Animal Reproduction Science 167 (2016) 51–58

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Systemic and local anti-Mullerian hormone reflects

differences in the reproduction potential of Zebu and
European type cattle
Anja Stojsin- Carter a , Kiana Mahboubi a , Nathalia N. Costa b , Daniel J. Gillis c ,
Timothy F. Carter a , Michael S. Neal d , Moyses S. Miranda b , Otavio M. Ohashi b ,
Laura A. Favetta a , W. Allan King a,∗
a
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road, Guelph, ON N1G 2W1, Canada
b
Instituto de Ciências Biológicas, Universidade Federal do Pará, Rua Augusto Corrêa 1, Belém, PA 66075-110, Brazil
c
School of Computer Science, University of Guelph, 50 Stone Road, Guelph, ON N1G 2W1, Canada
d
ONE Fertility, 3210 Harvester Rd, Burlington, ON L7N 3T1, Canada

a r t i c l e i n f o a b s t r a c t

Article history: This study was conducted to evaluate plasma anti-Mullerian hormone (Pl AMH), follic-
Received 31 August 2015 ular fluid AMH (FF AMH) and granulosa cell AMH transcript (GC AMH) levels and their
Received in revised form 28 January 2016 relationships with reproductive parameters in two cattle subspecies, Bos taurus indicus
Accepted 1 February 2016
(Zebu), and Bos taurus taurus (European type cattle). Two-dimensional ultrasound exami-
Available online 3 February 2016
nation and serum collection were performed on Zebu, European type and crossbreed cows
to determine antral follicle count (AFC), ovary diameter (OD) and Pl AMH concentration.
Keywords:
Slaughterhouse ovaries for Zebu and European type cattle were collected to determine FF
Anti-Mullerian hormone
AMH concentrations, GC AMH RNA levels, AFC, oocyte number, cleavage and blastocyst
Zebu cattle
European type cattle rate. Additionally GC AMH receptor 2 (AMHR2) RNA level was measured for European type
Plasma cattle. Relationship between AMH and reproductive parameters was found to be signifi-
Follicular fluid cantly greater in Zebu compared to European cattle. Average Pl AMH mean ± SE for Zebu
Granulosa cell and European cattle was 0.77 ± 0.09 and 0.33 ± 0.24 ng/ml respectively (p = 0.01), whereas
average antral FF AMH mean ± SE for Zebu and European cattle was 4934.3 ± 568.5 and
2977.9 ± 214.1 ng/ml respectively (p < 0.05). This is the first published report of FF and
GC AMH in Zebu cattle. Levels of GC AMHR2 RNA in European cattle were correlated to
oocyte number (p = 0.01). Crossbred animals were found more similar to their maternal
Zebu counterparts with respect to their Pl AMH to AFC and OD relationships. These results
demonstrate that AMH reflects differences between reproduction potential of the two cat-
tle subspecies therefore can potentially be used as a reproductive marker. Furthermore
these results reinforce the importance of separately considering the genetic backgrounds
of animals when collecting or interpreting bovine AMH data for reproductive performance.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction

The genetic background of cattle significantly impacts

their reproductive performance. It is believed that
∗ Corresponding author. increased inbreeding to achieve maximal milk yield has
E-mail address: waking@uoguelph.ca (W.A. King).

http://dx.doi.org/10.1016/j.anireprosci.2016.02.003
0378-4320/© 2016 Elsevier B.V. All rights reserved.
52 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58

negatively impacted reproductive efficiency in temperate,
Bos taurus taurus (European type cattle) animals (Bachelot
and Binart, 2007; Leroy et al., 2015). By comparison, the
tropical and subtropical subspecies of cattle, Bos taurus
indicus (Zebu), generally found in India and South America,
are known for lower milk yields, but greater reproductive
efficiency (Bó et al., 2003). In a direct in vitro production
(IVP) comparison with European type cattle vs. Zebu cattle
reported blastocyst rate was 25.6% and 32.1% respectively,
which ultimately gave rise to two thirds more pregnan-
cies (Viana et al., 2010). However, Zebu were found to
have lower breeding capacity due to delayed puberty,
decreased estrous duration and intensity, longer gestation
length and postpartum period, and lower twinning rate
compared to European type cattle (Abeygunawardena and
Dematawewa, 2004; Bó et al., 2003; Silva-Santos et al.,
2011). Taken together, the greater embryo and pregnancy
rate but lower breeding capacity make Zebu cattle an ideal
candidate for in vitro embryo production (IVP). To date,
there is an incomplete understanding of the underlying
endocrine, molecular and physiological factors necessary
to achieve optimal IVP protocols in Zebu and European type
cattle.
Anti-Mullerian hormone (AMH) is a 140 kDa glycopro-
tein dimer member of the TGF-beta protein subfamily
secreted by sertoli cells in testes, and by granulosa cells
(GC) in the ovary reviewed by (di Clemente et al., 2003).
In females AMH is believed to inhibit premature follicu-
lar growth and maturation, making it the ovarian reserve
“guardian” (Visser et al., 2006). Its minimal intra and inter
estrous variability makes it a useful predictor of ovarian
reserve (Fanchin, 2003; Ireland et al., 2007). The exact
mechanism by which AMH acts in the ovary is not certain,
but it is believed to be through decreasing GC sensitivity
to luteinizing hormone (LH) and follicle stimulating hor-
mone (FSH) (Visser and Themmen, 2005). Due to its link
with ovarian reserve, AMH is used extensively as a quan-
titative marker of fertility in humans, and is also being
increasingly examined as a potential biomarker of repro-
ductive efficiency in other species, including cattle (Ireland
et al., 2011; Monniaux et al., 2011; Rico et al., 2011). Various
human studies have reported correlation of AMH concen-
tration to a number of reproductive parameters related to
the oocyte developmental potential, such as fertilization,
implantation and pregnancy rates (Fanchin et al., 2007;
Majumder et al., 2010; Takahashi et al., 2008). In addition
to several reports of AMH in European type cattle (Ireland
et al., 2009; Ireland et al., 2008) there are three published
investigations involving Zebu cattle (Guerreiro et al., 2014;
Baldrighi et al., 2014; Batista et al., 2014). These studies
focused on comparing plasma AMH (Pl AMH) concentration
between Zebu and European type cattle and correlating to
the number of antral follicles (AFC) as a quantitative repro-
ductive parameter. The findings of these studies provide
evidence for plasma AMH (Pl AMH) to AFC correlation with
Zebu animals displaying greater AMH values for the same
AFC compared to European type animals.
The aim of the current study was to investigate and com-
pare the relationship between both systemic and local AMH
and reproductive parameters: AFC, ovary diameter (OD),
oocyte number, cleavage and blastocyst rate in both Zebu
and European type cattle, as well as a small cohort of cross-
bred animals. It was hypothesized that irrespective of its
source, AMH correlated to reproductive parameters with
Zebu having greater AMH value compared to European type
cattle.
2. Materials and methods
2.1. Animals and experimental design
2.1.1. Study group 1
B. taurus indicus or Zebu (Nelore breed, n = 30), B. taurus
taurus or European type cattle (Red Angus breed, n = 10),
and crossbred B. taurus taurus × B. taurus indicus (Bran-
gus breed, n = 10) cows of average reproductive age (mean
ages 8.6, 7.8 and 7.1 years respectively) raised on pasture
in Paraná state, Brazil were subject to ovarian examina-
tion of AFC and OD with the 2-D trans-rectal Mindray
adapted with a linear transductor ultrasound, and periph-
eral blood collection via puncture of the tail vein. All
sampling was done at once irrespective of the estrous

Table 1
Real Time PCR primers.

Gene GenBank accession Sequence Reference Size (bp)

number

AMH NM 173890.1 F-5 - (Ireland et al. (2009) 270

CAGGGAAGAAGTCTTCAGCA- Scheetz et al. (2012)
3
R-5 -
AAGGTGGTCAAGTCACTCAG-
3
GAPDH NM 001034034.2 F-5 -TTCCT Hamilton et al. (2011) 153
GGTACGACAATGAATTTG-
3
R-5 -GGAGATGGGG
CAGGACTC-3
AMHR2 NM 001205328.1 F-5 - Monniaux et al. (2011) 163
GTGCTTCTCCCAGGTCATAC-
3
R-5 -
AATGTGGTCATGCTGTAGGC-
3
 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58
cycle points (Rico et al., 2011). Blood was collected in
EDTA tubes (BD Vacutaner® , BD Franklin Lakes NJ, USA)
to allow for more consistent AMH ELISA measurement.
The Bovine AMH ELISA kit AL-114 (Ansh Labs, Texas, USA)
was used according to the manufacturer’s protocol with
the exception of an overnight separation of plasma at
room temperature following the MOFA® Minitube Canada
protocol for AMH testing. Samples were frozen at −20 ◦ C
following collection until ELISA measurement was per-
formed.
2.1.2. Study group 2
Post-mortem collection of ovaries from unsynchro-
nized, non-pregnant pasture raised Zebu (Nelore breed,
n = 12) and European type cattle (Holstein breed, n = 56)
raised under standard dairy farming conditions, was
performed in government-approved slaughterhouses in
Belem, Pará state, Brazil and Guelph, Ontario, Canada,
respectively. For the Zebu cattle, each animal had a match-
ing blood sample as well as a pair of ovaries. Blood was
collected during exsanguination at the time of slaughter
and was left overnight in EDTA tubes for separation of
plasma from blood cells at room temperature. For Euro-
pean type cattle, AFC was determined by visual counting
of the antral follicles per ovary. For both cattle subspecies,
antral follicles, 5–8 mm in diameter, for each ovary were
aspirated and pooled. After collection, pooled supernatant
FF was carefully removed without disturbing the cell pellet,
that has settled at the bottom of the tube by the time collec-
tion was completed, and frozen at −20 ◦ C until AMH ELISA
measurement. The pooled pellet was resuspended with
HEPES-buffered Ham’s F-10 plus 2% steer serum (Cansera;
Rexdale, ON, Canada) and snap frozen at −80 ◦ C until RNA
extraction was performed. The cumulus–oocyte complexes
(COC) were collected, counted and put into in vitro culture,
while the rest of the cells were frozen for qPCR analysis.
For investigations into mRNA expression, the total num-
ber of oocytes per ovary or ovary pair was counted. Samples
were grouped into “low” or “high” oocyte producing ovary
if they contained less than 9 or more than 19 oocytes
per ovary, respectively. These cut offs were determined by
using 25th and 75th percentiles of the ovary that contained
the highest number of oocytes in order to choose the sam-
ples with the potentially most obvious difference in AMH
expression.
2.2. IVF
Bovine ovaries from the local abattoir were transported
to the laboratory in a 0.9% saline solution at 33–37 ◦ C. IVP
was preformed as described by Ashkar et al. (2010) for
samples obtained in Canada and by Costa et al. (2013)
for samples obtained in Brazil with the exception that
cumulus–oocyte complexes (COCs) were kept separate per
each ovary. A maximum of 10 oocytes were matured, fer-
tilized and cultured per medium drop. Number of oocytes
was assessed at the time of collection, cleavage rate was
assessed on Day 2, and blastocyst rate on Day 8.
53
2.3. ELISA
AMH was measured using the Bovine AMH ELISA kit
AL-114 (Ansh Labs, Texas, USA) according to the manufac-
turer’s protocol. Before measurement, FF was first diluted
between 1:500 and 1:10,000× using the supplied dilu-
ent and placed in EDTA tubes at 4 ◦ C overnight. AMH was
measured using 50 ␮L of Pl or diluted FF sample. The pro-
vided analytical range of the assay was 0.156–10 ng/mL,
and the analytical sensitivity 0.011 ng/mL. The intra-assay
coefficients of variation were 2.92%, 2.54% and 3.65% for
quality control plasma samples containing 0.611, 1.259 and
2.56 ng/mL AMH, respectively.
2.4. RNA extraction and reverse transcription
RNA was extracted from the GC pellets using a TRI-
zol reagent (Invitrogen, Cincinnati, OH, USA) according
to the manufacturer’s instructions and previous publica-
tions (Favetta et al., 2004; Scheetz et al., 2012). In brief,
each pellet was homogenized in 0.5 mL of TRIzol Reagent.
Phase separation was achieved by adding chloroform, and
RNA was precipitated from the aqueous phase with iso-
propanol. Samples were incubated at −20 ◦ C overnight
and centrifuged at 10,000 × g at 4 ◦ C. The RNA pellet was
washed with 75% ethanol and dissolved in 20 ␮L of ster-
ile RNase-free water. Contaminants were removed with
DNase I treatment (Ambion, Huston, TX, USA) and total
RNA concentration was measured with a spectrophotome-
ter. RNA samples were stored at −80 ◦ C until further use.
Reverse transcription PCR was performed using the high-
capacity complementary DNA (cDNA) reverse transcription
kit (Applied Biosystems, Auckland, New Zealand) following
manufacturer’s protocol. Briefly, 500 ng of oligo(dT) was
first added to each RNA sample and incubated at 70 ◦ C for
2 min. A mixture of 4 ␮L of RT buffer, 1 ␮L of 0.1 M dithio-
threitol, 1 ␮L of 10 mM deoxyribonucleotide triphosphates
(dNTPs) mix, 0.5 ␮L of RNasin (40 U/␮L; Promega, Madison,
WI, USA), and 1 ␮L of Superscript II (200 U/␮L; Invitrogen)
was then added and the reaction was incubated further
at 42 ◦ C for 1 h, followed by a final incubation at 70 ◦ C for
30 minutes. cDNA samples were stored at −20 ◦ C until fur-
ther use.
2.5. Real time quantitative PCR
Real-time quantitative PCR (RT-qPCR) was carried out
to determine AMH transcript levels using the BIO-RAD
CFX96TM Real-Time PCR System and SsoFastTM Eva-
Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA)
according to a standard protocol that had been tem-
perature optimized for the AMH, GAPDH and AMHR2
primers (Table 1 . Prior to quantification, optimization
procedures were performed by running qPCRs with and
without the purified template to identify the melting
temperatures of the primer dimers and specific prod-
uct. Each reaction contained 5 ␮L of SsoFastTM EvaGreen
Supermix reaction mix, 1 ␮L of a mix of the forward
and reverse primers at a concentration of 2.5 ␮mol, 2 ␮L
of the cDNA of interest and was adjusted to a total
volume of 10 ␮L using H2 O. The standard curve was
54 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58

Summary of antral follicle count (AFC), ovary diameter (OD), plasma anti-Mullerian hormone (Pl AMH) from Study group 1, and follicular fluid AMH (FF AMH), oocyte, cleavage and blastocyst rate mean values
established using ovarian tissue cDNA template in five

Blastocyst rate/ovary

18.94 ± 2.96 (n = 32)

25.05 ± 6.34 (n = 24)
serial dilutions ranging from 1 ␮g/␮L to 0.03125 ␮g/␮L of
cDNA. The amplification program was as follows: 95 ◦ C

(± SE) from Study group 2 for European type (E), Zebu (Z) and crossbred (C) cattle and the P-values from their average value comparisons. Significant difference (P < 0.05) is indicated by the symbol (*).
for 10 min, followed by 50 amplification cycles of 95 ◦ C
for 10 s, 65 ◦ C for 10 s, 72 ◦ C for 10 s and acquisition of
fluorescence for 10 s. After the end of the last cycle, a melt-
ing curve was generated by taking measurements every
0.5 ◦ C from 72 ◦ C to 95 ◦ C. Glyceraldehyde-3-Phosphate
Dehydrogenase (GAPDH) transcript was selected as the

Cleavage rate/ovary

70.92 ± 4.55(n = 32)

76.77 ± 5.99(n = 24)
housekeeping gene, as it showed the most consistent
levels of mRNA expression among the samples tested
using geNorm software (Vandesompele et al., 2002). Other
housekeeping genes tested were histone (H2 A), tyrosine 3-
monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta (YWHAZ), hypoxanthine guanine phosphori-
bosyl transferase 1 (HPRT1) and succinate dehydrogenase
complex, subunit A (SDHA). Relative quantification of the

11.58 ± 1.83(n = 24)

17.5 ± 1.60 (n = 56)
transcripts of interest (Table 1) were calculated between

Oocyte #/ovary
two subspecies and between the low and high oocyte pro-
ducing ovaries.

0.064
2.6. Statistics

For statistical investigation Student’s t-test was used

to compare parameters between the subspecies, while

2977.9 ± 214.1(n = 56)

4934.3 ± 568.5(n = 24)
Spearman correlation, and multivariable linear models

(ng/mL)
were used to compare parameters within the subspecies

Study group 2
with P < 0.05 considered statistically significant. For exam-
ple, referring to Fig. 1Ai which outlines the relationship

*<0.0001
FF AMH
between Plasma AMH and the AFC, the following linear
model was considered: Pl AMH = Intercept + AFC + Breed.
Here the Breed is a categorical variable. This allows us to
model the Pl AMH for each breed, and compare the dif-
ferences between them. To compare differences between 0.77 ± 0.09(n = 30)
0.63 ± 0.07(n = 10)
0.33 ± 0.24 (n = 8)
Pl AMH (ng/mL)

breeds, we use a Tukey test that is an option within the

general linear hypothesis test (glht) from the multcomp
package in R (Team, 2009). The test allows us to estimate
*0.018
*0.010

0.532
the differences in intercepts between the breeds, assuming
that the slope (related to the AFC) is constant for all breeds.
This was done due to the available sample size. A similar
modeling exercise was performed comparing Pl AMH to
57.66 ± 1.17(n = 30)
66.20 ± 3.50 (n = 7)

59.03 ± 3.45 (n = 6)

OD (Fig. 1Aii), and FF AMH to oocyte # (Fig. 1Bii). Pl AMH

numbers were calculated using GraphPad Prism (GraphPad
Software, San Diego, CA, USA).
OD (mm)

*0.008
0.138
0.581

3. Results

3.1. Study group 1

27.37 ± 1.83(n = 30)
25.83 ± 10.55(n = 6)
17.14 ± 1.82 (n = 7)

Zebu and crossbred animals had significantly greater

Study group 1

mean Pl AMH concentration than their European coun-

terparts (P = 0.010, P = 0.018 respectively; Table 2. The Pl
AMH mean concentration for Zebu, European type cat-
*0.027
*0.006

0.782
AFC

tle and crossbred cattle were 0.77 ± 0.09, 0.33 ± 0.24 and
0.63 ± 0.07 ng/mL respectively (Table 2). The correlation
between Pl AMH and AFC was significantly different when
comparing either Zebu or crossbred to European type cat-
Crossbred (C)
European (E)
Zebu (Z)

tle (P = 0.004 and P = 0.049 respectively; Fig. 1Ai); the same

P-values

result was observed for Pl AMH and OD between Zebu

E vs. C
Z vs. C
E vs. Z
Table 2

and European type cattle (P = 0.009; Fig. 1Aii). The corre-

lation between Pl AMH and AFC in Zebu was significantly
 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58 55

Fig. 1. Correlations between anti-Mullerian hormone (AMH) and reproductive parameters in European type (E, light gray square), Zebu (Z, black triangle)
and crossbred (C, dark grey diamond) cattle. A) representing Study group 1 and B) representing Study group 2. Ai) Plasma AMH (Pl AMH) vs. antral follicle
count (AFC) for E (n = 7), Z (n = 30) and C (n = 6) animals. Z correlation, r = 0.54, p = 0.002; Z to E comparison, p = 0.004; C to E comparison, p = 0.049. Aii) Pl
AMH concentration vs. ovarian diameter (OD) in E (n = 6), Z (n = 26) and C (n = 6) animals. Z correlation, r = 0.36, p = 0.067; Z to E comparison, p = 0.009; C to E
comparison, p = 0.094. Bi) Pl AMH vs. average number of oocytes per pair in Z (n = 12; r = 0.818, p = 0.002); Bii) Follicular fluid AMH (FF AMH) concentration
vs. number of oocytes in Z (n = 24), and E (n = 59) cattle. E correlation, r = 0.37, p = 0.005; Z to E comparison, p < 0.0001. Biii) FF AMH concentration (ng/ml)
vs. AFC in E (n = 56; r = 0.35, p = 0.009).

positive (P = 0.002; Fig. 1Ai). The European type cattle had
significantly lower AFC compared to Zebu and crossbreed
(P = 0.006, P = 0.027 respectively; Table 2) and larger OD
(P = 0.008; Table 2) compared to Zebu animals.
3.2. Study group 2
In a subset of Zebu animals, matched plasma samples
and ovary samples were available and it was possible to
observe a significant and positive correlation between Pl
AMH and number of oocytes per ovary pair (P = 0.002;
Fig. 1Bi), with the Pl AMH mean value and the mean oocyte
number of 1.15 ± 0.27 ng/ml and 11.58 ± 1.83, respectively.
56 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58
Mean FF AMH concentration was found to be sig-
nificantly greater in Zebu compared to European type
cattle (P < 0.0001; Table 2). The FF AMH mean for
Zebu and European type cattle was 4934.3 ± 568.5 and
2977.9 ± 214.1 ng/mL respectively (Table 2). The corre-
lation between FF AMH and oocyte number per ovary
was significantly different between Zebu and European
type cattle (P < 0.0001; Fig. 1Bii), and only European
type showed significantly positive correlation (P = 0.005;
Fig. 1Bii). A positive correlation was not observed between
FF AMH and cleavage rate (70.92 ± 4.55 for European and
76.77 ± 5.99 for Zebu cattle Table 2). A positive correla-
tion was not observed between FF AMH and blastocyst rate
(18.94 ± 2.96 for European and 25.05 ± 6.34 for Zebu cat-
tle, Table 2). In a subset of European type animals where
ovaries were collected post mortem, it was also possible
to compare FF AMH concentration to AFC, and a significant
positive correlation was observed (P = 0.009; Fig. 1Biii).
The expression of AMH GC mRNA was not found to be
significantly different between either low or high oocyte
producing ovary groups, or between European type and
Zebu cattle (Fig. 2A). The expression of AMHR2 mRNA
was found to be significantly different between low and
high group for European type cattle (P = 0.035; Fig. 2B) and
positively correlated to oocyte number in this subspecies
(P = 0.01).
4. Discussion
The aim of the current study was to investigate and
compare the relationship between both systemic and local
AMH and reproductive parameters: antral follicle count,
ovary diameter, oocyte number, cleavage and blastocyst
rate in both Zebu and European type cattle, as well as a
small cohort of crossbred animals. Preliminary research
published by Batista et al. (2014) and Baldrighi et al.
(2014) examined and revealed for the first time that plasma
AMH and AFC relationship is greater in Zebu compared to
European type cattle. The current report expands their pre-
liminary research by providing evidence that AMH, from
both sources Pl and FF, is significantly different between
Zebu and European type cattle for antral follicle count,
ovary diameter and oocyte number with Zebu display-
ing greater AMH values. Plasma and FF AMH was found
correlated with two of the reproductive parameters exam-
ined, AFC and oocyte number. Additionally for the first
time in European type cattle GC AMHR2 RNA expression
was examined and found positively correlated to oocyte
number. The sequencing of the AMHR2 cDNA product was
performed and confirmed 100% homology to the bovine
AMHR2 (GenBank accession number NM 001205328.1).
Plasma AMH concentration was also measured in crossbred
cattle and found to more closely mirror their maternal Zebu
concentration.
The European type cattle FF AMH concentration dis-
played a significant positive correlation to the quantitative
reproductive markers: AFC and number of oocytes. A pos-
itive trend yet non-significant was also observed when
comparing Pl AMH to AFC. Our results for AMH in Euro-
pean type cattle are in general agreement with the current
literature, however none of the published work to our
knowledge directly compared AMH from both Pl and FF
between two subspecies of cattle. In the current study, FF
AMH in bovine subspecies was not found to have a signif-
icant correlation with cleavage nor blastocyst rate, which
is in agreement with previous bovine studies correlating
reproductive parameters to oocyte quality (Guerreiro et al.,
2014; Ireland et al., 2007). Nevertheless in human studies,
AMH from both FF and serum has been found to corre-
late to qualitative oocyte markers such as fertilization and
implantation rates (Fanchin et al., 2007; Majumder et al.,
2010; Takahashi et al., 2008). The study by Kawamura
et al. (2013) speculated that AFC as a quantitative marker
has a direct effect on the ovarian structure and morphol-
ogy and therefore should have an effect on the quality
of oocytes within the ovary. Further study looking at the
oocyte quality parameters would be required to determine
if the absence of the relationship between the quantitative
and qualitative markers is the result of inherent species
differences or factors related to in vitro culture conditions.
Granulosa expression measurements were performed
in the low and high oocyte yielding ovaries where the dif-
ference in the protein and transcript amounts would be
expected to be the greatest. GC AMH RNA expression mea-
surements from low and high oocyte yielding ovaries did
not appear to differ in the current study. Previously it has
been demonstrated that even a 1 mm size change in folli-
cle is associated with an exponential expression difference
in AMH level due to its crucial role in follicle growth and
maturation (Rico et al., 2011). This would lead to variability
of the AMH GC expression data within both high and low
yielding groups, and possibly result in the absence of the
expected difference. It could be speculated that other deter-
minants beside follicle size also need to be measured and
considered during comparison. Significant positive corre-
lation was observed between GC AMHR2 RNA levels and
number of oocytes, as well as significant difference in lev-
els between the low and high oocyte producing ovaries. The
measured levels of AMHR2 transcript reported here are in
accordance with previously published results in goat GCs
(Monniaux et al., 2011).
Zebu cattle are known for their greater in vitro embryo
and pregnancy rate but lower breeding capacity. Beyond
their different IVP rates and estrous cycle characteristics,
the underlying physiological and endocrine bases for their
different reproductive potential is not clear. In this study,
Zebu Pl AMH concentration was significantly positively
correlated with reproductive parameters: AFC and average
number of oocytes per pair of ovaries, as well as FF AMH
and number of oocytes, which indicates that AMH irrespec-
tive of being systemic or local can be used as a quantitative
reproductive marker. Overall this is in general agreement
with the current AMH model and patterns reported in pre-
vious human, European type cattle and Zebu experiments
by Baldraghi et al. (2014) and Batista et al. (2014). Impor-
tantly, although Zebu and European type cattle appear to
follow the same patterns of positive correlation, the AMH
values and correlated parameters were significantly differ-
ent in every case, with Zebu expressing significantly greater
AMH concentrations compared to European type cattle at
matched parameter values. This result demonstrates the
 A.S. Carter et al. / Animal Reproduction Science 167 (2016) 51–58
Fig. 2. A) Relative granulosa cell anti-Mullerian hormone (GC AMH) mRNA expression in low vs. high oocyte producing ovaries for European type (grey,
n = 56) and Zebu (black, n = 10) cattle. B) Relative GC AMH and anti-Mullerian hormone receptor 2 (AMHR2) mRNA expression in low (light gray) vs. high
(dark gray) oocyte producing ovaries for European type cattle (n = 56) was found to be significantly different (P = 0.035). Glyceraldehyde 3-phosphate
dehydrogenase housekeeping gene used to determine relative expression level, error bars denote the standard error of the mean (SEM) and significant
difference (P < 0.05) is indicated by the symbol (*).
importance of species and subspecies when interpreting
AMH data.
The crossbred cattle included in this study appeared
to be intermediate between the two subspecies, but more
comparable to their Zebu counterparts. The crossbred cows
were created using semen from European type bulls to
inseminate the Zebu cows despite being considered a 3/8
Zebu, 5/8 European type cattle cross on a genetic level. This
also underscores the importance of considering the genetic
background of animals when conducting and interpreting
bovine AMH studies.
5. Conclusion
The current experiments provide evidence of a com-
mon pattern in the relationships between AMH levels and
reproductive parameters in both bovine subspecies, with
AMH reflecting greater reproduction potential of Zebu sub-
species compared to European type subspecies. Also this
study demonstrates the importance of separately con-
sidering animals of different genetic backgrounds when
conducting AMH related experiments, analysis or data
interpretation. The results of this study will help to fur-
ther refine the AMH model and its utility as a reproductive
marker in future experiments and cattle IVP and breeding
programs.
Conflict of interests
Authors declare no conflict of interest.
Acknowledgements
The authors would like to acknowledge the help and
support of Tobias C. Sovernigo, Marcos A. Filho, and Paulo
R. Adona from Central Avançada de Biotechnologia em
Reproducion, Universidade Norte do Paraná and the fruit-
ful discussions and help in manuscript preparation of Dr.
Teresa Mogas Amorós of the Universitat Autonoma de
Barcelona.This work was supported by the OVC Doctoral
57
Scholarship, the Natural Sciences and Engineering Research
Council of Canada (NSERC), ONE Fertility, Canada Research
Chairs (CRC), and Coordenação de Aperfeiçoamento de Pes-
soal de Nível Superior Brazil (CAPES) and the Government
of Canada’s Department of Foreign Affairs and International
Trade (DFAIT).
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