Naturally occurring androgens, estregens and progestegens in the blood, urine and feces of young cattle
REPORT 88.60 April 1988
A LITERATURE SURVEY
P.L.M. Berende*, L.A. van Ginkel**, R. Schilt*, C.J.M. Arts***,
R.H. Stephany** and J.M.P. den Hartog*
*) RIKILT Postbox 230 6700 AE Hageningen The Netherlands **) RIVM Postbox 1 3720 BA Bilthoven The Netherlands ***)CIVO-TNO Postbox 360 3700 AJ Zeist The Netherlands
REPORT 88.60 April 1988
A LITERATURE SURVEY
P.L.M. Berende*, L.A. van Ginkel**, R. Schilt*, C.J.M. Arts***,
R.W. Stephany** and J.M.P. den Hartog*
*) RIKILT Postbox 230 6700 AE \olageningen The Netherlands **) RIVM Postbox 1 3720 BA Bilthoven The Netherlands ***)CIVO-TNO Postbox 360 3700 AJ Zeist The Netherlands
INTERN:
directeur RIKILT sectorhoofden
hoofd afdeling Blofarmaceutische Analyse produktcoördinator EXTERN: directie directie directie RVV VHI RIVH ILOB-TNO CIVO-TNO IVVO IVO VVDO DLO VKA VD
Produktschap voor Vee en Vlees Produktschap voor Veevoeder
voorzitter Begeleidingscommissie Kalvermelkonderzoek leden van de Werkgroep Hormooncontrole
prof. dr B. Hoffmann, Universität Giessen dr
w.
Daelman, EEG commissionEstrogeos in Progestogens Androgens in Estrogens in Progestogens Summary References Tables 1-11 Appendix 1 Appendix 2 the blood in the blood
the urine and feces the urine and feces in the urine and feces
6 9 10 10 15 16 19 25
Total steroid estrogen levels in the calf and cattle urine: an evaluation.
Results of the measurement (HPLC/RIA) of free e stradi-ol-17~, progesterone and free testosterone in blood plasma of male and female veal calves (a pilot study).
INTRODUCTION
The National Plan for Hormone Control in the Netherlands was set up in January 1988. Following the lines of the Common Market requirements,
the goal of this program is to control the illegal use of horrnanes for
fattening purposes in animal production. One part of this program con -sists of testing for a large number of exogenous horrnanes and thyreo-statics. Testing for naturally occurring horrnanes such as estrad
iol-17~, progesterone and testosterone is also included in the National Plan. The discrimination levels for these substances still have to be set with regard to illegal use. It is extremely important to have good
control procedures at hand and to be able to present them
internatio-nally. Nat only the government but also industry underlines this fact.
Cooperation between the Ministeries or Agriculture and of Public H e-alth in combination with the Research group of Calves'Milkreplacers
Producers of the Commodity Board of Feedstuffs has been initiated at an institutional level (ILOB/TNO, RIVN, RIKILT) to solve the problems
arising with veal calves. Befare actual experiments could be
initia-ted, however, it has been necessary to study not only the easily
ac-quired but also the less available literature (e.g. internal reports,
lab documents etc.).
The sourees discussed in this literature study give the levels of
an-drogens , estrogens , and progestegens found in the blood, urine and fe-ces of veal calves. Since there were not enough data available in the -se three matrices for veal calves, the literature data for male and female breeding calves in the age group ca. 7-11 months has also been added. The sourees used are:
- easily acquired literature, published in reno\~n journals; - data from internal reports of the RIV(M);
- data from Iabaratory documentation of RIV(M) (Appendix 1);
- data from internal reports of ILOB (and ILOB/TNO);
- data from experimental reports sent to members of the Commodity Board of Feedstuffs (veal calf section);
- research data from ILOB/CIVO/TNO/ (dr Arts) (soon to be published); - results of a recent pilot study by the ILOB/TNO (Appendix 2).
This last mentioned research project measured concentrations of free estradiol-17B, free testosterone and progesterone in the blood plasma of ca. 170 veal calves and 40 breeding calves. All the animals had
known backgrounds because they came from institutes and experimental farms.
Most of the analyses mentioned in this paper were conducted with a
ra-dioimmunochemical metbod (RIA). I f other methods \o,~ere used, this will be stated. Immunological measurements, e.g. RIA, are easily applied methods which are not only very sensitive, but also allow for large
numbers oE samples to be analyzed in a short period of time. One
im-portant shortcoming is the level of specificity for one component. Be-cause of the type of method, cross-reactions can occur. This means that material, whose chemical structure resembles that of the component to be measured, may cause a similar response. This is especially true for steroid analyses where, along with the component
to be measured, a number of very similar and cross-reactive compounds are present. To achieve valid results in such cases, very high
requirements on the sample preparatien (e.g. HPLC) are called for. It appears that in the literature studied, a number of authors describe
the quality aspects of testing (cross-reactions and sample
preparation) incompletely or not al all. This means that not all the
publisbed concentrations can be judged for their accuracy. In general it can be stated that analytica! methods, where not only extraction
but also chromatography are conducted, are the most reliable. Simple
purification using a sephadex column results in a less-pure extract than when HPLC is used. Table 11 gives a summary of the most important information from the analyses conducted by the cited researchers. This
report only uses the results of laboratorles with much experience in this field.
ANDROGENS IN THE BLOOD
Quantitatlvely, testosterone is the most important androgen. The next
two are androstenedione and dehydroepiandrosterone. 17a-Testosterone
(epitestosterone) is the most important metabolite found in ruminants (Rico 1983). Karg et al. (1976) have found that the levels of a second metabolite, Sa-dihydrotestosterone, are low. The analysis (RIA) they
used for testing testasterene showed a high cross-reactivity with Sa-dihydrotestosterone. However, since the level of this metabolite was so low, its contribution to the measured testasterene level was ignored. The results by Arts (CIVO/TNO) shmo~ed that this level ,.,as about 10% (8-100 pg/ml) of the testasterene found in ca. 5-month-old male veal calves. This sectien is mostly concerned with testosterone. Befare mentioning the factors that can influence testasterene levels in the blood plasma (or serum), we have given the (average) testaste-rene levels found in young bulls in Table 1. These values were all reached using RIA and most of the tests were performed on the plasma. In spite of this, the values are difficult to campare because some the samples were taken from a few animals once a week or month at one time period, whereas blood from ether animals was taken 8-10 times a day. The last column in the table has been added so that the values can be interpreted easier. The largest differences are seen in the average values stated by the different authors. Generally, one can conclude that the values increase slowly in animals up to an age of ca. 4 months. In animals ca. 4-7 months old, the levels increase more
quickly and after 7 months they vary greatly. These variations are not only found in the average testasterene levels between the different animals, but also in one animal from day to day or even in a 24-hour period. The literature mentions a few values to be aimed at:
<1
ng/ml forages 0-4 months; 1-4 ng/ml for age group 4-7 months; and 1-10 ng/ml and higher for animals older than 7 months. Karg et al. (1976) Here able to abserve these periods clearly. They do mention, hmo~ever,that the periods 1o1ere different for each animal, since the animals do not reach sexual maturity at the same age. Until this stage is
reached, the testasterene level is low, gradually increasing under the influence of the increasing development of the testes until the moment sexual maturity is reached.
From puberty onward, testasterene production is greatly increased. This does not mean, however, that from this moment on the level of testosterone in the blood remains high tbraughout the day.
Testasterene is released into the blood in pulses (Thibier, 1976), explaining to a great degree the large variability found in the given values.
When camparing the levels of different breeds one must keep in mind that eertaio breeds reach sexual maturlty earlier than others. There-fore at a eertaio age the testosterone levels of one breed will be different than another, meaning that a representative sample from all
breeds must be used as a starting point. For example, blood samples from a large number of animals should be taken at a eertaio time per i-ad or with a smaller number of animals, samples should be taken fre qu-ently per 24-hour period (e.g. every 15-20 min) (Hoffmann et al., 1986, 1987; Lacroix and Pelletier, 1979; Renaville et al., 1983; Sunby and Velle, 1980). With the once-a-day samples, it is possible that the animals from one herd show the same hormone profile; a so-called herd synchronization. The average values obtained by Hoffmann et al. (1986) and Renaville et al. (1983) point in this direction. Hoffmann and col-leagues found that in all breeds the lowest levels were reached at 15.00 hours, while Lacroix and Pelletier (1977) found these levels at ca. 12.00 hours and Renaville and colleagues between 9.00 and 12.00 hours. In contrary Hoffmann's group found that the highest values were reached between 9.00 and 12.00 hours, while Renaville's group found the highest values between 12.30 and 14.30 hours. In contrast to these
researchers, Karg et al. (1976) found two peaks in calves 20-40 weeks old, namely between 8.00 and 10.00 hours and between 14.00 and 16.00 hours. The reason for appearance of a peak at a eertaio time of the day is not yet known. Factors that can influence the release of testosterone in the blood are: time of sunrise; lengthof daylight; and the time of feeding and unrest such as appearance of farmer or stress which is possible when blood samples are taken. Along with these, the various authors list even more factors that can directly or indirectly (via influence of the time when sexual maturity is reached) be of influence. Bamberg et al. (1978) showed that testosterone levels in Fleckvieh did not rise as much during the first year as they did in Braunvieh. Giving more energy-rich feed did not result in higher
values. It was not possible to conclude from this report, however, if the bulls receiving the energy-poor feed ate more. This could mean that the difference in the energy intake between the two groups was
not very great. Feed is , however, mentioned as one of the factors that can influence hormone levels (Hemeida et al., 1985). It is doubtful if
this is true for West European circumstances. In extreme circumstan-ces, it will certainly have an influence upon the age when puberty begins. The influence of breed on the start of puberty is clearly illustrated by Lunstra et al. (1978). Various beef breeds and crosses of them were kept under the same conditlans with regard to feed, housing, care, etc. Significant diEferences in testasterene levels were found in theserum of calves in the age group 7-13 months. Season also has a definite influence on these levels (Hoffmann et al., 1986) and it is accepted that this is determined for the most part by the dark-light rhythm (i.e. daylight). Secchiari et al. (1976), however, could not actually prove that seasons had influence. Kozumplik (1981) mentioned the influence of sexual stimulation. The large individual differences, shown earlier in this report, are clearly shown in the experiments by Renaville et al. (1983) with double-muscled bulls. These animals were fed in the same way and kept in the same
conditions. They were divided into separate groups basedon the time of the testasterene peaks in the blood (130-140 etc. up to 190 and 191 days old and older). The animals in the first group had at an age of 130 days an average level of 1.5 ng/ml, while the 190-day group
reached this level at day 190. The low number of data for veal calves
show that testasterene levels lie in the same range as those found in breeding bull calves of the same age. Presently the veal calves are
kept langer befare slaughtering than those of which values are mentioned in Table 1.
In Table 2 (see also Appendix 2) the reader will find a summary of the most important analytica! results of a recently conducted ILOB/TNO ex-periment. It consistedof animals from experimental farms from the
campanles listed in the table and from 1~hich one of the authors (Be-rende) sampled blood, both on the farm and in the slaughterhouse. From one large herd of animals (Trouw Intern.), a large part of the samples were taken on the farm and a smaller part was collected from the same herd in the slaughterhouse but oot from the same animals. The levels of free estradiol-l7B, free testasterene and progesterone were measu
-red from this group. There was a large variatien found in the levels of testasterene in the individual male veal calves. Furthermore, a
blood samples taken· in the slaughterhouse and extremely high levels were not or seldom seen in this phase. A possible explanation for this could be that the animala are under great stress during transportation to and during the stay in the slaughterhouse. Stress is mentioned in the literature as one of the factors causing the fluctuation in testasterene levels. There is no mention, however, of the direction the levels are influenced. The authors think that the influence on the messurement of possible differences in testasterene levels from veneus blood or a mix of arterial and veneus blood is not very great.
Testasterene was demonstrated in blood plasma of female veal calves.
The levels were naturally much lower than these found in bull calves.
The average levels of the animals from two experimental farms were in the same range as these from breeding calves that were slightly older. Slightly higher levels were found in a few of the veal calves.
The experiments (25.30 and 25.040) conducted by ILOB showed that the average testasterene levels decreased after implantation of estradi-ol/testosterone. Four weeks after implantation the level had decreased to half that of day 0 (from ca. 1.0-1.7 to 0.4-0.7 ng/ml). The testos-terene level of the control group was 2.4 ng/ml four weeks after im-plantation in the experimental groups, with the highest and lewest va-lues being 6.0 and 0.2 ng/ml respectively.
ESTROGENS IN THE BLOOD
There ts less literature concerning estregens in male (breeding) calves than there is for androgens, even though male animals also produce estrogens.
Estregens are found in the blood in a free and a conjugated farm. Va-rious authors (Boursier et al. , 1983; Rattan and Riis, 1982; Richou-Bac et al., 1976; Silberzahn et al., 1982) found that levels of free estradiol-17P in blood plasma were less than 10 pg/ml. This agrees with the results of the ILOR experiments (25.25; 25.30; 25.35). In ILOB experiment 25.040 no free estradiol-17P could be measured in the blood plasma of male veal calves. These analyses were conducted using an HPLC/RIA technique with a detection limit of less than 10 pg/ml. This technique was also used in the previously mentioned pilot study, the data of which is shown in Appendix 2. All the free estradiol-17P
values for male veal calves were below the mentioned detection limit. In female veal calves only one animal had levels way above tltls limit (21 pg/ml). Since the levels of progesterone and testosterone were a
l-sa so high in this anima1, information was sought concerning beh
avio-ral, appearance and possible treatment with medicines. These factors could not explain the high hormone levels. Even the levels of older
breeding calves remained below the 10 pg/ml limit, except for two
ani-mals which were in heat on the day of sampling (16 pg/ml). In contrast
to the abovementioned authors, Schopper (1981) found levels above the
10 pg/ml limit in veal calves used in ILOB experiments. In his thesis
Schopper cited an average of 11 pg/ml with a range of 0-80 pg/ml. The
high levels were probably the result of an insufficient or omitted
prepurification. Richou-Bac et al. (1976) also found levels above the
100 pg/ml in a few percent of the blood samples taken from veal
calves. These samples were drawn from animals that were almost
certainly treated with anabolic compounds. If animals are treated with
a combination of anabolics , the chances are very good that the prepa -ration contains estradiol-17~. After treatment 1~ith a combination
preparation, for example estradiol/trenbolone acetate*, the level of
free estradiol-17~ is clearly higher. Hoffmann (1983) obtained values between 13 and 20 pg/ml. Richou-Bac et al. (1978), using the same pre
-paration, got much higher values, namely between 50 and 150 pg/ml. However , the levels measured in the control animals were also much
higher than those in Hoffman's experiment, namely between (30 and 60
pg/ml. In the !LOB experiments with male veal calves implanted with
estradiol/trenbolone at 11 weeks of age, tlte levels measured were not
as high (Table 4). The average levels in experiments 25.30, 25.039,
and 25.040 varied between 29-33, 20, and 15-43 pg/ml, 1, 3, and 5
*
All of the estradiol-containing implantation preparations used in Dutch, German, and French experiments contain free estradiol-17~. It will be clearly stated in this report when the ester form is used. Inthe rest of this paper the combination estradiol/trenbolone acetate is
given as estradiol/trenbolone. Furthermore, in the tables the
follo-~~ing abbreviations are used for implantation preparations: E=200 mg estradiol-173; E/Te=20 mg estradiol-17~/200 mg testosterone; E/Pr=20
mg estradiol-17~/200 mg progesterone; and E/Tr=20 mg estradiol-17~/140
weeks after implantatlon respectively. The levels measured in the con-trol animals were just a few pg/ml or were below the detection limit. There was , however, a large individual variation. This variatien can also be seen in the results from ILOB ex~eriment 25.25 (between 80 and 320 pg/ml) as determined by Schopper in samples taken a few weeks af-ter implantation. Higher levels were found by Beursier et al. (1983), 250 pg/ml six days after an intramuscular injection with estradial benzoate, while the levels of the control animals were just a few pg/ml. It is possible that the compounds are released in the blood
quicker after an injection than after implantation of tablets. After
reaching a higher maximum (shortly after injection), the levels should
then also decrease more quickly. lleitzman et al. (1979) reported low estradiol-17~ levels in female breeding calves. These levels were less than 10 pg/ml up to an age of 30 weeks. Trestment with
estradiol/trenbolone caused an abrupt increase in the levels with maximum levels between 110 and 170 pg/ml found four weeks after implantation.
A number of metabolites are found along with estradiol-17~ in various body fluids and tissues. Hoffmann (1983) and Schopper (1981) studied the conjugated and free farms of estradiol-17~; estradiol-17a and
estrone. This research was conducted in untreated and anabolically
treated veal calves. Five weeeks after trestment the following values (in pg/ml) were found.
Hoffmann Schopper
Control Treated* Control Treated** Estradiol-17~ free 13 20 19 78 conjugated 6 7 6 18 Estradiol-17a free 47 8 47 37 conjugated 87 157 87 273 Estron free 13 17 13 18 conjugated 8 80 8 37
*
Implantation with estradiol/trenboloneIn the ILOB experiment 25.039 the values for free and conjugated
estradiol-17~ were respectively (3 and 3 pg/ml for control animals and
32 and 12 pg/ml five weeks after estradiol/trenbolone implantation.
Meyer et al. (1985) measured the total steroid estragen level in the
blood of male and female veal calves 0-18 weeks old. They used
con-trols and animals treated with estradiol-containing preparations.
Their conclusion was that the total steroid estragen level in blood is
a mucl1 better standard than the levels found in the feces, because the
range in the levels found in the blood was smaller.
It is not actually possible to point out a single treated animal based
upon the total steroid estragen level in blood plasma. It is, however,
a fairly good indication that the calves have probably been treated if more animals in the herd are found with high levels ()1000 pg/ml
plasma).
PROGESTOGENS IN THE BLOOD
The most important (progestative) compounds are progesterone and, to a
lesser degree, 17a-hydroxyprogesterone. The most important metabolite
found in cattle is pregnandiol (Rico, 1983). It is also the most
im-portant metabolite of progesterone found in human endrocrinology.
Pregnantrial and etiocholanolone are also found. Very little has been
published about progesterone levels in male calves. There are,
howe-ver, publications available about progesterone levels in female
(bree-ding) calves. Based on their own research and on literature data,
Hoffmann and Karg (1975), Hoffmann et al . (1976) and HoEfman (1983) concluded that progesterone levels are below 0.1 ng/ml. Levels measu-red in comparative animals treated with progesterone 5-8 weeeks after
implantation varied between (0.1 and 0.5 ng/ml plasma (average 0.15
ng/ml). In fema1e veal calves (age unknown) the levels varied between
(0.1 and 0.35 ng/ml. In ILOB experiment 25.038 the progesterone levels
~o1ere measured in 18- and 22-~oleek-old male veal calves and found to be
0.05 and 0.08 ng/ml, respectively. The levels measured in groups of
animals implanted with estradiol/progesterone at 16 weeks of age were
0.23 and 0.28 ng/ml, respectively two and six weeks after implantati -on.
The levels in the blood in male and female veal calves measured in a recent ILOB/TNO study were on average higher than those found in the literature. In contrast to testasterene the levels were higher in bath
male and female veal calves in the samples taken in the slaughterhouse
(Table 2). The progesterone levels in plasma from breeding calves
(7-11 months old) agreed well with the expected levels (Table 3).
Af-ter the sampling the young cattle were controlled daily for 23 days
for signs of being in heat. The animals were kept in a loose house.
With regard to the measured and the expected values there were 2 e
x-ceptions, namely number 37 (in heat but not noted?) and number 38 that
should have shown low values 3 days after being in heat but did not.
ANDROGENS IN THE URINE AND FECES
Testesterene and epitestosterone (17a-testosterone) are excreted for
the most part via the feces (Rico et al., 1981; Schepper, 1981).
Schopper described an experiment with steers given testesterene where
90% of the hormone was excreted via the feces and 8.4% via the urine.
He also found that testesterene is excreted mostly in the free farm
via the feces. There is almast no literature results with regard to
veal calves . 17a-Testosterone and tetrahydrotestosterone have,
however, been measured in the urine of veal calves (Dept .
Biopharma-ceutical Analysis, RIKILT).
ESTROGENS IN THE URINE AND FECES
As in the blood, free and conjugated farms of estregens are found in
the urine and feces. The most important compounds are estradiol-17a,
estradiol-17B, estrone and estriol. According to Velle (1963, 1972),
estradiol-17a found in the urine of ruminants is quantitatively the
most important metabolite. Bouffault and Willemart (1987) have also
found this to be true for veal calves . Van Erb et al. (1977) showed
that at least 70% of the total amount of estregens found in the urine
of adult milk cows consists of estradiol-17a. These results agree with
these of ILOB experiment 25.34 using male veal calves. This experiment
showed the following results: estrone 15%; estradiol-17B 5%; and the
rest (80%) was probably estradiol-17a. The percentages found in
ani-mals implanted with estradiol/testosterone were not much different:
16.5%, 7.5% and 76%, respectively. Rico (1983) found that 30-50% of
the abovementioned estregens measured in the urine were in the
estradial was implanted in young calves, about 90% of the total acti-vity in the urine appeared as unconjugated steraids (Riis and Suresh, 1977). These results do not agree with those of the experiment conduc-ted by ILOB/TNO using steers and heifers (Arts, unpublished results). In these experiments no free estradiol-17B was found in the urine of the controls or in that of animals treated with estradiol/trenbolone. Free estradiol-17a was also not found in the controls, but could only be measured in the urine of treated animals which had a very highly conjugated estradiol-17a level.
Estrogeos are generally excreted via the feces in the free form (Vel-Ie, 1976). Research conducted by Huis in ' t Veld et al. (1976) using (only 2) veal calves also found this. According to Velle (1963) excretion via feces is quantitatively as important as that via the urine in ruminants. Huis in ' t Veld et al. (1976) reported that
estragen excretion via the urine is quantitatively the most important in veal calves, even though large differences can occur from one animal to the next. In a few animals estragen excretion via the feces was almast as high as via the urine. Bouffault and Willemart (1987)
also reported the irregular excretion of estradiol-17a via the urine in veal calves, i.e. large variations per and between the individuals. Riis and Suresh (1977) treated 6-12-\>leek-old calves \-lith radiolabeled estradiol. One-third of the labeled material was excreted via the urine and two-third via the feces.
In experiments with steers injected with radiolabeled estradiol., Ivie et al. (1986) found that 42% of the compound was excreted via the uri-ne and 58% via the feces. In ILOB experiment 25.32 the urine and feces were tested for estradiol-17B only. The feces of untreated male beef calves had 3 times as much estradiol-17B than the urine (average 0.17 and 0.05 ng/ml, respectively).
Karg's group in Munich (Meyer et al., 1985) also conducted experiments on the hormone levels in feces. Based upon the analysis results from 799 samples of feces from 0-18-week-old veal calves (male and female), this group conc1uded that the total steroid estragen level is not a good standard by which to test for illegal use of estrogen-containing preparations. They found that this level was raised in the first few weeks after treatment, but that there were also controls with high steroid estragen levels. The presence of high values (e.g. )300 ng/g
feces) in more than 5% of a herd can be used as a possible standard to
test for illegal treatment . The values from one individual cannot be
used as a basis for judgement. It is noteworthy, however, that the
me-conium of newborn calves contains very high estragen levels (15,800-67,000 ng/g).
The levels of the different horrnanes in the urines may vary depending
on how the sample was taken. In general, there are 3 types of urine
samples: (a) samples acquired when urine is being passed
(spontaneous-ly passed urine) that is produced at a eertaio time of day or taken
from the bladder at the slaughterhouse; (b) samples taken from an
amount of urine produced during 12 or 24 hours. The animals must stand
on balance cages for this type of sampling; and (c) samples taken
di-rectly from the bladder of the animals. This last sample is obtained
via a bladder catheter. According to Vogt et al. (1972) and Huis in ' t
Veld and Roth-Hiedema (1974) the varlation in estragen levels from day
to day is greater in the samples obtained from spontaneously passed
urine than in the 24-hour samples. The range in the spontaneous
sam-ples could be reduced if the levels were related to the level of the
excreted creatinine. RIVH research (Stephany, unpublished data) shmo~ed
that this frequently gives unsatisfactory results, partially because
of the instability of creatinine in bovine urine (see also Appendix 1).
Tables 5-10 show the results of estragen research on veal calf urine.
These data refer to research conducted on untreated and anabolically
treated male veal calves 11-17 weeks old. Even though there were
simi-larities in age, feed, and housing, the results cannot be compared.
The results in Tables 6-8 and 10 are based upon fluorimetric determi
-nations, while the data in the other tables were obtained using a RIA.
Despite the fact that the results in Tables 5 and 9 were achieved with
the same analytica! technique, they differ by a factor 10.
Bouffault and Willemart (1987), based upon their research shown in
Ta-ble 5, reached the conclusion that the long-accepted maximum level of
10 ng/ml total steroid estrogens in control animal urine is toa high.
At three time periods, one control even exceeded 20 ng/ml for es
tradi-ol-17a. Schopper's (1981) research did nat show this and in the rese
-arch by Huis in ' t Veld and Both-Hiedema (1974) the 10 ng/ml level was seldom exceeded.
Based upon a large number of (ILOB) experiments, the latter authors reached their average total steroid estragen levels of 2.1, 3.9, and
5.0 ng/ml in male veal calves 11, 14, and 17 weeks old, respectively. The varlation was great, even in the 24-hour urine, ranging from 2 to
15 ng/ml, with a tendency toward the higher levels in older animals.
The steroid estragen levels from a number of ether ILOB experiments
were also measured (Table 1 in Appendix 1). This table shows the
ave-rage levels found in untreated animals of the control groups. Then the
levels of the groups treated with anabolica, the averages and range, were the same as the control levels. The average, minimum and maximum levels for these "untreated" male veal calves Here respectively 9.5, 2.3, and 15.9 ng/ml. These values are higher than these given above by Huis in ' t Veld, possibly because the last-mentioned levels were
mea-sured years later.
Up to no~o~ the estragen levels in urine from "untreated" control calves have been discussed. Tables 6-10 give the values from animals treated with anabolics. The total steroid estragen level is clearly raised du-ring the first few weeks after treatment. After implantation of
estra-diol/testosterone, estradiol/progesterone, and estradial alone, the total steroid estragen is significantly raised, especially in the
first 2-3 weeks after implantation. Then a rapid decrease is seen in the levels found in untreated animals. Implantation with estradi-ol/trenbolone causes lower peak levels, but the levels remain higher langer than control levels. This points to a more gradual release of estradial from the pellets, possibly in combination with a lowered me-tabolism.
The results from Huis in ' t Veld and Both-Miedema (1974) also show this trend. Other experiments by these authors, not shown in the
Ap-pendix tables, gave the follmo~ing results (ILOR experiments):
Tot al steroid estragen levels (ng/ml)
Days after trestment 3 21 40
estradial 5-40 <10 <10
estradiol/testosterone 5-75 1-70 <10
estradiol/progesterone 5-40 (10
In another ILOB experiment where 24-hour urine was tested, the highest values (ca. 15.5 ng/ml) were measured ca. 7 days after estradial
im-plantation. The levels for the groups implanted with estradiol/
testasterene and estradiol/trenbolone '~ere 93-133 ng/ml 7 days after
implantation and 27-35 ng/ml 21 days after implantation, respectively. The latter results are approximately the same as the average values
found for female animals in ILOB experiment 25.20, namely 6, 15, 20, and 15 ng/ml 0, 2, 3, and 4 weeks after implantation with estradiol/-trenbolone. The average (minimum and maximum) levels found in these animals at 17 weeks of age, 6 weeks after implantation were: placebo 2
(1-2) ng/ml; estradial 2(1-3) ng/ml; estradiol/progesterone 4.5 (1-11) ng/ml; estradiol/testosterone 4 (1-14) ng/ml; and estradiol/trenbolone
11 (1-26) ng/ml. These values coincide with those obtained from ILOB experiments conducted in the early 1980's (Table 2 in Appendix 1 and
Tables 7 and 8). In the experiments shown in Table 2 of Appendix 1, the animals were implanted with estradiol/testosterone and
estradiol/progesterone. About 6 weeks after implantation the urine was tested for steroid estrogen. The average, minimum and maximum levels of total steroid estragen were 20.4, 9.2 and 28.3 ng/ml, respectively.
Despite the distinct increase, especially the first few weeks after treatment, Bouffault and Willemart (1987) stated that the
estradiol-17a level in the urine (and thus the total steroid estragen
level) was not a good standard to use for testing for the use of
estrogen-containing preparations. These authors based their conclusion
on research conducted on urine collected 6 and more weeks after
implantation. This resembles the results of other researchers. One exception has to he made for the implantation of estradlol/trenbolone;
this combination shows higher levels in ehe urine langer than 6 weeks
after treatment.
The above inforrnation proves that the total steroid estragen level is
not a good standard in all circumstances for testing the presence of estragen compounds. For this reason, other researchers have also tested the estradiol-17B level in urine. Schopper (1981) found that the average levels from ILOB experiments on untreated male veal calves 11-18 week old were: for an experiment with 48 analyses 1.9 ng/ml;
with 68 analyses 1.0 ng/ml; and with 40 analyses 1.3 ng/ml. These
directly after feeding. Hoffman et al. (1976) also found a great
variatien in estradiol-17~ levels in untreated female veal calves. The values acquired using a RIA varled from 4.6 to 14.3 ng/ml and from 2.4 to 12.6 ng/ml for animals 12 and 17 weeks old, respectively. The sam-ples were taken from spontaneously passed urine. These values resemble
those found by Kroes et al. (1977) from female calves in ILOB experi-ment 25.20. They were obtained fluorimetrically from samples of spon-taneously passed urine and varled between 1 and 8 ng/ml for 11- and
14-~-Teek-old animals. The reasen ~o~hy estradiol-17(3 is determined instead of estradiol-17a is that it is assumed that exogenously administered estradiol-17(3 is not or hardly metabolized into
estradiol-17a in the animal and that estradiol-17(3 as such is passed in the urine. The levels in both male and young female veal calves were low: (0.1 ng/ml (Boursier et al., 1983); 0.156 (0.111-0.199) ng/ml (Richou-Bac et al., 1979); 0.05 (0.03-0.08) ng/ml (ILOB) and always (5 ng/ml (Bouffault and Willemart 1987).
The estradiol-17(3 level was clearly higher after trestment with an estradiol-17(3-containing preparatien in an experiment conducted by Bouffault and Willemart (Table 5). The experiment conducted by Beur-sier et al. (1983) also showed this increase after an injection with estradiol-17(3. After 6 days the level varled between 12 and 20 ng/ml and after 43 days it was still 0.2 ng/ml (control (0.1 ng/ml). Forty-three days after implantation the estradiol-17a level was equal to that of the controls. This is in contrast to the findings of Richou-Bac et al. (1979) who showed that the estradiol-17(3 level was approxi-mately equal to that of the controls three weeks after implantation.
Even ten days after implantatlon, the estradiol-17(3 levels were alrea-dy not sifnificantly different Erom those of the controls, though they were higher (0.4 and 0.2 ng/ml, respectively).
PROGESTOGENS IN THE FECES AND URINE
Progesterone is excreted in the feces by ruminants mainly in the form of pregnandiol (Rico et al., 1981; Rico, 1983). As far as the authors
kno~-1, there is no literature covering the excretion of progesterone and its metabolites in the feces and urine in veal calves.
SUMNARY
Before summing up the information on the levels of testosterone,
estrogens and progesterone in blood, urine and feces, the following must be mentioned. Besides the large individual variatlons in these
levels, there are factors which can influence the levels. Three of them are: (a) method of analysis \oJith e.g. manoer of pre-purification, possibly pre-concentration and specificity of the measurement; (b) m
e-thod of sample-taking: frequency, site where blood is drawn (carotid
vein, tail vein, arterial blood), 24-hour urine and feces or a
one-time sample, amount of stress during sample-taking, sample-taking in the stall or in the slaughterhouse; and (c) transportation and
sto-rage conditions of sample material.
Testosterone in blood
In general, the level slowly increases in young bulls to the age of 4
months (<1 ng/ml). From 4-7 months of age the level increases more rapidly (1-4 ng/ml) and in bulls older than 7 months it varies much (1-10 ng/ml and higher). In male calves the release of testosterone
into the blood occurs in pulsatile peaks. Because of this a large varlation cao be seen in the levels in a 24-hour period. This has even
been seen in 20-week-old animals (Karg 1976).
Trestment with testosterone-containing preparations usually causes a
decrease in the testosterone level. Because of the large varlation of
testosterone levels in the blood of untreated veal calves, it is im-possible to show a clear, significant change in this level after
treatment with testosterone-containing preparations . For example the
ratio testosterone and epitestosterone can be used for treatment
con-trol with testosteron as is now used in sports world. More research is
needed on fema1e calves to sho\oJ if there is a change in testosterone levels in the blood after administration of testosterone.
Estrogeos in blood
The most import (quantitatively) estrogens in the blood are estradi-ol-17~, estradiol-17a, estriol and estrone. These estrogens appear in both free and conjugated forms . Most of the research has been conduc-ted on estradiol-17~ and the levels that have been found were usually
less than 10 pg/ml. The level of estradiol-17a might be somewhat
higher. The estradiol-17P concentration in the blood can possibly be
used as a parameter to test for the use of estradiol-containing
prepa-ratlans in male and possibly in female calves. This is especially true
for animals examined on the farm; that is the first weeks after
treat-ment. The total steroid estragen level is less suitable for testing
individual animals treated with anabolics. To test suspected herds,
however, this level does seem suitable, at least in the first weeks
after treatment .
Progesterone in the blood
According to the literature results, these levels should be below 100
pg/ml for male veal calves and between (100 and 350 pg/ml for female
veal calves. A recent !LOB/TNO experiment has put the progesterone
level in male veal calves at 10-675 pg/ml. For female veal calves this
level varied between 25 and 2400 pg/ml. Implantation with a
progeste-rone-containing preparation showed a distinct increase the first few
weeks after treatment. There are as yet insufficient data available to
state the length of time after treatment in which statistically signi
-ficant increases can still be shown. There are also insufficient data
available on progesterone levels in the blood to test a suspect
animal; not only on the farm but also in the slaughterhouse.
Testosterone in the urine and feces
BAt the moment there are no literature data available on testosterone
levels in urine and feces of veal calves. lt is possible that the
testosterone concentration in urine and/or feces or the relationship
between the concentration of testosterone and, for example,
epitesto-sterone in urine and/or feces are good parameters for showing that
treatment has been given.
Estrogeos in the urine and feces
There are four types of estrogens found in these matrices : estradiol
-17a; estradiol-17P; estrone; and estriol. Of these estradiol-17a is
quantitatively the most important in cattle. All four compounds occur
opinions with regard to the total steroid excretion in cattle. Certain authors state that quantitatively more is excreted via the feces, while ethers claim that more leave the body via the urine.
Much research has been conducted on the influence of implanting
estro-gen-containing preparations on the total steroid estragen excreted via
the urine of veal calves. Most researchers report that in male veal
calves levels less than 10 ng/ml have been found in untreated animals.
Older untreated female veal calves have somewhat higher levels and
sho'~ more varlation in levels. Implantation of estrogen-containing
preparations causes distinctly higher steroid estragen levels in male
calves during the first few weeks after treatment. The amount of in-crease and the length of time in which this increase can be seen is
dependent on for example the compounds and carriers used and the
me-thod of administration. It is possible that measuring the amount of estradiol-17B in the urine and in the feces in the first few weeks af-ter treatment could he used to track down suspect animals. With enough
tested animals (5%?) from one herd, the total steroid estragen level
could he used in the first few weeks after treatment.
Progesterone in the urine and feces
As far as is known, there are no literature data available on veal calves.
REFERENCES
Bamberg, E- Kanout, A.G. , StHckl,
w.
Ceska, M. und Choi, H.S.Gehalt an Testosteron und Luteinisierungshormon im Blutplasma von Stieren während des ersten Lebensjahres.
Zbl. Vet. Med. A 25 (1978) 773-780.
Basn , J.J. Peterson, A.J. and Payne, E.
Plasma concentrations of luteinizing hormone, testasterene and andro-stenedione in castrated and adrenalectomized bull calves.
J, Endocr. 79 (1978) 137-138.
Bass, J.J., Peterson, A.J. , Payne, E. and Jarnet, M.P.
The effect of neonatal estragen treatment on plasma hormone levels and
behaviour in pre- and post-pubertal bulls. Theriogenology 8 (1977) 59-71.
Bouffault, J.C. and Willemart, J.P.
Excrétion urinaire d'estradiol après différents traitements anaboli-sants. Bull. Acad. Vét. de France 60 (1987) 81-87.
Boursier, B., Ledoux, M. et Richou-Bac, L.
Teneurs plasmatiques et urinaires en estradial 17a et en estradial 17~
chez le veau traité au benzoate d'estradiol (Applications au
contro-le).
Réc. Méd. Vét. 159 (1983) 419-423.
Erb, R.E., Chew, B.P. and Keller, H.F.
Relative concentrations of estragen and progesterone in milk and
blood, and excretion of estragen in urine.
J. Anim. Sci. 46 (1977) 617-626.
Heitzman, R.J., Harwood, D.J., Kay, R.M., Little,
w.,
Mallinson, C.B.and Rcynolds, I.P.
Effects of implanting prepubertal dairing heifers with anabolic
ste-raids on hormonal studies, puberty and parturition.
J, Anim. Sci. 48 (1979) 859-866.
Hemeida, N.A., El-Baghdady, Y.R. and El-Fadaly, M.A.
Serum profiles of androstenedione, testasterene and LH from birth through puberty in buffalo bull calves.
J, Reprod. Fert. 74 (1985) 311-316.
Hoffmann, B.
Natural occurrence of steraids horrnanes in food producing animals.
In: Public health aspects, analytica! methods and regulation.
Symposium O&E, Paris 15-17 February 1983. Hoffmann, B., and Karg, H.
Metabolic fate en anabolic agents in treated animals and residue levels in their meat.
Anabolic Agents in Animal Production. FAO/WHO Symp. Rome, Harch 1975.
Hoffmann, B . . Heinritzi, K.H., Kyrein, H.J. Oehrle, K.L. Oettle, G., Rattenberger, E. , Vogt, K und Karg, H.
Untersuchungen ilber Hormonkonzentratlonen in Geweben, Plasma und Urin von Mastkilbern nach Behandlung mit hormonenwerksamen Wirkstaffe An
a-bolika. Beihefte
z.
Tierphys. Tierernähr und Futtermittelkunde, (1976) 80-90.Hoffmann, S., Leuthold, G. und Reinecke, P.
Untersuchungen zur Eignung der Plasmakonzentrationen von Testosteron, Insulin und der Schilddrilsenhormone als Hilfsmerkmal filr die
Selection auf Fleischleistung heim Rind.
1. Mitt. Die Abhängigkeit der Plasrnahormonkonzentrationen von inte r-nen und externen Faktoren.
Arch. Tierz. , Berlin 29 (1986) 553-564.
Hoffmann,
s.,
Leuthold, G. und Reinecke, P.Untersuchungen zur Eignung der Plasmakonzentrationen von Testosteron, Insulin und der Schilddrilsenhormone als Hilfsmerkmal filr dle Selection
auf Fleisch1eitung beim Rind.
2. Mitt. Die Beziehungen zwischen den Plasmahormonkonzentrationen und Leistungsmerkmalen.
Arch. Tierz., Berlin 30 (1987) 223-232.
Huis in ' t Veld, L.G., Both-Miedema, R. , Schuiler, P.L.,
Stephany, R.W. Bosch. D. van de en Kroes, R.
Onderzoek naar de invloed van combinatiepreparaten van mannelijke en
vrouwelijke hormonen op mannelijke mestkalveren. (Een biologisch, che-misch en histologisch onderzoek). RIV-rapport nr. 18/73 Path,
16 april 1973.
Huis in ' t Veld, L.G. en Both-Miedema, R.
Onderzoek over het verloop van de uitscheiding van steraidoestrogenen
in de urine van kalveren, welke behandeld zijn met oestradiol-17B of oestradiol-17B bevattende combinatlepreparaten.
Tijdschr. Diergen. 99 (1974) 155-165.
Huis in ' t Veld, L.G., Both-Miedema, R., Smit, P.J. en
Stekelenburg, P.
Opsporing van exogene estrogenen bij levende dieren en bij slachting. Berichten uit het RIV, Utrecht (1975) Versl. Rapp. (1976) 183-187. Huis in ' t Veld, L.G., Berg, R.H. van de, Wieten C.A. en
Kroonenberg, W.M.
Radio-immunologische bepaling van estradio1-17B in bloedserum, muscu
-latuur en organen van onbehandelde runderen en van runderen welke met
estradlol-17B (al of niet in combinatie met andere hormonale stoffen)
zijn behandeld. RIV-rapport nr. 152/76 Endo, september 1976.
ILOB proef 25.22
Jong, J. de. Intern verslag December 1980. ILOB proef 25.25
ILOR proef 25.30 Intern verslag 1981. ILOB proef 25.35
Internverslag 1983-12··13.
!LOB proeven 25.30. 25-32, 25.34.
Vries P.H.U. Ontwikkeling van bepalingsmethoden voor enkele anabool werkende verbindingen in dierlijke matrices.
ILOJ3-rapport 510 (1982·-l0-12). ILOB proef 25-38
Intern verslag 22-5-1985. ILOB proef 25.039.
Arts , C.J.M., Baak. M.J. van, Huisman, J. en Weerden, E.J. van. Onder-zoek met getmplanteerd oestradiol/trenbolon bij mannelijke vleeskalve-ren.
-Effecten op groei en voederconversie.
- Residue niveau's.
ILOB-rapport 579 (20 oktober 1986).
ILOB proef 25.040 (vertrou1o1elijke gegevens van C.J.M. Arts). Nog niet in rapport vastgelegd. Wordt gepubliceerd.
Ivie, G.W., Christopher,
1ftJ., Munger, C.E. and Coppock, C.E.
Fate and residues of [4- ] estradiol-17B after intramuscular
injec-tion into Holstein steer calves. J. Anim. Sci. 62 (1986) 681-690.
Karg, H. Giménez, T., Hartl, M., Hoffmann, 8., Schallenberger, E. and
Schams,
o.
Testosterone, Luteinizing hormone (LH) and follicle stiualating
hormone (FSH) in peripheral plasma of bulls: levels from birth through puberty and short term varitions.
Zbl. Vet. Med. A 23 (1976) 793-803. Kozumplik, J.
The level of plasma testasterene during the prenatal and postoatal period of development in bulls.
Acta Vet., Brno. 50 (1961) 27-32.
Kroes, R., Huis in ' t Veld, L.G., Stephany, R.\-1., Berende, P.L.H. en
Terluin,
R.w
.
1 De invloed van anabole stoffen op vrouwelijke mestkalveren (een zo8
-technisch en histologisch onderzoek).
2 Bepaling van het uitscheidingspatroon van zeranol bij mannelijke
kalveren.
RIV-rapport no 88/77 Path (1977).
Kroes, R., Huis in ' t Veld, L.G. en Stephany, R.W.
Onder zoek naar de invloed van combinatie preparaten van mannelijke en vromlelijke hormonen of hormonaal lolerkende stoffen op mannelijke m
est-kalveren (!LOB-proef 25.14, P 72/82) (Een biologisch, chemisch en histologisch onderzoek). RIV-rapport nr. 193/76 Path (1976).
Kroes, R. en Huis in ' t Veld, L.G.
Onderzoek naar de invloed van combinatie-preparaten van mannelijke en
vrouwelijke hormonen of hormonaal werkende stoffen op mannelijke
mest-kalveren (!LOR-proef 25.16, P 72/83) (Een biologisch, chemisch en
histologisch onderzoek). RIV-rapport nr. 194/76 Path (1976).
Kroes, R., Huis in 't Veld, L.G. , Berg, R.H. van de, Both-Miedema, R. en Stephany, R.W.
Onderzoek naar de invloed van het combinatie-preparaat 17B
oestra-diol/trenbolone acetaat op mannelijke mestkalveren, welke een
ver-schillend eiwitrantsoen ontvingen (ILOB-proef 25.22). RIV-rapport nr.
213/76 Path (1976).
Kroes R., Huis in ' t Veld, L.G. en Both-Niedema, R.
Onderzoek naar het effect van het toedienen van het
combinatie-prepa-raat 17B-oestradiol en trenbolone acetaat op mestkalveren gevoederd
met verschillende eiwitrantsoenen (!LOB-proeven 25.18, 25.19 en 61.05)
(Een histologisch en chemisch onderzoek). RIV-rapport nr. 199/76 Path
(1976).
Kroes . R., Huis in ' t Veld, L.G. en Both-Niedema, R.
Onderzoek naar de invloed van het combinatie-preparaat 17B
oestra-diol/trenbolone acetaat op mannelijke mestkalveren (!LOB-proef 25.21,
P 74/58) (Een histologisch en chemisch onderzoek). RIV-rapport nr.
214/76 Path (1976).
Lacroix, A. and Pelletier, J,
Short-term variations in plasma LH and testasterene in bull calves from birth to 1 year of age.
J, Reprod. Fert. 55 (1979) 81-85.
Lacroix, A., Garnier, D.H. and Pelletier, J,
Temporal fluctuations of plasma LH and testasterene in Charolais bull
calves during the first year of life.
Ann. Biol. anim. Bioch. Biophys. 17 (1977) 1013-1019.
Lunstra, D.D., Ford, J,J, and Echternkamp, T.E.
Pubertyin beef bulls: Hormone concentrations, growth, testicular de
-velopment . sperm production and sexual aggressiveness in bulls of
dif-ferent breeds.
J, Anim. Sci. 46 (1978) 1054-1062.
Meyer, H.H.D., Herges, C., Landwehr M. und Karg, H.
M8glichkeiten der Erkennung des illegalen Einsatzes von Ostradiol
benzaat in der Kälbermast durch Steroid-östrogenbestimmung in Kot und
Blutplasma.
Arch. Lebensmittelhyg. 36 (1985) 27-31.
Pellegrini,
s
.
,
Secchiari, P., Fassorra, C. , Levanti, C. and Luisi, M.The determination of testasterene in the plasma of calves by
radio-immoassay.
Rattan, P.J.S. and Riis , P.M.
Influence of estradial and trienbolene acetate treatments on the circulating level of plasma estradial in Jersey calves.
J. Nuclear Agrlc. Biol. 11 (1982) 63-65.
Renavillea, R., Fabry, J., Halleux, V. and Buarny, A.
Testosterone plasma proflles, as a function of age in young bulls from the bovine double-muscled belgian white blue breed. A preliminary report.
Theriogenology 19 (1983) 159-168.
Richou-Bac, L . . Pochard, M. L. et Boursier, B.
Niveaux plasmatiques et tlssulaires en estradial 17 B, estrone et
D.E.S. chez des jeunes bovins traitês aux anabolisants. Aspects
zoo-techniques et qualitatifs (1). Rec. Mêd. Vêt. 154 (1978) 441-450.
Richou-Bac, L., Mollei, M.F., Boursier, B. et Cumont, G.
Recherche des oestrogènes par radioimmunologie dans le plasma et les tissus des jeunes bovins. Premiers Resultats.
Bull. Acad. Vét. de France 49 (1976) 409-415. Richou-Bac, L., Pochard, M.F. et Boursier, B.
Evolution des teneurs en estradial 17B du muscle, du foie, de l'urine
de veaux traités au benzoate d'estradiol par injection intramusculai-re.
Bull. Acad. Vét. de France 52 (1979) 173-180.
Rico, A.G.
Metabolism of endogenous and exogenous anabolic agents in cattle.
J. Anim. Sci. 57 (1983) 226-232.
Rico, A.G., Burgat-Sacaze,
v.,
Braun, J.P. and Benard, P.Metabolism of endogenous and exogenous anabolic agents in cattle. In: Anabolic agents in beef and veal production. Proc. EC Workshop,
Brussels , March 5th and 6th. (1981) pp. 45-56.
Riis, P.M. and Suresh, T.P.
The effect of a synthetic steroid (trenbolone) on the rate of release
and excretlon of subcutaneously administered estradial in calves. Steraids 27 (1977) 5-15.
Schams, D., Gombe, S., Schallenberger, E., Reinhardt, V. and Claus, R. Relationships between short- term variations of LH, FSH, prolactio and testosterone in peripheral plasma of prepubertal bulls.
J. Reprod. Fert. 54 (1978) 145-148. Schopper, D.
Messung von RUckständen von Ostradiol-17B, Testosteron und Progesteron im Plasma und Telh<~eise im Urin von ~1ast rindern nach Anabolika~ehand
lung sowie Untersuchungen zum Stoffwechsel von Trenbolonacetat . Dissertation (1981), München.
Secchiari,P., Martorana, F., Pellegrini,
s
.
and Luisi, M.Varlation of plasma testosterone in developing friesian bulls.
J, Anirn. Sci. 42 (1976) 405-409.
Silberzahn, P., Moreau, J., Chauvet, P., Dehennin, L. et Scholier, R.
L'estradiol-17B plasmatique chez le veau traité par les anabolisants
mesure par dosage radio-immunologique et par spectrométrie de masse.
Rec. Méd. Vét. 158 (1982) 529-534.
Sundby, A. and Velle, W.
Plasma concentratien of testasterene in young bulls in relation to
age, rate of weight gain and stimulation with human chorionic
gonadotrophin.
J, Endocr. 86 (1980) 465-469.
Thibier, ~1.
Peripheral plasma testasterene concentratien in bulls around puberty.
J. Reprod. Fert. 42 (1975) 567-569.
Thibier, H.
Diurnal testasterene and 17a-hydroxyprogesterone in peripheral plasma
of young post-pubertal bulls. A study by frequent sampling.
Acta Endocr. 81 (1976) 623-634.
Velle,
w.
Metabolism of estrogenic horrnanes in dornestic animals.
Gen. and Compar. Endocr. 3 (1963) 621-635.
Velle, W.
Levels and metabolisrn of cestragenie horrnanes in dornestic animals.
Exposée d'une journée d'étude consacrée à l'utilisation des
oestro-gènes en élevage.
Paris 14 Mai 1972, 31-36.
Vogt, K., Waldschmidt, H. und Karg, H.
Bestirnmung von Ausscheidungsverlauf und Rückständen von Ostradiol beim
Kalb nach intramuskulärer bzw. subcutaner Implantation von Presslingen
mit entsprechende Ostrogengehalt.
Arch. Lebensmittelhyg. 23 (1972) 70-76.
Zerobin, K. and Thun, R.
Testosteron beim wachsenden Stierkalb.
Wien. Tierärtzl. Mschr. 70 (1983) 198-202.
Table 1: Average testosterone levels (ng/ml) in blood plasma of young bulls Author (year) Bamberg et al. (1978) Bass et al. (1978) Bass et al. (1977) Hoffmann et al. (1986, 1987) Karg et al. (1976) Kozumplik (1981)
Lacroix and Pelletier (1979) Lacroix et al. (1977)
Lunstra et al. (1978)
Renaville et al. (1983) Secchiari et al. (1976) Sunby and Velle (1980) Zerobin and Thun (1983)
Hoffmann et al. (1976)b ILOB (exp. 25.3Q)b ILOB (exp. 25.35)b ILOB (exp. 25.040)b 1 2 ±
o
.
s
1.1 1.5 0.1 0.1 ± 0.3 ± 0.3 0.32 0.31 0.26 ± 0.2 ± 0.2 0.1 0.2 0.60 0.60a Average in 7-13 month period: 6.65 ng/ml b Veal calves
c In the period 11-17 weeks old
d In the period 15-19 weeks old e In the period 17-22 weeks old
table.1 Age in months 3 4 5 6 ± 1.0 ± 2.0 ± 2.0 ± 3.0 1.8 2.0 3.0 2.0 0.2 0.4 5.5 2.5 4.4 ± 0.3 ±
o.s
±o.s
1-2.5 0.79 0.43 0.91 2.32 1.19 ± 0.3 ± 0.6 ± 1.2 ± 1.8 0.85 1.04 1.68 0.3 0.6 1.0 0.9 0.9 2.0 2.8 4.0 0. 50-0. 90c 1l
l
I
0.41-o.sd 1.5-2.4e 7 8 ± 4.0 ± 4.5 2.0 1.3 4.9 5.2 1-3 2-4 0.50-26.53 ida 0.47 ± 1.2 ± 2.2 4.3 4.1 0.8 1.8 4.8 5.9I
I
9 ±s
.
o
1.5 5.2 2-4 ida 0.70 ± 3.0 5.2 3.5 4.8I
Number of samples takenf 39 5 5 320g 12g 57 96g 10 62 2610h 18 574i 48j 5 4 6sok
f Approximate number of samples taken upon which the levels were based (no. animals x no. per animal)
g 4 animals h 29 animals i 82 animals j 6 animals k 9 animals
I
I I I IOrigin of the animals Sex Age (wks) Number Progesterone level Testosterone level
(Company) average lowest-highest average lowest-highest
Trouw Intern. (farm) male 22 75 39 10 - 118 2812 141 - 9239
Trouw Intern.(slaughter- male 23 24 237 53 - 679 747 210 - 6961
house)
Denkavit (slaugtherhouse) female 26 38 370 46 - 2412 36 12 - 262
Navobi (farm) female 24.5 35 62 28 - 339 22 1 - 70
I
- - - -
Table 3: Progesterone and testosterone levels (pg/ml) in HF young cattle
between ca. 30 and ca. 50 weeks of age
Probably not in oestrus Unsure
1 Indivi- Age Proges- Testos- Indivi- Age Proges- Testos-
Indivi-I
dual terone terone dual terone terone dualnumber level level number level level number
9 29 190 16 18 37 91 23 35 l3 30 45 20 8 37 72 22 22 ll 30 84 18 16 38 66 18 23 17 31 169 18 15 38 88 25 5 1 31 90 24 26 38 62 36 28 2 31 60 22 3 38 93 24 25 4 32 93 18 14 38 43 18 38 19 34 72 37 12 38 53 26 21VV 7 34 155 21 36 39 51 21 34VV 10 39 67 16 24 6 39 188 18 40 31 40 43 46 20 27 40 43 31 30 33 41 58 26 39 42 46 13 37 45 3463 24 29 45 83 28 32 46 569 29 Av.level 106 22 288 25 Min.-max. 60-190 16-37 43-3463 13-46 ~eve_!_ __ --- - - - -- -- -- - - -~--- - - --- --- - - - --- - - --- ~ table.3 In oestrus
Days Age Pro ges-
Testos-around terone terone
oestrus level level
-12 38 50 35 + 4 39 0 l l - 5 40 434 17 + 5 40 109 13 -13 41 136 28 -10 41 4115 14 + 3 42 3775 22 0 42 326 26 0 44 327 38 -12 44 83 19 I -12 46 3114 22 i - 2 46 272 19 -12 56 4887 29 1356 23 0-4887 ll-38
Experiment Experiment Experiment
Weeks 25.23C 25.30 25.35
af ter
treatmentb Controld E Control E/Tr E/Te Control E/Te
0 2 6 25 13 18 1 2 83 10 33 48 4 29 2 2 220 3 38 38 3 2 135 n.d. 20 20 2 l l 4 2 85 5 2 32 5 43 25 1 l l 6 2 14 7 65 18 7 2 4 ~ -
-a Respectively 6, 4, 6, 6, 9 animals per experimental group b Implantation at ca. 11 weeks of age
c Analyzed by Dr. Schopper (TU-München) d Average value of 64 samples
e 3 days f 10 days g 33 days n.d. Not detectable table.4 Experiment Experiment 25.039 25.040
Control E/Tr Control E/Te
n.d. n.d. n.d. l l n.d. 33e n.d. 39f n.d. 20 n.d. 20 n.d. 19 n.d. 32 n.d. 15g n.d.
-n.d. 35 n.d. - - - --- --- - - -I ITable 5: Average estradiol-17B and estradiol-17a levels (ng/ml) in the urine of male veal calves in the experiment conducted by Bouffault and Willemart
Age 8 week sa 12 weeks
Controle E/Pr E/Tr Control E/Pr E/Tr Control
estradiol-17B 0.38 2.54 0.37b 0.64 1. 81 2.21 0.63
estradiol-17a 8.72 36.52 4. nb 10.68 30.41 49.79 11.78
a E/Pr implanted at 4 weeks of age, E/Tr implanted at 8 weeks of age
b Considered as control group at this time period
c 4 Animals per group
17 ~'leeks
E/Pr 0.97 12.99
Table 6: Average total steroid estrogens (ng/ml) in urine of male veal calves in ILOB experiment 25.14 (Huis in 't Veld and Both-Miedema, 1974)
Age in weeks Control a EC E/Te E/Tr
llb 4 17 37 13 11.5 5 40 62 15 12 5 155 110 10 13d 3 64 33 19 14 3 7 6 30 15e 5 2 9 16 17 6 3 6 20 a 3 Animals/group
b Average of 24-hour urine, week 11 is day 1
c Implanted at 11 weeks of age
d 12 Days after implantation
e 30 Days after implantation
table.5
E/Tr 2.07
group)
Days after E/Tr at 11 weeks E/Tr at 14 weeks E/Tr at 17 weeks implantation Control Treated Control Treated Control Treated
0 5 8 4 6 6
,
..
1 4 19 3 18 4 16 2 2 22 4 31 9 22 3 5 74 6 49 10 18 4 4 46 8 31 5 28 5 7 29 8 70 6 20 6 3 36 4 34 16 26 7 4 16 6 84 13 25 8 3 25 7 70 10 29 9 3 22 7 82 15 43 10 2 24 7 104 10 52 11 4 20 9 40 10 82 12 3 32 6 42 6 80 13 12 59 5 131 6 80 14 6 8 6 105 5 84 15 4 12 12 106 8 64 16 5 20 6 64 8 34 17 4 10 6 9 12 32 18 3 4 4 50 12 39 19 4 7 9 84 7 18 20 6 6 10 27 10 26 21 8 10 5 34 22 8 8 6 17 23 4 9 16 26 24 6 5 13 26 25 7 8 10 33 26 7 8 15 44 27 7 9 10 28 28 9 6 10 22 29 6 10 6 24 30 5 14 31 6 8 6 24 32 12 7 5 24 33 6 5 8 28 34 6 6 8 8 35 4 6 12 10 36 9-
12 10 37 10-
7 6 38 5 5 10 7 39 6 4 40 16 5 41 13 8 42 10 7 43 15 14 44 10 9 45 10 5 table.7Table 7 cont.
Days after E/Tr at 11 ~-1eeks E/Tr at 14 ~-1eeks E/Tr at 17 weeks
implantation Control Treated Control Treated Control Treated
46 6 7 ll7 6 7 48 5 5 49 8 10 50 8 4 51 12 6 52 12 7 53 7 10 54 10 8 table.7a
times after implantation.
Experiment Treatment Number Weeks after implantation
animals 0 2 3 4 25.18 control 3-1-2b E/Tr 8-6-5 E/Tr 9-7-1 25.22 control 5 6(4-9) 7(2-17) 7(3-14) 2(2-4) E/Tr 5 6(4-11) 21(12-36) 21(9-34) 6(5-9) control 5 5(2-11) 8(3-9) 8(3-12) 1 E/Tr 5 6(2-16) 15(3-29) 20(5-35) 15(6-31) 25.21 control 2 E/Tr 5 w 2 E/Tr 8 w 2 E/Tr 11 w 2 control 2 25.16 control 2 E/Tr 11 25.13 control 11 2(2-04)C 4(1-9) E 12 14(3-41)C 7(2-12 E/Te l l 24(4-74)C 19(5-70) E/Tr l l
-
3(5-56) E/Pr 12-
17(4-38)a Average (lowest-highest) levels
b 5, 6 and 7 weeks after implantation, respectively 3, 1, 2 animals/group
c After 3 days table.8 5 6 8(5-12) 4 18(6-41) 16(4-31) 18(9-38) 11(5-24) 6(4-8) 16(16-17) 0(0-1) 11(2-16) 5(1-14) 3(0-8) 3(0-6) 13(2-40) 4(1-12) Comment 7 9 12 5(3-8) 6(2-9) 5 I ! 3.5 Week 16 . 3.5 Week 12 8(7-10) 3(2-4) 2(2-3)
Table 9: Average total steroid estrogen levels (ng/ml) in urine of male veal
calves in ILOB experiments
Treatment Age in weeks llb 12 13 14 15 16 17 18 19 Control8 (n=4) 0.57 0.62 1.14 0.98 1. 94 1.80 1.56 1. 72 2.38 (n=5)& 0.52 3.28 3.19 7.82 2.80 3.67 7.24 E/Trc (n=6) h 0.23 2.20 2.23 2.92 0.87f 0.97f 1. 78f EC E (n=5) (n=5)i 1. 95 2.04 8.81 4.83 14.42 5.69 15.15 10.49 8.08
I
4.19 4.151 9.82 2.20 10.66 2. 71 2. 20ea Not all experimental groups had same control group
b Average 24-hours urine week 11 is day 0
c Implantation at 11 weeks of age
d Estradiol-17~ infusion 1 mg/d
e After discontinuation estradiol infusion
f After removal of implantation
g ILOB experiment 25.22 h ILOB experiment 25.25 i ILOB experiment 25.23 table.9 inf.d (n=4) h 0.89 7. 53 4.95 4.99 5.42 8.66 3.96e 1.23e 1.98e
(modified version of Table 10 from Appendix 1)
Experi- Treatment Age at Age at urine Comments No. of
ment treatment sampling animals
number (\o7eeks) ("~>7eeks) (n)
25.23 control
-
18.5 2 E 10 idem (8.5) 4 E inf. 10 idem (2.5) 5 25.24 control-
20 2 E 11 idem (9) 4 E inf. 11 idem (l) 5 25.25 control-
19 2 E/Tr 10 idem (9) 2E/Tr 10 idem (6) rem. 2
E inf. 10 idem (3) 2
25.27 control
-
17 6E/Tr 11 idem (6) 6
E/Tr(n) 11 idem (2) rem. 7
E/Tr(n) 12 idem (2) rem. 7
E/Tr(n) 13 idem (2) rem. 7
25.29 control
-
18 and 22 6E/Te 11 18, 22 and 28 6
(7, 11 and 17)
E/Te 11 18 and 22 rem. 7
(3 and 7)
E/Te(2x) 11 18, 22 and 28 rem. 7
(3) 11 and 17)
81.24 control
-
21 and 23 2E/Tr 12 idem(9 and 11) 14
81.25 control
-
24 and 26 3E/Tr 12 idem(12 and 14) 16
Average level 8.4 7.8 6. L1 4.2 5.3 3.9 4.2 12.6 16.8 6.4 14.0 32.2 9.8 10.1 17.6 5.0 and 7.8 16.5, 15.4 and 4.2 5.0 and 7.3 10.1, 17.6 and 12.6 3.3 and 12.9 12.6 and 16.~ 2.9 and 2.6 10.0 and 8.8
*
number in parentheses=
1o1eeks after implantation, remaval of implantation, ordiscontinuatien infusion
E E inf. Tr Te E/Tr (n) rem. 2x table.10 20 mg estradiol-17~
= infusion with 1 mg estradiol-17~
140 mg trenbolone acetate
200 mg testosterone
40 mg estradiol-17B/287,9 trenbolone acetate as a noseclip
remaval (surgery or noseclip removed)
