CSIRO PUBLISHING

Reproduction, Fertility and Development

http://dx.doi.org/10.1071/RD11290

Determination of anti-Müllerian hormone concentrations

in blood as a tool to select Holstein donor cows for embryo
production: from the laboratory to the farm

Charlène RicoA, Laurence Drouilhet A, Pascal Salvetti B, Rozenn Dalbiès-TranA,

Peggy Jarrier A, Jean-Luc Touzé A, Elodie PilletB, Claire PonsartB,
Stéphane FabreA and Danielle Monniaux A,C
A
Physiologie de la Reproduction et des Comportements, UMR 085 INRA-UMR 7247
CNRS-Université de Tours-IFCE, Centre INRA de Tours, 37380 Nouzilly, France.
B
UNCEIA, Department of Research and Development, 13 rue Jouet, 94704
Maisons-Alfort, France.
C
Corresponding author. Email: dmonniaux@tours.inra.fr

Abstract. High between-animal variability in the number of embryos produced by multiple ovulation and embryo
transfer (MOET) and ovum pick-up and in vitro production (OPU–IVP) methods remains a major limit to the development
of embryo biotechnologies in cattle. The measurement of anti-Müllerian hormone (AMH) endocrine concentrations in
cows can help to predict their follicular and ovulatory responses to gonadotrophin treatment. The present study aimed to
provide practical information for a simple prognostic method based on AMH measurement in Holstein cows. Accurate
AMH concentrations could be measured with ELISA in blood or plasma. In cows undergoing repeated OPU protocols over
1 year, the AMH concentrations measured in plasma samples collected before each gonadotrophin treatment were found to
be highly repeatable and were tightly correlated with follicular responses. From data obtained at both an experimental
station and farm settings, it was possible to propose AMH cut-off values to identify low-responding cows. Gonadotrophin-
stimulated cows producing fewer than 15 large follicles at oestrus and fewer than 10 embryos in MOET protocols could be
discarded efficiently with plasma AMH concentrations below 87 and 74 pg mL
1, respectively. In conclusion, we propose
a prognostic method based on a single AMH measurement to improve the results of embryo biotechnologies.

Additional keywords: assisted reproductive technology, bovine species, FSH, ovulation.

Received 19 November 2011, accepted 17 February 2012, published online 20 March 2012

Introduction OPU–IVP appears even more critical to produce large numbers

Nowadays two different methods can be used to improve the
number of offspring produced by genetically valuable cows.
The first method, so-called multiple ovulation and embryo
transfer (MOET), developed in the early 1970s, involves
administering gonadotrophins together with prostaglandins to
donor cows in order to induce multiple ovulations (i.e. super-
ovulation), fertilising the ovulated oocytes after routine artificial
insemination and recovering the embryos by uterine flushing.
The second method, so-called ovum pick-up and in vitro
production (OPU–IVP), is more recent and involves ultrasound-
guided transvaginal aspiration of oocytes with or without
pretreatment of the donor cows by gonadotrophins, followed by
in vitro oocyte maturation, fertilisation and early embryo
development. The development of genomic selection has
recently induced dramatic changes in the management of
genetic selection schemes, but the efficiency of MOET and
of animals to be genotyped (Humblot et al. 2010). In this con-
text, the existence of high between-animal variability in the
number of embryos produced by both methods remains a major
limit to the development of embryo biotechnologies (Driancourt
2001; Mapletoft et al. 2002; Merton et al. 2003; De Roover et al.
2005; Pontes et al. 2011).
The embryo production capacity of donor cows has been
shown to be a repeatable and heritable trait in MOET (Asada and
Terawaki 2002; Benyei et al. 2004; Eriksson et al. 2007;
Monniaux et al. 2010) and recently in OPU–IVP protocols
(Merton et al. 2009). Thus, predicting the capacity of each cow
to respond to gonadotrophin treatment could improve the results
of MOET and OPU–IVP protocols by identifying in advance
and discarding low-responding cows. Recent results indicate
that anti-Müllerian hormone (AMH), also known as Müllerian
inhibiting substance (MIS), specifically expressed by the

Journal compilation Ó CSIRO 2012 www.publish.csiro.au/journals/rfd

B Reproduction, Fertility and Development
granulosa cells of small growing follicles, is an endocrine
marker for the size of the pool of ovarian gonadotrophin-
responsive follicles in the cow and can help to predict the
ovulatory responses of individuals (Rico et al. 2009, 2011;
Monniaux et al. 2010; Ireland et al. 2011).
The development of a prognostic method to determine the
intrinsic capacity of a potential donor cow to produce an
expected number of embryos might be based on the measure-
ment of circulating AMH concentrations. However, several
questions have to be answered before validating a prognostic
test. The first question concerns technical problems related to
AMH determination in blood. Presently, AMH concentrations
can be measured accurately in the plasma of cows using the
commercially available Active MIS/AMH ELISA kit supplied
by Beckman Coulter for the quantitative measurement of AMH
in human serum. The use of this kit has been recently validated
for AMH measurement in various female mammals such as
mouse (Kevenaar et al. 2006), rat (Yeh et al. 2007), cow (Ireland
et al. 2008; Monniaux et al. 2008), goat (Monniaux et al. 2011),
horse (Almeida et al. 2011), dog and cat (Place et al. 2011). In
the cow, plasma AMH concentrations are ,10-fold lower than
in humans, so high sensitivity and repeatability of the assay are
of crucial importance for an accurate AMH measurement;
moreover, the practical conditions of blood sampling of cows
and blood storage before AMH assay remain to be defined.
A second important point for developing a diagnostic test is how
to schedule the optimal time for carrying out the blood test on
cows. From our results, plasma AMH concentrations show only
small variations during the oestrous cycle (Rico et al. 2011) and
between-animal differences in AMH concentrations were found
to be unchanged after a 3-month delay (Rico et al. 2009);
whether circulating AMH concentrations could be characteristic
of each animal on a longer period has not been assessed. A third
important question concerns the possibility of defining a thresh-
old (cut-off) AMH concentration for discarding low-responding
cows, that could be used in cows bred under farm conditions. In
humans, from receiver-operating characteristic (ROC) analysis
of data, different AMH cut-off values have been recently
proposed to predict ovulation and conception in women with
polycystic ovary syndrome (Amer et al. 2009), to evaluate its
diagnostic value in adolescent and young adults with this
syndrome (Li et al. 2010) and to predict nonpregnancy and
cycle cancellation in patients of advanced reproductive age in
the context of assisted reproductive treatments (Fadini et al.
2011; Lee et al. 2011).
The present study aimed to provide practical information for
a simple prognostic method based on AMH measurement, in
order to characterise the capacity of each cow to respond to
gonadotrophin treatment and to produce embryos. Experiments
were designed to: (1) better define the technical conditions of
blood sampling and storage, and of the assay itself for optimal
AMH determination in bovine samples, (2) evaluate the indi-
vidual repeatability of AMH concentrations and the follicular
responses to gonadotrophins in the long-term, and (3) propose
AMH cut-off values for discarding low-responding cows, by
ROC analysis of data obtained in two independent groups of
Holstein cows, bred in one experimental station and under farm
conditions.
C. Rico et al.
Materials and methods
Animals
A first group of 64 Holstein dairy cows, 4–9 years old, was used
for Experiment 1 (n ¼ 13) and Experiment 2 (n ¼ 51). The
experiments were conducted at the Experimental Unit UEPAO
in Nouzilly (France). Animals were housed in free stalls and
provided with feed and water ad libitum. Their average milk
production during lactation before the experiment was
8516  249 kg, after normalisation to 305 days of milk pro-
duction. None of the cows was lactating at the time of the
experiment. All procedures were approved by the agricultural
and scientific research agencies (approval number C37–175–2)
and conducted in accordance with the guidelines for the Care
and Use of Agricultural Animals in Agricultural Research and
Teaching.
A second group of 34 Holstein dairy cows, 3–10 years old,
was submitted to embryo production under farm conditions
(Experiment 3). The 34 animals were raised in different farms
scattered in north-west and eastern France (in the area of the
breeding companies Amelis and Franche-Comté Embryon).
Animal breeding conditions were different for each animal
raised in a different farm. In most cases, animals were housed
in free stalls and fed with hay, concentrate or corn silage. Water
was provided ad libitum. All the 34 enrolled animals were
healthy cows.
Experiment 1
This experiment aimed to assess the within-animal repeatability
in plasma AMH concentrations and follicular responses after
repeated gonadotrophin treatments and ovum pick-up (OPU).
During 2008, 13 cows were submitted to repeated ovarian
gonadotrophin treatments (up to 11 repetitions), each followed
by OPU. Before each treatment, the cows received subcutaneous
progesterone implants (Crestar; Intervet, Angers, France)
during 10 days, and an intramuscular injection of 10 mg
Buserelin (Receptal; Intervet) was administered at implant
insertion to induce ovulation of all large follicles present on the
ovaries. Eight days after implant insertion, cows received a
total of 32 mg FSH (Stimufol; Merial, Lyon, France, gift from
Dr J. F. Beckers), corresponding to a superovulatory treatment,
which was given as twice-daily injections over 4 days on a
decreasing dose schedule (Mapletoft et al. 2002). An intra-
muscular injection of 22.5 mg prostaglandin F2a (Prosolvin;
Intervet) was administered at the time of the fifth FSH injection
to induce luteolysis, and progesterone implants were removed at
the time of the sixth FSH injection. Forty-eight hours after the
last FSH injection, the large follicles (diameter .6 mm) that had
developed in response to the treatment were counted by ovarian
ultrasonography scanning using transrectal endocavitary probe
EC123 (5.0/7.5-MHz transducer, MyLab 30 Vet; Esaote, Saint-
Germain-en-Laye, France) and punctured by OPU before
ovulation. OPU was performed by means of a long needle
(18G  11/20 ) introduced through the vagina, under ultrasound
control. At each repetition of treatment, blood (2 to 4 mL) was
collected from the jugular or the tail vein in vacuum tubes
containing sodium heparin (BD Vacutainer NH; Becton,
Dickinson and Co., Le Pont de Claix, France) just before the
AMH and embryo production in the Holstein cow
first FSH injection (before treatment) and at the day of OPU (at
OPU), then plasma was recovered after centrifugation (3200g
for 10 min at 48C) and immediately stored at
208C until AMH
assay.
Cows entered the experimental protocol in January (6 cows),
March (4 cows) or May (3 cows). Animals were then treated and
submitted to OPU repeatedly (between 4 and 11 repetitions per
cow) until December. No treatment and OPU were carried out
on cows during the hot season (July and August). The average
interval between two successive OPU was 31 days, but varied in
the range 11–252 days. Five cows with low follicular response
(,15 large follicles at each puncture) received only four to six
repetitions of the treatment and left the protocol within 3 months
after entry.
Experiment 2
This experiment aimed to assess the between-animal variability
in plasma AMH concentrations, follicular and ovulatory
responses to gonadotrophin treatment, and to propose AMH cut-
off values for discarding low-responding cows. Between 2006
and 2009, 51 cows were submitted to gonadotrophin treatment
as described in Experiment 1, except that no Buserelin was
administered. Each cow was treated once only. At the time of
oestrus, ovaries were scanned by ultrasonography as described
above, and all large follicles (LF, diameter .7 mm) induced by
the treatment were counted. The number of ovulations was
assessed using ultrasonography by counting the corpora lutea
(CL) on ovaries 7 days after oestrus during the luteal phase
following treatment. Blood samples were recovered before
treatment as described in Experiment 1 and the plasma samples
were immediately stored at
208C until AMH assay.
Experiment 3
This experiment aimed to assess the between-animal variability
in plasma AMH concentrations and embryo production in
response to gonadotrophin treatment, and to propose AMH
cut-off values for discarding cows with a low capacity to pro-
duce embryos under farm conditions. Between January and
April 2010, 34 cows were submitted to gonadotrophin treatment
as described in Experiment 1 and each cow was treated once
only. Cows were inseminated twice 12 h and 24 h after oestrus
detection. Seven days after oestrus, embryos were non-surgically
collected through flushing of the uterine horns via a catheter.
Embryos were recovered on a filter, examined in a petri dish
using a binocular magnifying glass and counted. Blood samples
were recovered at the time of embryo collection as described in
Experiment 1 and the plasma samples were immediately stored at

208C until AMH assay.
Testing of blood storage and sampling conditions
for AMH quantification
The effect of blood storage conditions (temperature and dura-
tion) before plasma centrifugation was tested on blood samples
collected on six randomly chosen cows in vacuum tubes
containing sodium heparin (BD Vacutainer NH; Becton,
Dickinson and Co.), stored at 48C and at 208C for times varying
between 4 h and 48 h, with and without preservative (sodium
Reproduction, Fertility and Development C
azide, 5 mg mL
1; Merck Serono, Lyon, France). Blood was
then centrifuged (3200g, 10 min, 48C) and the recovered plasma
was stored frozen before AMH assay.
The effect of the blood-sampling conditions (heparin vs
EDTA) was tested on nine randomly chosen cows by compari-
son of the AMH concentrations measured in blood and in
plasma, both collected in vacuum tubes containing sodium
heparin (1.7 IU, final concentration, BD Vacutainer NH) or
EDTA (1.8 mg mL
1, final concentration, BD Vacutainer K2E).
The AMH assay was carried out on these samples after one or
two freeze–thaw cycles. In a further experiment, the effect of
EDTA was tested by adding EDTA (1.8 mg mL
1, final con-
centration) before AMH assay to heparinised plasma samples
recovered from the 51 cows of Experiment 2.
AMH assay
Plasma and blood concentrations of AMH were determined
using the Active MIS/AMH ELISA kit (Beckman Coulter
France, Roissy CDG, France), as described previously for the
bovine species (Monniaux et al. 2008; Rico et al. 2009), with
some modifications in order to improve the sensitivity and
repeatability of the assay. The standard curve was adapted to
the measurement of the low AMH concentrations present in
bovine blood samples (range of the reduced standard curve:
15–1000 pg mL
1), and dilutions of the standard points were
made in steer plasma. AMH concentrations were determined in
50-mL aliquots of undiluted samples. Just before assay, the
samples were thawed in a warm water bath, vortexed and cen-
trifuged (3200g, 10 min, 48C) to remove any cell fragments that
could interfere with the reagents of the assay. Afterwards the
samples were incubated overnight at 48C in the presence of
the primary antibody, then for 1.5 h at room temperature in the
presence of the secondary antibody. With these conditions,
the limit of detection of the assay was found to be 15 pg mL
1.
For further validation of the assay, serial dilutions of different
bovine plasma samples were analysed. Results showed that
dilution curves were linear and parallel to the standard curve
(Fig. 1). The intra-assay and the inter-assay coefficients of
variation (s.e.m./mean) were between 3% and 5%, and between
6% and 14%, respectively, for quality control bovine plasma
samples containing between 80 and 800 pg mL
1 AMH.
Data analysis
Experimental data are presented as mean  s.e.m., except for
correlation studies. Data were analysed using t-tests or one-way
ANOVA followed by Newman–Keuls multiple-comparison
tests for comparisons between two or several means, respec-
tively. Repeated-measures ANOVA were used to analyse the
effects of blood storage conditions and those of blood-sampling
conditions on AMH concentrations. When variances were het-
erogeneous, data were analysed by the nonparametric Kruskal–
Wallis test. For correlation studies, the significance of the value
of the correlation coefficient was considered according to the
Bravais–Pearson r critical values. For all analyses, differences
with P . 0.05 were considered as not significant.
For Experiment 1, five periods of time were compared
in order to evaluate a seasonal effect: January–February,
D Reproduction, Fertility and Development
1000
Beckman SC
Reduced SC
Optical density (1000)
Cow 445
Cow 473
100
Cow 3723
Cow 466
10
1 91.2, respectively) for Experiment 3. From these ROC analyses,
0.1 1.0 10.0 100.0
AMH (dilution factor)
Fig. 1. Dilution curves for plasma of four Holstein cows, and the standard
curves (SC) of the AMH ELISA proposed by the manufacturer of the kit
(Beckman SC) and adapted to the measurement of AMH concentrations in
bovine plasma samples (Reduced SC).
March–April, May–June, September–October and November–
December. The effects of the repetition of treatments on the
number of large follicles and AMH concentrations were ana-
lysed with one-way repeated-measures ANOVA. The repeat-
ability (r2) of each parameter was calculated as the ratio of the
between-animal variance to the sum of the between-animal and
the residual variances.
For Experiments 2 and 3, data were submitted to receiver-
operating characteristic (ROC) analysis for diagnostic tests
(Greiner et al. 2000) in order to determine AMH cut-off values
for discarding cows with low capacity to respond to gonadotro-
phin treatments (i.e. cows with several LF at oestrus fewer than
Nf, or cows with several embryos collected fewer than Ne). For
a given value of Nf or Ne, each AMH value on the original scale
was selected as a decision threshold (cut-off value) to define
positive and negative test outcomes, i.e. cows with a high and a
low response to the gonadotrophin treatment, respectively.
Comparison of the dichotomised test results against the true
status of individuals (as determined by their numbers of LF at
oestrus, and their numbers of embryos collected, for Experi-
ments 2 and 3, respectively) allowed estimation of the diagnostic
specificity (Sp, probability of a positive test outcome in a high-
responding individual) and sensitivity (Se, probability of a
negative test outcome in a low-responding individual). ROC
analysis assessed the diagnostic performance of the system in
terms of Se and (1–Sp) for each possible AMH cut-off value of
the test. The resulting pairs ((1–Sp), Se) were plotted in a unit
square, then the area under the resulting ROC curve (AUC) was
estimated by non-parametric approach (Wilcoxon-area esti-
mate) and compared with the expected value (AUC ¼ 0.5) under
the null hypothesis of a non-informative test using the two-
sample Mann–Whitney rank-sum test (Greiner et al. 2000).
When the AUC was significantly different from 0.5, the
AMH cut-off value was chosen to maximise the Youden index
(J ¼ Se þ Sp – 1), which corresponds to the point of the ROC
curve closest to the upper left corner of the unit square and
optimises prevalence-independent summary measures of Se
and Sp. The effect of the prevalence (Pr) of the character in
C. Rico et al.
the population of cows (i.e. the percentage of individuals with
several LF at oestrus fewer than Nf, and the percentage of
individuals with several collected embryos fewer than Ne, in
Experiments 2 and 3, respectively) on the diagnostic perfor-
mance was assessed by calculating the efficiency (Ef ¼ Pr 
Se þ (1
Pr)  Sp) of the cut-off choice. All analyses were
carried out for values of Nf varying between 3 and 30 (corre-
sponding to prevalence values between 2.0 and 90.2, respec-
tively) for Experiment 2, and values of Ne varying between 3
and 18 (corresponding to prevalence values between 8.8 and
1000.0 the same AMH cut-off value could be found for a range of
different values of Nf or Ne. Finally, we determined the values
of Nf and Ne (for Experiments 2 and 3, respectively) corres-
ponding to the optimisation of all the studied parameters
(Sp, Se, J and Ef ) for each AMH cut-off value resulting from
the ROC analyses. For each AMH cut-off value determined,
the predictive positive value (PPV, i.e. the probability of a high
response in an individual with a positive test outcome) and the
predictive negative value (PNV, i.e. the probability of a low
response in an individual with a negative test outcome) were
calculated and expressed as percentages.
Results
Effects of blood storage and blood-sampling conditions
on AMH concentrations
The management of the blood samples recovered from cows in
farm conditions raises some practical problems, such as the
distance to access to a centrifuge required to prepare the plasma.
Thus, we tested the effect of different conditions of storage of
unfrozen blood samples recovered in vacuum tubes containing
sodium heparin, on AMH concentrations measured in plasma.
The storage of unfrozen blood samples for several hours before
centrifugation induced an increase in the AMH concentrations
measured in the recovered plasma samples (Fig. 2). The increase
was significant after 8 h storage at 208C (P , 0.01, n ¼ 6 cows)
and 24 h storage at 48C (P , 0.05, n ¼ 6 cows). It was highly
variable between samples, and the addition of a preservative
(sodium azide) before storage for 24 h and 48 h at 208C did not
permit stabilisation of plasma AMH concentrations (Fig. 2).
These results indicate that the storage of unfrozen blood samples
can lead to overestimation of plasma AMH concentrations and
to bias the results of the AMH assay.
As immediate freezing could be an alternative for storage of
blood samples, the effect of freezing and thawing was tested on
blood and plasma. After one freeze–thaw cycle, AMH concen-
trations were similar in blood and plasma samples, and a second
freeze–thaw cycle had no effect on AMH concentrations in
plasma (Fig. 3a). However, repeated cycles of freezing and
thawing of plasma samples induced a 1.2- to 1.7-fold average
increase in plasma AMH concentrations, significant after three
freeze–thaw cycles (P , 0.05), and highly variable between
samples (data not shown). These results indicate that three or
more cycles of freezing and thawing should be avoided to
measure AMH accurately in plasma samples.
The effect of the anticoagulant present in the vacuum tube
used for blood sampling was also tested. AMH concentrations
AMH and embryo production in the Holstein cow Reproduction, Fertility and Development E

500 (a) 200 b

* b

400 150 b

AMH (pg mL1)

300 **
AMH (%)

100
a
* *
200 a
50
** a

100
0
BH P1H P2H BE P1E P2E
0 Heparin EDTA
T0 4°C 20°C 4°C 20°C 20°C 20°C
T8 T24 T48 (b) 750
With preservative

AMH (pg mL1) in plasma

Fig. 2. Effect of blood storage on AMH concentrations measured in bovine
plasma. Blood samples were collected from six Holstein cows in vacuum
500
tubes containing sodium heparin, unstored (T0) or stored for 8 h (T8), 24 h

with EDTA
(T24) or 48 h (T48), at 48C or at 208C, in the absence or presence of a
preservative (sodium azide, 5 mg mL
1). Afterwards, the plasma samples
were recovered by centrifugation (3200g, 10 min, 48C) and stored frozen
before AMH assay. Data are expressed as the percentage of AMH concen- 250
trations measured in the corresponding plasma samples recovered without
blood storage (T0). *P , 0.05, **P , 0.01, compared with T0.

were compared in blood and plasma samples recovered from 0

nine cows in the presence of heparin or EDTA, immediately
frozen at
208C for storage, and thawed just before the assay.
Blood and plasma samples containing EDTA had higher AMH
concentrations than the corresponding blood and plasma sam-
ples containing heparin (blood, BH vs BE, P , 0.05; plasma after
one freeze–thaw cycle, P1H vs P1E, P , 0.001; plasma after two
freeze–thaw cycles: P2H vs P2E, P , 0.001; n ¼ 9 cows,
Fig. 3a). Nevertheless, AMH concentrations with heparin and
EDTA were highly correlated (BH and BE, r ¼ 0.98, P , 0.0001;
P1H and P1E, r ¼ 0.98, P , 0.0001; P2H and P2E, r ¼ 0.92,
P , 0.001). Furthermore, AMH concentrations in blood and
plasma were highly correlated (with heparin, BH and P1H,
r ¼ 0.99, P , 0.0001; with EDTA, BE and P1E, r ¼ 0.98,
P , 0.0001). The addition of EDTA to plasma samples recov-
ered from 51 cows in vacuum tubes containing heparin induced
a 1.5-fold increase in AMH concentrations; AMH concentra-
tions in plasma with and without EDTA were still highly
correlated (r ¼ 0.91, P , 0.0001, n ¼ 51 cows, Fig. 3b). These
results indicate that AMH measurement can be made accurately
either on total blood or on plasma, in the presence of heparin
or EDTA, but the blood-sampling conditions can affect the
measured AMH concentrations.
Within-animal repeatability in AMH concentrations
and follicular responses to gonadotrophin treatments
A total number of 91 gonadotrophin treatments, each followed
by OPU, were carried out in the 13 cows of Experiment 1. The
AMH concentrations measured in heparinised plasma increased
following treatment (176.6  11.8 AMH before treatment vs
0 250 500 750
AMH (pg mL1) in plasma
without EDTA
Fig. 3. Effect of blood-sampling conditions on AMH concentrations
measured in bovine blood and plasma. (a) Blood and plasma were collected
in vacuum tubes containing sodium heparin (1.7 IU, final concentration) or
EDTA (1.8 mg mL
1, final concentration) from nine Holstein cows, then
frozen 1-fold (BH, blood with heparin; BE, blood with EDTA; P1H, plasma
with heparin; P1E, plasma with EDTA) or 2-fold (P2H, plasma with heparin;
P2E, plasma with EDTA) before AMH assay; a vs b: P , 0.05. (b) Blood was
collected in vacuum tubes containing sodium heparin (1.7 IU, final concen-
tration) from 51 Holstein cows, and the recovered plasma samples were
stored frozen. After thawing, AMH concentrations were measured in the
plasma samples with or without the addition of EDTA (1.8 mg mL
1, final
concentration). The regression line between AMH concentrations with and
without EDTA follows the equation: y ¼ 1.5x þ 5.3.
253.7  19.8 pg mL
1 AMH at OPU, P , 0.01) and a highly
significant correlation was observed between plasma AMH
concentrations measured before treatment and at the time of
OPU (r ¼ 0.88, P , 0.0001). The numbers of large follicles at
OPU were significantly correlated with plasma AMH con-
centrations before treatment (r ¼ 0.56, P , 0.0001, Fig. 4a) and
at the time of OPU (r ¼ 0.65, P , 0.0001). Moreover, the
average AMH concentrations per cow were significantly cor-
related with the average numbers of large follicles and oocytes
recovered at OPU per cow (r ¼ 0.75 and r ¼ 0.70, respectively,
both P , 0.01, n ¼ 13 cows). As the oocyte yield per cow was
F Reproduction, Fertility and Development C. Rico et al.

(a) 40
low (44.4  4.4%, percentage of recovered oocytes relative to
punctured follicles, n ¼ 13 cows) and variable between OPU
sessions for technical reasons, the data concerning oocyte 30
numbers were not submitted to further analyses. Cow 445
No seasonal effect was observed for either the number of 20
Cow 6452
large follicles on ovaries at OPU or plasma AMH concentrations Cow 466
(data not shown). There was no significant effect of OPU Cow 1518
repetition number on the numbers of large follicles on ovaries 10

at OPU or plasma AMH concentrations before treatment

(Fig. 4b). When the data were analysed for four (n ¼ 13 cows),

Number of follicles
0
five (n ¼ 10 cows), six (n ¼ 9 cows), seven (n ¼ 6 cows), eight 0 100 200 300 400
(n ¼ 5 cows) or 11 repeated OPU (n ¼ 3 cows), a highly signifi- AMH before treatment
cant repeatability was observed in all analyses for plasma AMH (pg mL1)
concentrations before treatment (P , 0.0001) and the numbers
of large follicles at OPU (P , 0.001; Fig. 4c). Similar results (b) 60 400
were observed for plasma AMH concentrations at the time of (3)

AMH before treatment

50
OPU (data not shown). Altogether, these results indicate that (3) 300
plasma AMH concentrations and follicular responses to gonad- 40 (13)

(pg mL1)
(5)
otrophin treatment were correlated, highly variable between (13) (13) (13) (10) (9) (6)
30 200
individuals, but repeatable for each cow. (3)

20
100
Between-animal variability in AMH concentrations, 10
follicular and ovulatory responses to gonadotrophin
treatment, and determination of AMH cut-off 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12
values for discarding low-responding cows
OPU repetition number
In Experiment 2, the follicular and ovulatory responses to the
gonadotrophin treatment administered to the 51 cows were
highly variable between animals as illustrated by the prevalence (c) 1.0
Coefficient of repeatability (r2)

AMH
curves of the numbers of CL and of LF at oestrus (Fig. 5a). The 0.9 Number of follicles
AMH concentrations measured in heparinised plasma before
treatment varied in the range 5–244 pg mL
1 for 50 of the 51 0.8 (10)
(13) (9)
studied cows; one cow had a very high AMH concentration of
413 pg mL
1 (Fig. 3b and 6a). The numbers of LF at oestrus 0.7 (6)
(3)
were significantly correlated with plasma AMH concentrations 0.6 (5)
before treatment (r ¼ 0.46, P , 0.001, n ¼ 51) and the correla-
tion coefficient was higher when the cow with a very high 0.5
AMH concentration was removed from the analysis (r ¼ 0.60, 0.4
P , 0.0001, n ¼ 50, Fig. 6a). The numbers of CL were also 3 4 5 6 7 8 9 10 11 12
significantly correlated with plasma AMH concentrations Number of OPU per cow
before treatment (r ¼ 0.43, P , 0.01, for both n ¼ 51 and
n ¼ 50). No correlation was found between the plasma AMH Fig. 4. Individual repeatability of AMH concentrations and follicle num-
concentration of the cows and their milk production during bers at OPU during repeated sessions of OPU. Holstein cows (n ¼ 13) were
lactation before the experiment (r ¼
0.04, n.s., n ¼ 32 cows in treated repeatedly (n ¼ 4 to 11 repetitions per cow) by gonadotrophins, each
which individual milk production was registered). treatment being followed by OPU. (a) Relationship between AMH concen-
tration before treatment and number of large follicles at each individual OPU
The data of the 51 cows were analysed in order to determine
session in four cows, taken as examples. (b) Effect of OPU repetition number
the AMH cut-off values for discarding cows with a low follicular
on AMH concentration before treatment (black circles, right scale) and
response to treatment. As an example, the determination of the number of large follicles at OPU (black bars, left scale). (c) Value of the
AMH cut-off value for discarding cows with several LF fewer coefficient of repeatability (r2) of AMH concentration before treatment
than 15 at oestrus is illustrated in Fig. 7. For each AMH (black circles) and number of large follicles at OPU (empty squares). The
concentration taken as a possible cut-off value, the diagnostic number of studied cows is indicated in brackets.
sensitivity and specificity corresponding to Nf ¼ 15 were deter-
mined (Fig. 7a). The area under the ROC curve was highly
significantly different from the area corresponding to the null experimental prevalence (Pr ¼ 31.4%) observed from the 51
hypothesis of a non-informative test (P , 0.0001, Fig. 7b, studied cows (Table 1). For prevalence values between 10% and
Table 1). The plasma AMH concentration that maximised the 75%, this AMH cut-off value was found to be optimal, the
Youden index was found to be 87 pg mL
1. It corresponded to efficiency of the test varying between 0.833 and 0.863, respec-
high PPV and PPN values and a high efficiency of the test at the tively (Fig. 7c).
AMH and embryo production in the Holstein cow Reproduction, Fertility and Development G

(a) 100 (a) 50

Number of LF at oestrus
80 40

60 30

40 20
CL
LF at oestrus
20
10
Prevalence (%)

0
0
0 10 20 30 40 50
0 100 200 300 400 500
Number of ovarian structures
AMH before treatment (pg mL1)
(b) 100
(b) 30
80

Number of embryos
60
20

40

20 10

0
0 5 10 15 20
Number of embryos
Fig. 5. Prevalence curves for ovarian sensitivity to gonadotrophin treat-
ment and embryo production in two populations of Holstein cows. The
prevalence was defined as the percentage of individuals with several ovarian
structures (LF, large follicles .7 mm at oestrus or CL, corpora lutea) or
collected embryos fewer than N in the population of cows. Prevalence
curves were obtained with N values varying between 1 and Nmax þ 1, with
Nmax ¼ the highest number of ovarian structures or embryos in the studied
population of cows. (a) Prevalence curves for the numbers of LF and CL
observed in response to gonadotrophin treatment. The curves were obtained
from the results of Experiment 2 conducted on 51 Holstein cows at the
Experimental Unit UEPAO in Nouzilly. (b) Prevalence curve for the number
of embryos collected after administration of gonadotrophin treatment. The
curve was obtained from the results of Experiment 3 conducted on 34
Holstein cows in farms.
The AUC was significantly different from 0.5 in all analyses
carried out for Nf values between 4 and 26 (corresponding to
prevalence values between 5.9 and 86.3, respectively, Fig. 5a).
Finally, five AMH cut-off values (35, 42, 87, 109 and
123 pg mL
1) were found from the ROC analyses, and they
were then associated with the five Nf values (8, 10, 15, 20 and
26) that optimised all the studied parameters (Sp, Se, J and Ef ).
The results of these analyses are presented in Table 1. For each
pair (Nf value, AMH cut-off value) that was determined, the
Youden index and the efficiency of the test reached values
higher than J ¼ 0.56 and Ef ¼ 0.72, respectively. For increasing
values of Nf, the PPV and the PNV values decreased and
increased, respectively. Except for the PPV at Nf ¼ 26 (corre-
sponding to AMH cut-off ¼ 123 pg mL
1), Sp, Se, PPV and
PPN were all clearly higher than 50% (Table 1). Each AMH cut-
off value divided the population of cows into two groups with a
25 30 0
0 100 200 300 400 500
AMH at embryo collection (pg mL1)
Fig. 6. Plasma AMH concentration and response of Holstein cows to
gonadotrophin treatment. (a) Relationship between AMH measured before
treatment and number of large follicles (LF, large follicles .7 mm) at
oestrus after treatment (n ¼ 51 cows). (b) Relationship between AMH
measured at the time of embryo collection and the number of collected
embryos (n ¼ 34 cows). Each circle represents data from one cow.
high significance (P , 0.001) and an average between-group
difference of 8.1 to 14.1 LF at oestrus, according to the AMH
cut-off value that was used (Fig. 8a).
The five AMH cut-off values were also applied to the dataset
of the number of CL in the population of 51 cows. Each AMH
cut-off value divided the population of cows into two groups
(P , 0.01 to P , 0.001), with an average between-group differ-
ence of 7.5 to 9.6 CL, according to the AMH cut-off value that
was used (Fig. 8b).
Between-animal variability in AMH concentrations and
embryo production in response to gonadotrophin treatment,
and determination of AMH cut-off values for discarding
low-responding cows bred under farm conditions
The number of embryos collected from the 34 cows under the farm
conditions of the Experiment 3 was highly variable between ani-
mals as illustrated by the prevalence curve (Fig. 5b). In these cows,
the AMH concentrations measured in heparinised plasma at the
time of embryo collection varied in the range 14–340 pg mL
1.
The number of embryos collected was significantly correlated
with plasma AMH concentrations measured at the time of embryo
collection (r ¼ 0.46, P , 0.01, n ¼ 34, Fig. 6b).
H Reproduction, Fertility and Development C. Rico et al.

87 pg mL1 The dataset of Experiment 3 was submitted to a systematic

(a)
100
analysis as used for the dataset of Experiment 2 in order to
80 determine the AMH cut-off values for discarding cows with a
Sensitivity and
specificity (%)

low embryo production capacity in response to treatment. As an

60 example, the determination of the AMH cut-off value for
discarding the cows producing fewer than 10 embryos is
40
Specificity illustrated in Fig. 9. For each AMH concentration taken as a
20 Sensitivity possible cut-off value, the diagnostic sensitivity and specificity
corresponding to Ne ¼ 10 were determined (Fig. 9a). The area
0 under the ROC curve was significantly different from the area
0 100 200 300 400 corresponding to the null hypothesis of a non-informative test
AMH (pg mL1) (P , 0.01, Fig. 9b, Table 2). The plasma AMH concentration
that maximised the Youden index was found to be 74 pg mL
1.
(b) 87 pg mL1 It corresponded to high PPV and PPN values and a high
100 efficiency of the test at the prevalence (Pr ¼ 41.2%) existing
80
in the 34 studied cows (Table 2). For prevalence values between
Sensitivity (%)

10% and 75%, this AMH cut-off value was found to be optimal,
60 the efficiency of the test varying between 0.791 and 0.736,
respectively (Fig. 9c).
40
The AUC was significantly different from 0.5 for Ne values
20 between 4 and 17 (corresponding to prevalence values between
17.6 and 88.2, respectively, in the studied population of
0 cows, Fig. 5b). Finally, four cut-off values (74, 79, 83 and
0 20 40 60 80 100 177 pg mL
1) were found from the ROC analyses, and they were
100-specificity (%) then associated with the four Ne values (Ne ¼ 10, 12, 14 and 17)
that optimised all the studied parameters (Sp, Se, J and Ef ). The
87 pg mL1 results of these analyses are presented in Table 2. For each pair
(c) 1.00 (Nf value, AMH cut-off value) that was determined, the Youden
index and the efficiency of the test reached values higher than
0.75 J ¼ 0.51 and Ef ¼ 0.76, respectively. For increasing values of
Nf, the PPV and the PNV values decreased and increased,
Efficiency

0.50
0.25
0 population of cows.
0 100 200 300 400
AMH (pg mL1)
Fig. 7. Example of ROC analysis of the dataset of Experiment 2:
determination of the AMH cut-off value for discarding cows with several
large follicles fewer than 15 at oestrus. Data were obtained from 51 Holstein
cows. (a) Sensitivity and specificity curves. Each AMH concentration was
selected as a decision threshold (cut-off value) to define positive and
negative test outcomes, i.e. cows with several LF $ 15 and ,15 at oestrus,
respectively. The diagnostic specificity, Sp, was defined as the probability of
a positive test outcome in an individual with several LF $ 15 at oestrus. The
diagnostic sensitivity, Se, was defined as the probability of a negative test
outcome in an individual with several LF , 15 at oestrus. Data for Se and
Sp are expressed as percentages. (b) ROC plot representation of the data.
The AMH cut-off value of 87 pg mL
1 that maximised the Youden index
(J ¼ Se þ Sp
1) corresponds to the point of the ROC curve closest to
the upper left corner of the unit square. The dotted line corresponds to the
null hypothesis of a non-informative test. (c) Influence of the prevalence on
the efficiency of the diagnostic test. The curves illustrate the changes in the
efficiency (Ef ¼ P  Se þ (1
P)  Sp) of the cut-off choice with AMH
concentration, for a prevalence P varying between 10 and 75% in the
population of cows. The prevalence corresponding to several LF , 15 at
oestrus was 31.4% in the studied population of cows.
P  75%
respectively. Except for the PPV at Ne ¼ 17 (corresponding to
P  50% AMH cut-off ¼ 177 pg mL
1), Sp, Se, PPV and PPN were all
P  31.4% clearly higher than 50% (Table 2). It was not possible to
P  25% determine AMH cut-off values for Ne values below 10, corre-
P  10% sponding to prevalence values below 41.2% in the studied
When the five AMH cut-off values determined from the
dataset of Experiment 2 were applied to the dataset of the
number of embryos in the population of 34 cows of
Experiment 3, the three lowest AMH cut-off values divided
the population of cows into two groups (P , 0.05 to P , 0.01)
with an average between-group difference of 6 to 6.8 embryos
according to the AMH cut-off value that was used, but the two
highest AMH cut-off values were not efficient at discriminating
cows producing high and low numbers of embryos (Fig. 10a).
However, when the four AMH cut-off values determined from
the dataset of Experiment 3 were applied to this dataset, each
AMH cut-off value divided the population of cows into two
groups (P , 0.05 to P , 0.001) with an average between-group
difference of 5.5 to 6.6 embryos, according to the AMH cut-off
value that was used (Fig. 10b).
Discussion
The results of this study reinforce the hypothesis that the mea-
surement of AMH concentration in the plasma of cows can help
to predict their capacity for embryo production in response to
gonadotrophin treatment; furthermore they give new practical
AMH and embryo production in the Holstein cow Reproduction, Fertility and Development I

Table 1. Determination of AMH cut-off values for the numbers of LF at oestrus by ROC analyses of the data obtained
from 51 Holstein cows undergoing gonadotrophin treatment
*P , 0.05, ***P , 0.001, compared with the expected value (AUC ¼ 0.5) under the null hypothesis of a non-informative test

LF , 8 LF , 10 LF , 15 LF , 20 LF , 26

Prevalence (%) 11.8 15.7 31.4 66.7 86.3

AMH cut-off (pg mL
1) 35 42 87 109 123
AUC 0.941*** 0.959*** 0.927*** 0.830*** 0.769*
Youden index 0.789 0.828 0.704 0.647 0.562
Efficiency 0.941 0.941 0.843 0.804 0.725
Specificity (%) 95.6 95.3 82.9 88.2 71.4
Sensitivity (%) 83.3 87.5 87.5 76.5 70.5
PPV (%) 97.7 97.6 93.5 65.2 27.8
PNV (%) 71.4 77.8 70.0 92.9 93.9

(a) 30 importance for an accurate measurement of AMH concentra-

tions in bovine plasma or blood, (2) there is a high individual
Number of LF at oestrus

25 repeatability of AMH concentrations and subsequent follicular

*** *** ***
****** responses to gonadotrophins in cows undergoing repeated OPU
20
protocols, and (3) from data obtained in both experimental
15 station and farm settings, it is possible to propose AMH cut-off
values for discarding the lowest-responding cows.
10 From our results, its appears that storing unfrozen blood
samples induced time- and temperature-dependent increases in
5
AMH concentrations measured by the Active MIS/AMH ELISA
0 kit. Moreover, recovering blood samples in vacuum tubes
containing EDTA, a potent chelator of divalent cations, or
(b) 20 ** adding EDTA to plasma samples also induced an increase in
*** ** AMH concentrations measured by the assay. These increases in
15
****** AMH concentrations were unexpected and remain unexplained.
They could be due to the extent of processing of the AMH
Number of CL

protein in blood. Prior to secretion, AMH is produced as a

10
5
0 mature domains, generating 110-kDa N-terminal and 25-kDa
0 25 50 75 100
AMH cut-off value (pg mL1)
AMH  cut-off AMH  cut-off
Fig. 8. Ability of different AMH cut-off values to discriminate cows with
high and low follicular or ovulatory response to gonadotrophin treatment.
The AMH cut-off values were determined by ROC analyses of the dataset
(numbers of LF at oestrus) obtained in the 51 Holstein cows of Experiment 2.
(a) Numbers of LF at oestrus in the cows with AMH concentrations higher
and lower than the cut-off values; ***P , 0.001, compared with the group
with AMH concentration , AMH cut-off value. (b) Numbers of CL in cows
with AMH concentrations higher and lower than the cut-off values;
**P , 0.01, ***P , 0.001, compared with the group with AMH concentra-
tion , AMH cut-off value.
information about the use of AMH concentration measurement
as a tool for discarding the lowest-responding cows. In this
study, we have shown that: (1) the technical conditions of blood
sampling and storage, and of AMH assay are of crucial
144-kDa dimer composed of identical disulfide-linked 72-kDa
monomer subunits, each monomer containing a large
N-terminal domain (the pro-region) and a C-terminal domain
(the mature region). After secretion, a small proportion (5–20%)
of the full-length precursor is cleaved upstream of the
125 150 C-terminal homodimers, which remain associated in a biologi-
cally active noncovalent complex (Pepinsky et al. 1988; Wilson
et al. 1993; di Clemente et al. 2010). The proteolytic processing
of AMH can be completed in the extracellular environment by
plasmin (Pepinsky et al. 1988; Wilson et al. 1993; Skaar et al.
2011) or at its target site by proprotein convertases (Nachtigal
and Ingraham 1996). Interestingly, it has been shown that the
fibrinogen present in EDTA-anticoagulated plasma can stimu-
late the tissue-type plasminogen activator-catalysed conversion
of plasminogen to plasmin (Haddeland et al. 1994; Haddeland
et al. 1995). Thus, we suggest that the proteolytic processing of
the full-length AMH precursor may continue under plasmin
activation during blood storage or in the presence of EDTA in
plasma, generating increasing amounts of the biologically active
noncovalent complex whose epitopes are recognised by the
antibodies of the Active MIS/AMH ELISA. Alternatively, the
accessibility of the AMH epitopes to the antibodies might be
enhanced by other conformational changes of the complex, or by
changes in its interactions with binding proteins present in blood
J Reproduction, Fertility and Development C. Rico et al.

(a) 74 pg mL1
100 accurately either on total blood or on plasma, in the presence
of heparin or EDTA, since AMH concentrations in all sampling
80 conditions were found to be highly correlated. However, storing
Sensitivity and
specificity (%)

unfrozen blood samples should be avoided since the time- and

60 Specificity temperature-dependent increases observed in AMH concentra-
Sensitivity tions during storage can bias the results of the AMH assay.
40
This study provides new experimental evidence that AMH
20
concentrations are highly variable between cows but show little
variation within individuals. The concentrations of AMH mea-
0 sured in plasma before each repetition of the OPU protocol were
0 100 200 300 400 highly repeatable over a one-year period of time and no seasonal
AMH (pg mL1)
effect was observed. Moreover, the numbers of follicles that
developed after treatment and were available for follicular
puncture were also observed to be highly repeatable, confirming
(b)
100 74 pg mL1 the results of previous studies (Boni et al. 1997; Machado et al.
2006). AMH has been shown to be a reliable endocrine marker
80 of the population of small antral healthy follicles in bovine
Sensitivity (%)

ovaries (Ireland et al. 2008; Rico et al. 2009), and a strong

60 within-animal repeatability in the numbers of these follicles has
been reported (Burns et al. 2005; Ireland et al. 2007). Moreover,
40 we have recently shown that AMH concentrations in plasma are
independent of the occurrence of waves of terminal follicular
20 development in the cow (Rico et al. 2011). In the present study,
administration of each gonadotrophin treatment induced an
0
increase in plasma AMH concentrations, likely reflecting the
0 20 40 60 80 100
enhanced growth of small follicles; this increase is transient
100-specificity (%)
since AMH concentrations return to their initial values a few
days after ovulation in superovulated cows (Rico et al. 2009).
74 pg mL1
(c) 1.00 Altogether, these results reinforce the idea that each cow could
P  75%
be characterised by its own circulating AMH concentration,
P  50%
which reflects the size of its population of gonadotrophin-
0.75 P  41.2% responsive follicles, and they indicate that AMH measurement
P  25%
Efficiency

in plasma can be valid in the long-term, at least for 1 year.

0.50
0.25
0 ment of large follicles at oestrus, which is the primary ovarian
0 100 200 300
AMH (pg mL1)
Fig. 9. Example of ROC analysis of the dataset of Experiment 3:
determination of the AMH cut-off value for discarding cows producing
fewer than 10 embryos. Data were obtained from 34 Holstein cows in farms.
(a) Sensitivity and specificity curves. Data are expressed as percentages.
(b) ROC plot representation of the data. The AMH cut-off value of
74 pg mL
1 maximised the Youden index. The dotted line corresponds to
the null hypothesis of a non-informative test. (c) Influence of the prevalence
on the efficiency of the diagnostic test. The curves illustrate the changes in
the efficiency of the cut-off choice with AMH concentration, for a preva-
lence P varying between 10 and 75% in the population of cows. The
prevalence corresponding to several embryos ,10 was 41.2% in the studied
population of cows.
and plasma. The Active MIS/AMH ELISA kit is a commercial
immunoassay using a pair of antibodies whose target epitopes
are not specified by the manufacturer of the kit. Despite these
unsolved questions, AMH measurements can be made
P  10% Our results have confirmed that measuring AMH concentra-
tion in the plasma can predict the capacity of an individual cow
to respond to gonadotrophin treatment; furthermore, this study
provides the first evidence of a possible prognostic test for cows
bred under farm conditions. Data concerned both the develop-
400 response to gonadotrophins, relevant to OPU protocols, and
embryo production, which is a more integrated response rele-
vant to MOET protocols. They were obtained in two indepen-
dent groups of Holstein cows, in experimental station and in
farm settings, and were both submitted to ROC analysis for
diagnostic tests in order to define the AMH cut-off values for
discarding low-responding cows. The quality of the estimation
of the cut-off values and their predicting performance were
evaluated by different optimality criteria. Several of them (the
area under the ROC curve, AUC; the Youden index, J) are
independent from, and another (the efficiency, Ef ) dependent on
the prevalence of the character in the population (Greiner et al.
2000). In our analyses, AMH cut-off values were retained
(1) when the AUC for the number of follicles (or embryos)
was significantly different from the AUC under the null hypoth-
esis of a non-informative test, (2) at the maximal value of J and
(3) after pairing each AMH cut-off value with the number of
follicles (or embryos) that gave the highest specificity, sensitiv-
ity and efficiency values in the test. The AUC values found from
AMH and embryo production in the Holstein cow
Table 2. Determination of AMH cut-off values for embryo production by ROC analyses of the data obtained from 34 Holstein cows undergoing
embryo production on farms
*P , 0.05, **P , 0.01, compared with the expected value (AUC ¼ 0.5) under the null hypothesis of a non-informative test
Embryos , 10
Prevalence (%) 41.2
AMH cut-off (pg mL
1) 74
AUC 0.768**
Youden index 0.514
Efficiency 0.765
Specificity (%) 80.00
Sensitivity (%) 71.43
PPV (%) 80.00
PNV (%) 71.43
these analyses permitted us to classify the predicting perfor-
mances as highly or moderately accurate, according to an
arbitrary guideline that distinguishes between non-informative
(AUC ¼ 0.5), less accurate (0.5 , AUC , 0.7), moderately ac-
curate (0.7 , AUC , 0.9), highly accurate (0.9 , AUC , 1)
and perfect tests (AUC ¼ 1; Swets 1988). The predicting per-
formances of the AMH cut-off values were all higher for the
number of follicles than the number of embryos. These differ-
ences are likely due to differences in the nature (more or less
integrated) of the response analysed, the breeding conditions
(experimental station or farm settings) and the numbers of
animals studied, and also to the possible bias related to technical
problems at embryo collection. For example, from data obtained
under farm conditions, it was not possible to propose AMH cut-
off values to discard cows producing fewer than 1 to 9 embryos,
because either the test was statistically non-informative, or
when it was informative the AMH cut-off value was found to
be the same (74 pg mL
1) as for cows producing fewer than
10 embryos, but with a lower predicting performance. Further
studies with higher numbers of cows are needed to assess
accurately the AMH cut-off values associated to the production
of very low numbers of embryos in farm breeding conditions.
The AMH cut-off values found in the two independent
groups of Holstein cows for the characters analysed, i.e. the
number of follicles at oestrus in the first group and the number of
embryos collected in the second one, were consistent. In
Experiment 2, the 87 pg mL
1 AMH cut-off value was found
for cows with fewer than 15 large follicles at oestrus, and in
Experiment 3, the 74 and 79 pg mL
1 AMH cut-off values were
found for cows with fewer than 10 and 12 embryos, respectively.
Using these cut-off values, the predictive positive and negative
values were all above 70%, i.e. more than 70% of the cows were
adequately categorised as high- or low-responding cows when
using these cut-off values in a test. In practice, we propose that
an AMH cut-off value of between 75 and 80 pg mL
1 can be
applied to discard Holstein cows with a low capacity to respond
to gonadotrophin treatments.
The highest AMH cut-off values may be used to select cows
with an exceptionally high capacity of embryo production.
However, in both groups of cows, the highest AMH cut-off
values were associated with low predictive positive values,
indicating that 50% or more of the cows with AMH
Reproduction, Fertility and Development K
Embryos , 12 Embryos , 14 Embryos , 17
58.8 70.6 88.2
79 83 177
0.821** 0.842** 0.875*
0.607 0.650 0.683
0.794 0.794 0.912
85.71 90.00 75.00
75.00 75.00 90.00
70.59 60.00 50.00
88.24 94.74 96.43
concentrations higher than these cut-off values develop fewer
than 26 follicles at oestrus or produce fewer than 17 embryos.
Indeed, a small number of cows do not display exceptional
responses to gonadotrophin treatment, despite their very high
AMH concentrations in plasma. The hypothesis that the proba-
bility of selecting highly responding cows would rise with AMH
up to a maximum before decreasing for higher AMH values
should be confirmed with a higher number of animals. If true, the
characteristics of AMH secretion, follicular growth and gonad-
otrophin sensitivity in the cows with very high plasma AMH
concentrations should be further investigated.
In conclusion, it appears possible to discard individuals
with a low capacity for embryo production in a population of
Holstein cows from the determination of AMH concentration
in a single blood or plasma sample, taken from animals
several months before their entry into MOET or OPU protocols.
Blood or plasma samples must be recovered and frozen
quickly after sampling, and kept frozen until AMH assay. When
AMH concentrations are measured on heparinised plasma,
gonadotrophin-stimulated Holstein cows producing fewer than
15 large follicles at oestrus and fewer than 10 embryos in MOET
protocols could be discarded efficiently with plasma AMH
concentrations below 87 and 74 pg mL
1, respectively. Today,
the assessment of AMH concentration in plasma may represent a
very promising tool for the selection of embryo donor heifers
with equivalent genetic value, but the integration of AMH levels
into genomic evaluation requires further physiological and
genetic investigations. The possible consequences of excluding
cows with low AMH concentrations from MOET and OPU
schemes are not known. From our results, there was no relation-
ship between AMH concentration and milk production or body-
weight (data not shown), suggesting that excluding cows with
low AMH concentrations would not concomitantly select cows
against high milk yield or heavy carcass. There is no reason to
think that AMH is related to important agronomic traits, except
for reproduction. Recently, AMH was proposed as a diagnostic
marker for fertility (Ireland et al. 2011), but AMH concentra-
tions have not been found to differ between two groups of dairy
cows contrasted at one female fertility quantitative trait locus
(Coyral-Castel et al. 2011). The relationship between AMH
concentration and fertility should be further investigated and the
consequence of the selection of cows on the basis their plasma
L Reproduction, Fertility and Development C. Rico et al.

(a) 20
company Franche-Comté Embryon, France) and the ‘ruminant’ team of the
Experimental Unit UEPAO (Nouzilly, France) for animal management and
15 ** participation in the blood sampling.

* *
References
10
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Mathewson, L., Stanley, S. D., and Moeller, B. C. (2011). Biological
5 and clinical significance of anti-Müllerian hormone determination in
blood serum of the mare. Theriogenology 76, 1393–1403. doi:10.1016/
Number of embryos

0 Amer, S. A., Li, T. C., and Ledger, W. L. (2009). The value of measuring
0 25 50 75 100 125
AMH cut-off value (pg mL1)
(b) 20
* Asian-Australas. J. Anim. Sci. 15, 944–948.
15
*****
**
10
5 Follicular dynamics, repeatability and predictability of follicular recruit-
0 Burns, D. S., Jimenez-Krassel, F., Ireland, J. L., Knight, P. G., and Ireland,
50 75 100 125 150 175
AMH cut-off value (pg mL1)
AMH  cut-off AMH  cut-off
Fig. 10. Ability of different AMH cut-off values to discriminate cows
producing high and low numbers of embryos in response to gonadotrophin
treatment administered on farms. (a) The AMH cut-off values determined by
ROC analyses of the dataset obtained in the cows of Experiment 2 were
applied to the dataset of Experiment 3 (n ¼ 34 cows). Data represent the
number of embryos for cows with AMH concentrations higher and lower
than the cut-off values determined from the ROC analyses carried out on the
numbers of LF at oestrus; *P , 0.05, **P , 0.01, compared with the group
with AMH concentration , AMH cut-off value. (b) The AMH cut-off values
determined by ROC analyses of the dataset obtained from the cows of
Experiment 3 were applied to the same dataset (n ¼ 34). Data represent the
numbers of embryos for cows with AMH concentrations higher and lower
than the cut-off values determined from the ROC analyses carried out on the
numbers of embryos; *P , 0.05, **P , 0.01, ***P , 0.001, compared with
the group with AMH concentration , AMH cut-off value.
AMH concentrations should also be assessed on other important
traits linked to reproduction such as sexual precocity and
reproductive lifespan.
Acknowledgements
The so-called PREDICTOV pre-valorisation project was supported by
specific funding from INRA, by the REGATE INRIA/INRA Large Scale
Initiative Action and by a French fellowship from the Région Centre to
Charlène Rico. The authors thank Dr Patrice Humblot (Department of
Clinical Sciences, SLU, 750–07 Uppsala, Sweden) and Dr Eric Schmitt
(IMV Technologies) for helpful discussions and additional support to this
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