 012

9

0

27

300 1082 COO05 (183pgml-’ 100

90

45

27

’ Lmut of detection = 8 pg ml-’

b After lqlectlon

veal calves The data are displayed m the results tables (Tables 5-121, as “control” values and “treated” values, wlthout Qstmctlon of the samplmg tune Data from injected bulls were analysed separately at each time of samplmg

RESULTS

For each hormone studied, the results are pen m Tables 5-13 and Illustrated m Frgs l-5 They show the lowest (mmunum) and the highest (maxTABLE 9 Estrone m trues of male and female veal calves treated or not urlth natural stero~d-contammg implants Estrone m tisues (pg g-l)

Controls Mmlmum Maxunum Treated Mmmmm Maxmmm CJtest (p) Deculon level Score (%o)

Muscle

tier

Gdney

Fat

n=3 15 78 n=18 3 72 0255 ND ND

n=3 170 198 n = 18 73 284a 0 021 ,170pgg-‘b 83

n=3 23 166 n=18 34 144 0093 ND ND

n=3 80 94 n = 18 29 149 0 113 ND ND

a Three values out of eighteen were above 170 pg g-’ b The declslon level determmed for the estrone content in hver IS a mmmmm level

unum) hormone concentration found m control and 111treated ammals, the result of the statistical test (Wdcoxon test, also called U-test), the deaslon level estabbhed accordmg to the values obtamed from the control ammals, and the score of the decision level When measurmg 17/3-estradlol, 17a-estra&ol and estrone concentrations m both urme and tissue veal calves samples, no sign&ant Merence was found between female and male veal calves No &stmction was therefore made between male and female ammals m the analysis of these data 17@Estradwl Veal calves (Table 5, Fig lA) In urme, the 17/3-estradlol concentrations were slgmficantly higher m treated than m control ammals A deaslon level of 1 ng ml-’ 1s proposed, which allows 95% of the treated ammals to be declared posltive In tissues, the 17@estradlol levels were srgmficantly higher m treated ammals The normality lrrmts, fixed as the highest levels found m control ammals, display scores varymg from 75 to 81% dependmg on the tissue Bulls (Table 6, Fag 1B and C) In plasma of bulls, both free and coqugated forms of 17/3estradlol are slgnifxantly higher after the treatment until the seventh day for the free form and

ML Suppo et al /AnaL CJwn. Acta 275 (1993) 57-74

63

TABLE 10 Eatrone m plasma of bulls before kontrols) and after treatment by an m~ect~on of a testosterone-estradol

(n = 33) Mlmmum Maximum

Uncoqlugated estrone m plasma ofbulls(pgml-‘)

GmJugated estrone m plasma ofbulls@gml-‘1



cocktad

Day2 b

Day3 b

Day5 b

Day7 b

Day2b

Day3 b

Day5 b

Day7 b

Treated (n = 11) Mullmum Maxunum U test (p) Dension level





58 110 <0005


-=.LD 108 <0005

score (o/o)

64

ND

73

64


27

54

72

’ ID = hnut of detetion

= 7 pg ml-’

b After uuectlon

untd the fifth day for the coaugated form Declslon levels were set at 41 and 32 pg ml-’ for the free and coqlugated forms of 17/3-estradol, respectlvely The score of the free 17&estradlol deaslon hnut IS better than that of the conJugated form It ranges from lOO%,2 days after the treatment, to 45%, 7 days after the treatment

17ct-Estradwl Veal calves (Table 7, Fig 2A) A higher 17ar-

estradlol level was found m both urme and hver from treated anunals than from the controls Declslon levels of 23 ng ml-’ m urme and 761 pg g-’ m hver make possible the detectlon of 58% and 75%, respectwely, of the treated animals

TABLE 11 Testosterone

m urme and tissues of male and female veal calves treated or not wth natural steroids contammg unplsnts

Veal calves

Testosterone m urme (ng ml-‘)

Testosterone m hssws (pg g-l) Muscle

Lrver

I(ldney

Fat

Male

Controls MmmlUm Manmum Treated MlIUItlUIll MaXmlum U test (p) Decuuon level Score (%o)

n = 26 3 36 n==40 03 25 <00005 &25ngml-’ 100

n = 10 34 773 n = 10 42 418 0065 ND ND

n=5 96 324 n==5 21 95 0005 .%pBg_’ 100

n=5 580 10169 n-5 167 310 0005 .58Opgg-’ 100

Ii=5 270 3879 n=5 292 2981 0 174 ND ND

Female

Controls Mllumum Mzomum Treated Mmunum Maxnnum U test (p) Declslon level Score (o/c)

n=4

n = 30 6 422 n=L?tJ 64 310 0250 ND ND

n = 10

n = 10

n = 10

12 121 n - 21 4 317 0199 ND ND

58 375 n=26 195 870 COO005 <375pgg-’ 73

21 16.5 n = 26 263 1742 <00005 <165pgg-’ 100

02 04 n-28 006 78 0004 ~045ngml-’ 90

64

ML Scrppo et al /AnaL Chun. Acta 275 (1993) 57-74

TABLE 12 Testosterone m plasma of bulls before (controls) and after treatment by an m~ect~on of a testosterone-estrahol

Controls (n = 33) Mmmmm Maximum

Treated (?I = 11) Mmmmm MaXmluIll V test(p) Decwon level Score (o/o)

Uncoqugated testosterone in plasma of bulls (pg ml - ‘)

Coqugated testosterone m plasma of bulls (Pg ml-‘)



cocktad

Day2 b

Day3 b

Day5 b

Day7 b

Day2b

Day3 b

DaySb

Day7 b

572 5027 0228 ND ND

349 4081 0 318

676 36% 0119

514 3566 0253




ND

ND

ND


ND

ND

ND

’ LD = lmut of detechon = 50 pg ml-’

b After mjectlon

Bulls (Table 8, Fag 28 and C) 17cr-estradlol levels are higher (p < 0 OS) m the plasma of bulls until 7 days after the steroid qection than before

treatment The score of the deaslon lnmt f=ed for the conjugated form (183 pg ml-‘) 1s better than that of the non-conjugated 17a-estradlol(70

I I

I 0

Fig 1 (pi, Dlstnbution of 17/3-estrtiol concentrations m urme and hssues of treated and untreated veal calves (male and female) (B) Dlstnbutlon of 17@-estradlol concentrations hmcoqugated forms) m plasma of treated and untreated bulls (0 Dlstnbutlon of the 17@-estradlol concentrations koqugated forms) m plasma of treated and untreated bulls D L = decnlon hut

ML hppo

et al. /Ad

65

Chm Acta 275 (1993) 57-74

I:

I

ecommls

l

Tmatd

l

.

i l

.

.

. l

I

8 .

l . 0

l

0 8

i

+ 0

0 Fu

1 (contmucd)

Day 2

bY2

Day 3

Day 5

i

.

l

0 l .

bY3

hY5

Day 7

:

l

Day 7

alter the injecmn

l

D.L. . 32

pglml

66

ML Sctppo et al /Anal Chm Acta 275 (1993) 57-74

pg ml-‘), It ranges from lOO%, 2 days after the treatment, to 27%, 7 days after the treatment Estrone Veal calves (Table 9, Ftg 3A) In muscle, lud-

ney and fat, no dtierence m estrone levels was found between treated and untreated ammals Only m liver were the concentrations obtamed from the treated veal calves ngmficantly lower than those observed m the control annnals A declslon level established at 170 pg g-’ (the mmlmum hormone concentration found m the control anunals) led to a score of 83% Bulls (Table 10, F& 3B and C) In contrast to the 17/3- and 17a-estradtol results, the free estrone levels decrease (p < 0 05) after the sterord treatment (with the unexplained exception of the plasma samples on the thud day after the qectlon) The decision hmit was then set at the mmunum level found 111control samples, 7 pg ml-’ The score of the free estrone declslon limit

ranges from 64 to 73% W-73% of the bulls display a free estrone concentration lower than 7 pg ml-‘) Coqugated estrone first increases (p < 0 05) m plasma of bulls (the second and third days after the nqectlon) and decreases (p < 0 OS) on the fifth and seventh days after the treatment. Hence a mmnnum lmt of 19 pg ml- ’ and a maxnnum level of 96 pg ml-’ are proposed That means that bulls dtsplaymg a coqugated estrone concentration m then plasma lower than 19 and higher than 96 pg ml- ’ would be declared posltlve This 1sthe case for 72% of the bulls, 7 days after the steroid mjectlon Testosterone Veal calves (Table 11, Fag 4A and B) In both male and female veal calves, the testosterone concentrations were signScantly dtierent m treated annnals and m controls For male veal calves, the values are lower m the treated am-

1ooO:

100;

10 7

l-.

Fig 2 (A) Dlstnbutlon of 17a-estradlol concentrations m urme and her of treated and untreated veal calves (male and female) (B) Distribution of 17a-estradlol concentrations huxmwgated forms) m plasmaof treated and untreated bulls (C) Detnbuhon of 17a-estrahol concentrations (coqlugated forms) m plasma of treated and untreated bulls D L = decwon hut

ML

Scrppo et d/AnaL

67

Chvn Acta 275 (1993) 57-74

+ 70 :

!

.

3

.

DL pglml

t t 8

. .

10 0

bY2

Day3

hY5

Day7

aftmthaln@7ion

El

+

1

q

Day 2

Day3

Day7

a#lheinjecth

D.L : 183 pglml

68

hi L Sappo et al /Am!

Chm Acta 275 (1993) 57-74

TABLE 13 Epltestosterone

m plasma of bulls before (controls) and tier Uncoqlugated epltestosterone plasma of bulls (pg ml- ‘)

Controls (n = 33) Mmmmm Maxmmm

treatment by an ~qlectlon of a testosterone-estrahol

m

Coqugated epitestosterone plasma of bulls (pg ml-‘)

134 974

Treated (n = 11) Mmlmum Maxmwm u test(p) Decwon level Score (%I

cocktail a

m

219 1750

Day2 b

Day3 b

Day5 b

Day7 b

Day2b

Day3 b

Day5 b

Day7b

361 1970 004 974pgml-’ 27

200 1950 001

337 820 0114

309 1196 0 197

524 1297 0 337

382 951 0 130

333 865 0 053

27

ND

ND

450 1386 01% ND ND

ND

ND

ND

a Lmut of detection = 50 pg ml-’

b After uuection

mals, and a declslon level of 2 5 ng ml-’ (which 1s a nununum acceptable testosterone concentration) gives a sc43re of 100% In female veal calves, the values are higher 111the treated than m the

untreated ammals. A decision lmt estabhshed at 0 45 ng ml-’ allows the detection of 90% of the treated ammals Concerning the tissues, the bver and the lod-

El

.

I 100:

I l

; 0

I n

l

(’ minimum

.

vslue)

Fig 3 (A) Dlstnbutron of estrone concentrations m tawues of tre&ed and untreated veal calves (male and female) (B) Dlstnbution of estrone concentrations (uncoqugated forms) m plasma of treated and untreated bulls (C) Dlsmbutlon of estrone concentrations (cowugated forms) m plasma of treated and untreated bulls D L = decuuon llrmt

ML Sctppo et aL /AnaL Chum Acta 275 (1993) 57-74

69

DL’ *

7

PrW

(’ minimum value)

0

Day 2

Day 3

Day 5

Day 7

altar the inpdion

-D

96

(

1

I

a

.

+_

1

$ .

:

m

Fw 3 (cmtmued)

I

bY2

value)

19

DL’ pglml

(’ mlnimum value)

3

0

maximum

l

lolj( l!

I).

DL l * pglml

hY3

-

hY5

Day7

afmrtheinjacUon

ML Scrppoet al. /AML Chm Acta 275 (1993) 57-74

l

10000

q

q

q

I

1000

I DL’ 2 nglml I

ii. 8 .

.

i

’ 8 n

l

100

10

1 (’ mrdmum value) ,I Fat

1000

n

m q

01 -I

pq TISSUES F% 4 (A) Dmtnbution of testosterone concentrations m urme and hssues of treated and untreated male veal calves (B) Dsmbuhon of testosterone concentrations III urme and hssues of treated and untreated female veal calves (0 Dutnbutlon of testosterone concentrations (uncoqlugated forms) m plasma of treated and untreated bulls (D) Dwtnbutlon of testosterone concentrations (coqlugated forms) m plasma of treated and untreated bulls D L = decwon tit

ML Scrppo et d/Anal

Chum Acta 275 (1993) 57-74

71

l .

l

t

: .

; i

8

.

0

. . .

.

. .

l

NO DECISION

LIMIT

a

Day 2

Day 3

Day 5

Day 7

alter the injudion

. . .

.

.

l

. . .

I

l

.

l i l . l . l

NO DECISION

Day 2

Treated

l

l +

0

c0nbde

.

. . . .

.

Fu 4 kontmued)

l

l

t .

.

I

Day 3

LIMIT

Day 5

Day 7

after the mjectm

72

ML Scppo et aL/AnaL Cham.Acta 275 (1993) 57-74

x

l

+

l

OL. 974 pglml

i

8 .

Day 2

i

i

.

Day 3

Day 5

NO DECISION

Day 7

after the injection

LIMIT

l

. 1000:

i

I . I8 8 j

i :

a t

f

:

:

!

i

!

Day 2

.

Day 3

bY5

Day7

alterthelnjsdon

F~J 5 (A) Dlsmbutlon of epltestosterone concentrations (uncoqlugated form) III plasma of treated and untreated bulls (B) Dlsmbuwn of epltestosterone concentrattons (cowugated forms) IIIplasma of treated and untreated bulls D L = decuaon hut

73

ML Soppo et aL /AMI! Chm Acta 275 (1993) 57-74

ney of treated male ammals contam srgmficantly lower amounts of testosterone than those of control male veal calves (Table 11) Decrsron levels of 96 and 580 pg g-l, respectrvely, would make It possible to declare posrtrve 100% of the treated ammals However, these values were obtamed on a very lmuted serres of ammals (n = 5) In the female veal calves, only the testosterone contents of fat and krdney are srgmficantly hrgher m treated than m control ammals, 100% of the treated ammals could be detected wrth a decrsron lnmt of 165 and 375 pg g-l, respectrvely (Table 11) Bulls (Table 12, Fag 4C and 0) The plasma testosterone concentration was not srgmficantly drfferent m bulls before and after the steroid mjection Epztestosterone VTbulls (Table 13, Fzg 5A and B) A shght Increase was obtamed 2 and 3 days after the treatment for the non-conjugated epnestosterone only The decrsron lrmrt of 974 pg ml-l (the maxmmm free eprtestosterone concen-

tratron before the treatment) allows only 27% of the treated ammals to be detected DISCUSSION

From 1 January, 1988, the use, as growth promoters 111meat productron, of anabohc sterords and related substances mcludmg the strlbenes and zeranol was banned m the EEC The control of the rllegal use of these substances IS based on the analysrs of blood, trssues or excreta for the presence of synthetrc compounds (residues of parent drugs or metabohtes) not occurrmg naturally m the body The composrtion of several “hormone cocktails” cuculatmg on the black market mvolved “natural” sex steroid hormones (17/3-estradrol, testosterone, progesterone) or therr ester derrvatrves [2] These hormones and therr metabolites occur naturally m both male and female meat-producmg ammals The drscnmmatron between physrologrcal levels and elevated concentratrons due to hormone admmrstratron

TABLE 14 Decwon levels for 17&estradlol, 17a-estradlol, estrone, epltestosterone and testosterone 111urme and tissues of male veal calves and m plasma of bulls a Sterord hormone

Declslon level III Plasma of bulls

Veal calves Urme

Muscle

Leer

fidney

Fat

Uncoqlugated form

Conjugated form

17/3-Eatradio (male and female veal calves)


<34pgg-’ (81%)

(53pgg-’ (80%)

<69pgg-’ (75%)

<5Opgg-’ (80%)

<4Opgml-’ (loo%, 45%)

<32pgml-’ (loo%, 0%)

17a-Ekadlol (male and female veal calves)

(23ngml-’ (58%)

ND

<761pgg-’ (78%)

ND

ND

Gi7opgml-’ (36%, 27%)

<183pgml-’ (lOO%,27%)

Estrone (male and female veal calves)

ND

ND

3 17opgg-’ (83%)

ND

ND

.7pgln-’ (64%, 64%)

<%pgml_’

Testosterone Male veal calves Femalevealcalves Ep~testosterone

>19pgnl-’ (82%, 72%)

*25ngmi-’ (100%) (045ngml-’ (90%)

ND ND

.%Pgg-’ (100%) ND

.58oPgg-’

ND

(100%) <375pgg-’ (73%)

c165pgg-’ (100%)

ND

ND

<974pgml-’ (27%, 0%)

ND

’ The percentages of treated ammals detected ( = the score of effiaency of the decwon level) are mdwated m parentheses (2 and 7 days, respectwely, after the m~ect~onfor the bulls)

14

requires quantrtatwe analysis and the estabhshment of lnnlts at which one can decide whether or not an ammal has been treated Nevertheless, physlologlcal levels are mfluenced by nutrition, breed, sex, age, clrcardlan rythms, environmental factors, stress, etc [8] Age is an unportant parameter when controllmg veal calves As lllustrated by Massart et al [9], large changes m blood plasma testosterone are observed around puberty and delayed puberty can occur Many data have already been published, but the large varlablllty reported m the literature needs further studies before the furatlon of consensus reference values that could be used as decision levels The data presented here for the veal calves were obtained from several expernnents m which male and female calves had been subnutted to different treatments with estradlol and/or testosterone using implants or inJections This expenmental context contrasts with that of Arts and co-workers [3,4], where blood, urine or tissue samples were obtained from animals untreated or injected with oily cocktail containing 17/3-estradlol and testosterone or then ester derwatlves In addition, Arts and co-workers used an analytical procedure that was time consuming, namely hquld chromatography (LC) followed by RIA In our hands, the analytical results on test samples were not slgmflcantly different when RIA was applied without prehmmary LC to those obtained usmg the LC-RIA procedure Despite these experimental differences, we reached the same conclusion as Arts and coworkers concerning 17/3-estradlol m veal calves, namely that Its level m urine 1s a good criterion for detecting female and male veal calves treated with natural steroids A decision lnnlt of 1 ng ml-’ allowed 95% of the implanted veal calves to be detected m this study (Table 14) and 100% of the veal calves, 5 days after a steroid mJectlon, m the study of Arts and co-workers It 1s considered that the testosterone concentration in urine or plasma is not a good criterion for detecting cattle treated by mJectlon In this study, it was found that m addition to the 17&estradlol m urme, the unnary testosterone level 1s also a good cntenon for detecting implanted veal calves, 100% of the implanted

ML Scppo et al /Anal Chum Acta 275 (1993) 57-74

male veal calves (aged 16-20 weeks) dlsplayed a urmary testosterone level lower than the mmlmum value found m untreated ammals, 2 5 ng ml-’ In the study of Arts and co-workers, the testosterone concentration was sometimes lower than 2 5 ng ml-’ m the urine of male veal calves aged less than 20 weeks From the age of 20 weeks, 97% of the values were higher than 2 5 ng ml-’ Because of the restrlctlon of age for the testosterone decision level, the criterion of the 17j3estradlol appears more suitable for detecting natural steroid-treated veal calves Table 14 summarizes the decision levels proposed to detect bulls treated by an inJection of natural steroids It can be seen that the best criterion is either the plasma non-conJugated 17/3-estradlol concentration or the plasma conjugated 17a-estradlol The authors thank IRSIA (Instltut pour l’Encouragement de la Recherche Sclentlfique pour 1’Industne et I’Agrxulture) for financial support They are grateful to Roussel-Uclaf for provldmg the samples of treated animals

REFERENCES 1 G Maghum-Register, Mxrotuol Ahments Nutr , 9, (1991) 13 2 G Maghum-Roglster, m L Fiems and B Cottyn (Eds), Animal Biotechnology and the Quahty of Meat Prodution, Elsewer, Amsterdam, 1991, pp 179-189 3 C J M Arts, M J Van Baak, H Van Den Berg, R Schdt, P L M Berende and J M P Den Hartog, Arch Lebensmittelhyg ,41 (1990) 53 4 CMJ Arts, MJ Van Baak and JMP Den Hartog, J Chromatogr ,564 (1991) 429 5 P Gaspar and G Maghum-Roglster, J Chromatogr , 328 (1985) 413 6 P Gaspar, P , Evrard and G Maghum-Romter, m Proceedings of the 2nd World Congress on Foodbome Infectlons and Intoxications, 26-30 May 1986, Institute of Vetermary Me&me-Robert von Ostertag-Institute (FAO/ WHO collaborating Centre for Research and Trammg m Food Hygiene and Zoonoses), Berhn, p 1206 7 K.1 H Wdhams, D H Henry, D C Collins and D S Layne Endocnnology, 83 (1968) 113 8 S Hoffman, G Leuthold and P Remecke, Arch Tierz , 29 (1986) 553 9 S Massart, R Renawlle, M Sneyers, A Bumy and D Portetelle, Arch Int Physiol Ebochim Blophys , 99 (1991) B68