J. Dairy Sci.

97:6888–6900
http://dx.doi.org/10.3168/jds.2014-7908
© American Dairy Science Association®, 2014.

Plasma anti-Müllerian hormone in adult dairy

cows and associations with fertility
E. S. Ribeiro,* R. S. Bisinotto,* F. S. Lima,* L. F. Greco,* A. Morrison,† A. Kumar,† W. W. Thatcher,*
and J. E. P. Santos*1
*Department of Animal Sciences, University of Florida, Gainesville 32611
†Ansh Labs Inc., Webster, TX 77598

ABSTRACT ated positively with maintenance of pregnancy at the

first postpartum AI and with pregnancy rate in cows
The objectives of this study were to identify fac- inseminated after detection of spontaneous estrus.
tors associated with concentrations of anti-Müllerian Synchronization of ovulation might override positive
hormone (AMH) in plasma of dairy cows and to in- associations between AMH and fertility.
vestigate the relationships between plasma AMH and Key words: anti-Müllerian hormone, dairy cow, fertil-
fertility responses during a 100-d breeding season. Lac- ity, ovarian reserve
tating cows, 1,237 in 2 seasonally calving herds, had
estrous cycles presynchronized and were enrolled in a
timed artificial insemination (AI) protocol. All cows INTRODUCTION
were inseminated on the first day of breeding season,
considered study d 0. Blood was sampled on d −8 and Female mammals are born with a variable number of
analyzed for concentrations of AMH and progesterone. healthy follicles in their ovaries (Block, 1952; Erickson,
From d 19 to 35, detection of estrus was performed 1966). They constitute the ovarian reserve from which
daily and cows detected in estrus were reinseminated. primordial follicles will be activated and recruited into
On d 36, bulls were placed with cows for 65 d of natu- the pool of growing follicles to eventually undergo atre-
ral service breeding. Factors identified to be associated sia or ovulation (Scaramuzzi et al., 2011). Whether this
with concentrations of AMH in plasma were breed of reserve is renewable or not in postnatal life has been
the cow and lactation number. Concentrations of AMH the focus of extensive debate in the last decade and
were greater for Jerseys, followed by crossbreds, and little is known about the renewal or lack thereof of
then Holsteins. Cows on lactations 2 and 3 had greater the ovarian reserve in livestock species (Woods et al.,
concentrations of AMH than those on lactations 1 and 2013). Nevertheless, recent findings have associated the
≥4. Although pregnancy per AI at the timed AI was ovarian reserve in cattle with number of follicles at all
not associated with concentrations of AMH, cows with stages of folliculogenesis, ovarian function, and some
low AMH had greater detection of estrus at timed AI, measures of fertility (Ireland et al., 2011). These find-
and the latter benefited pregnancy per AI, particularly ings are important for the dairy industry considering
in cows that had low progesterone at the beginning of the effect of reproductive performance on the profit-
the synchronization protocol. Pregnancy loss between ability of dairy herds (Ribeiro et al., 2012b).
gestation d 30 and 65 was greater in cows with low Antral stages in folliculogenesis in cattle can be
AMH compared with those with intermediate or high evaluated by ultrasonography with no need for ex vivo
AMH. Return to estrus in cows that failed to become histology to reliably estimate their population. Re-
pregnant from the timed AI was not associated with cently, it has been evidenced that the number of antral
AMH, but pregnancy rate in cows bred on estrus (rein- follicles [antral follicle count (AFC)] was highly corre-
semination + natural service) was associated positively lated with the total number of follicles across all stages
with AMH. In conclusion, breed and lactation number of development (Ireland et al., 2008). In addition, AFC
were identified to be associated with concentrations of was found to be highly variable among adult cattle, but
AMH in plasma. Concentration of AMH was associ- repeatable within individuals over time, which allows
its use for phenotyping (Singh et al., 2004; Burns et
al., 2005; Ireland et al., 2007). In a model comparing
cows with low versus high AFC, the later was associ-
ated positively with fertility traits such as progesterone
Received January 6, 2014.
Accepted June 29, 2014. concentration in plasma, endometrial thickness early
1
Corresponding author: Jepsantos@ufl.edu in the luteal phase (Jimenez-Krassel et al., 2009), and
6888
 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6889

pregnancy rates in beef heifers (Cushman et al., 2009) MATERIALS AND METHODS
and lactating dairy cows (Mossa et al., 2012). Further-
Cows, Pasture, and General Management
more, differences have been reported for secretion of
gonadotropins and sensitivity of follicular and luteal The study was conducted on 2 commercial dairy
cells to gonadotropins according to the AFC in cows farms located in Trenton, Florida. Both herds were
(Singh et al., 2004; Ireland et al., 2008; Jimenez-Krassel seasonally calving and had similar genetics and man-
et al., 2009; Mossa et al., 2010). agement practices. A total of 1,237 lactating dairy cows
Antral follicle count is positively and highly associ- (457 Holsteins, 185 Jerseys, and 621 Holstein-Jersey
ated with concentrations of anti-Müllerian hormone crossbreds) were enrolled in the study. Crossbred cows
(AMH) in plasma of cattle (Ireland et al., 2008), and population was mostly composed by F1 (50/50) and F2
AMH has also been used as a marker for the ovarian re- (25/75) generation of crossbreeding between Holstein
serve (Ireland et al., 2008; Rico et al., 2009). The AMH and Jersey genetics. In both herds, different genetic
is a dimeric glycoprotein member of the transforming groups were managed together in a pasture-based sys-
growth factor β superfamily produced exclusively by tem. Cows were maintained under irrigated pasture
granulosa cells of growing follicles in females (Monniaux paddocks of 2.7 ha and managed in a daily rotational
et al., 2013). Similar to AFC, concentration of AMH in method, allowing 15 d for the resting period. Stocking
plasma varies little throughout the estrous cycle, which rate for the total area of pasture was approximately
facilitates its use for phenotyping cattle using a single 10 cows/ha. The pasture was composed of annual
measurement at random stages of the estrous cycle ryegrass (Lolium multiflorum Lam.) during winter and
(Ireland et al., 2008, 2011; Rico et al., 2009). Concen- early spring (cool season) and perennial Bermudagrass
tration of AMH has been successfully used as a marker Tifton 85 (Cynodon spp.) during late spring, summer,
to predict responses to multiple ovulation treatments and fall (warm season). Cows were milked twice a day
and number of oocytes recovered by ovum pick-up in and the average milk production per cow was approxi-
cows (Singh et al., 2004; Ireland et al., 2007; Rico et mately 6,000 kg/lactation. During and immediately
al., 2012), but it has not been tested as a predictor of after each milking, cows were offered variable amounts
fertility responses in cows after breeding. of concentrate (7 to 13 kg as fed/cow per day) accord-
Recently, it has become evident that folliculogenesis ing to forage availability and stage of lactation. The
is controlled not only by endocrine actions of gonadotro- concentrate was based on corn meal, corn gluten feed,
pins and steroids, but also by paracrine and autocrine soybean meal, soybean hulls, citrus pulp, whole cotton-
activity of factors secreted by the oocyte and follicular seed, cottonseed hulls, molasses, and a mineral-vitamin
cells (Scaramuzzi et al., 2011). Among them, AMH is premix, and designed to contain approximately 15%
suggested to reduce the rate of progression through the CP, 4.5% fat, and 28% NDF.
gonadotropin responsive phase of folliculogenesis and
number of ovulations in sheep (Campbell et al., 2012), Reproductive Management
as well as reduce activation of primordial follicles and
sensitivity of growing follicles to FSH in rodents and All cows in both herds were enrolled in a preplanned
humans (Durlinger et al., 2002; Visser and Themme, breeding season of 100 d. All cows received a timed AI
2014). The expression of AMH in granulosa cells is in the first day of breeding season (study d 0) preceded
timely and spatially dynamic during follicle develop- by estrous cycle presynchronization and synchroniza-
ment, and inversely related to aromatase expression and tion programs (Figure 1). The presynchronization
estradiol production (Ireland et al., 2009; Monniaux et consisted of 2 injections of PGF2α (Lutalyse sterile
al., 2013). Whether the potential roles of AMH on fol- solution; 5 mg/mL of dinoprost tromethamine, Zoetis,
liculogenesis are linked with subsequent reproductive Madison, NJ) administered 14 d apart. The timed AI
performance warrants investigation. protocol was initiated 11 d after the second PGF2α of
We hypothesized that concentration of AMH in the presynchronization program and involved an injec-
plasma of lactating dairy cows in seasonally calving tion of GnRH (Cystorelin; 50 μg/mL of gonadorelin
herds would be variable among individuals, but posi- diacetate tetrahydrate, Merial Ltd., Inselin, NJ) on d
tively associated with fertility in the breeding season. −8, PGF2α on d −3 and −2, and GnRH and AI on d 0.
Therefore, our objectives were to characterize the On d −3, tailheads were painted using an oil-based tail
concentrations of AMH in plasma in a large number paint (Donaghy tail paint, Donaghy, Dunedin, NZ) for
of dairy cows, identify factors associated with AMH detection of estrus on the day of AI based on removal
concentrations, and investigate associations between of tail paint. The first postpartum insemination was
AMH and fertility responses in dairy cows during a performed by the same 9 technicians in on both farms;
breeding season. semen from 12 sires was used.
Journal of Dairy Science Vol. 97 No. 11, 2014
6890 RIBEIRO ET AL.

Figure 1. Diagram of activities throughout the experiment. Blood samples were collected for analyses of concentrations of anti-Müllerian
hormone (AMH) and progesterone in plasma using chemiluminescence and radioimmunoassays, respectively. Presynchronization consisted of 2
injections of PGF2a on d −33 and −19. The timed AI program was initiated 11 d after the presynchronization protocol and was comprised of
GnRH on d −8, PGF2α on d −3 and −2, and GnRH on d 0 concurrent with AI. Pregnancy diagnoses were performed on d 30 and 65 after timed
AI, on d 70 ± 7 after reinseminations, and each 35 d after introduction of bulls during the natural service period.

On d 18, tailheads were painted and from d 19 to 156 to 10,000 pg/mL including 8 calibrators, and the
35 cows were observed once daily, after the morning analytical sensitivity of the assay was 11 pg/mL, which
milking, for signs of return to estrus. Cows in estrus was calculated by the interpolation of the mean and 2
were inseminated on the same morning. Tail paint was SD below the mean from 14 replicates of known sample
reapplied as needed in all cows during this period. On containing 39 pg/mL (sample calibrator B diluted 1 to
d 36, Holstein and Jersey bulls were placed with all 4), using a known sample containing 0 pg/mL (calibra-
cows for an additional 65 d of natural service to com- tor A) to detect the optical density for no AMH in
plete the 100-d breeding season. Bulls between 18 and the sample. Imprecision, calculated on 3 samples and
36 mo of age were used at a ratio of 1 bull for every 24 replicates, was 2.9 (sample averaged 610 pg/mL),
20 nonpregnant cows at the beginning of the natural 2.5 (sample averaged 1,260 pg/mL), and 3.7% (sample
service period. For statistical analyses, cows were also average 2,560 pg/mL). Linearity was measured accord-
categorized according to DIM at first AI as low (<60 ing to the National Committee on Clinical Laboratory
DIM), medium (60 to 80 DIM), or high (>80 DIM). Standards EP6-P guidelines (Passey et al., 1986), using
4 dilutions of 3 pools. The dilution factors were 2, 4,
Blood Samples and Analysis of AMH 8, and 16 and the expected concentrations for the 3
and Progesterone in Plasma pools ranged from 283 to 7,468 pg/mL. The percentage
recovery ranged from 97 to 109% and averaged 103.3%.
On d −8, concurrent with the first GnRH of the Comparison to a commercial kit, using 180 bovine se-
timed AI protocol, blood was sampled by puncture of rum samples in the range of 10 to 1,100 pg/mL, yielded
the coccygeal vein or artery into evacuated tubes con- a correlation coefficient greater than 0.8 and a slope of
taining K2 EDTA (Becton Dickinson, Franklin Lakes, 2.86 with an intercept of 0.01 ng/mL.
NJ). Samples were immediately placed on ice and Samples were analyzed in 4 assays according to the
transported to the laboratory within 6 h of collection. manufacturer’s instructions. Fifty microliters of cali-
Tubes were centrifuged at 2,000 × g for 15 min (4°C) brators, controls, and unknowns were pipetted in their
for plasma separation. Plasma samples were frozen at respective wells in microtitration strips. The intraas-
−30°C and later analyzed for their concentrations of say and interassay CV were 5.0 and 5.9%, respectively.
AMH and progesterone. Concentration of progesterone was analyzed by radio-
Concentration of AMH was analyzed using a com- immunoassay using a commercial kit (Coat-a-Count,
mercial chemiluminescence immunoassay (AnshLite Siemens Healthcare Diagnostics, Los Angeles, CA).
Bovine AMH CLIA, Ansh Labs, Webster, TX) devel- Samples were analyzed in 4 assays and the sensitiv-
oped for specific binding and accurate quantitation of ity of the assays calculated at 2 SD below the mean
bovine AMH. The monoclonal antibody pair used in counts per min at maximum binding of 0.05 ng/mL.
this assay detects pro-mature covalent and noncova- The intraassay and interassay CV were 5.9 and 5.5%,
lent bovine AMH complexes and does not have cross respectively.
reactivity to other proteins of the transforming growth Values of AMH concentrations in plasma were ranked
factor family, such as inhibins A and B and activins and categorized as low (20% lowest values), high (20%
A, B, and AB. The assay had an analytical range from highest values), and intermediate (60% intermediate

Journal of Dairy Science Vol. 97 No. 11, 2014

 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6891

values) to mimic suggested relevance of ovarian reserve described previously, and ovulation was confirmed by
to fertility (Ireland et al., 2011). Progesterone concen- the presence of a mature corpus luteum (>20 mm) on
tration in plasma on d −8 was categorized as ≥1 ng/ ultrasonography examination of ovaries 7 d after the
mL or <1 ng/mL as an indicator of cows in diestrus final GnRH injection of timed AI protocol. The day of
(≥1 ng/mL) or with low progesterone (<1 ng/mL; an- the final GnRH of the timed AI protocol was consid-
ovular or not in diestrus) because of the relevance to ered d 0 of the estrous cycle. Blood samples were then
fertility responses (Ribeiro et al., 2011). collected on d 7 and 15 of the estrous cycle for measure-
ments of AMH concentrations in plasma, as described
Pregnancy Diagnoses and Calculation previously. Measurements were used to estimate the
of Reproductive Responses correlation and coefficient of variation between samples
from the same cow.
Pregnancy was diagnosed in all cows on d 30 via
ultrasonography of the uterus and its contents, and Statistical Analyses
was characterized by visualization of an embryo with
heartbeat. Cows diagnosed as pregnant on d 30 were All data were analyzed by the SAS statistical soft-
reexamined by transrectal palpation 35 d later. Cows ware version 9.3 (SAS/STAT; SAS Institute Inc., Cary,
reinseminated after the first AI were examined by NC). To identify factors associated with concentrations
transrectal palpation on d 70 ± 7 after reinsemination. of AMH in plasma, a multivariable model including
Pregnancies from natural service were diagnosed each farm (A or B), breed of the cow (Holstein, Jersey, or
35 d after introduction of bulls; age of pregnancy was crossbred), lactation number (1, 2, 3, or ≥4), and DIM
estimated based on size of the amniotic vesicle either by at first AI (<60, 60 to 80, or >80) were used to explain
ultrasound (28 to 35 d of gestation) or by transrectal the observed variability in plasma AMH concentrations
palpation (36 to 63 d of gestation). by ANOVA using the GLIMMIX procedure of SAS.
Pregnancy per AI (P/AI) was calculated for the first Values of AMH were transformed by square root to
AI as the number of pregnant cows on d 30 and 65 after meet the assumptions for a Gaussian data distribu-
an insemination divided by the total number of cows tion. Explanatory variables that resulted in P < 0.05
inseminated. Pregnancy loss for first AI was calculated were considered to be associated with concentrations of
as the number of cows that lost pregnancy between AMH in plasma.
gestation d 30 and 65 divided by the number of preg- To investigate if the concentrations of AMH differed
nant cows on d 30. Reinsemination rate was calculated from cows experiencing a binary reproductive response
as the number of nonpregnant cows on d 30 after the or not, variables found to be associated with AMH
first AI that was reinseminated before introduction of concentration in the previous model were used to build
the bulls. Pregnancy per AI for the reinsemination was a second multivariable model that included a binary
calculated as the number of pregnant cows on d 70 ± reproductive response as an additional independent
7 after an insemination divided by the total number variable to explain the variability of AMH concentra-
of cows reinseminated. Pregnancy during natural ser- tions by ANOVA. Reproductive responses investigated
vice was calculated as the number of cows pregnant included progesterone ≥1 ng/mL on study d −8, detec-
to bulls divided by the number of cows that did not tion of estrus at timed AI, P/AI on d 30 and 65 after
become pregnant to AI. Finally, pregnancy at the end timed AI, pregnancy loss from gestation d 30 to 65 for
of the study was calculated as the number of pregnant timed AI, reinsemination from study d 19 to 35, P/AI
cows divided by the total number of cows enrolled in on d 70 ± 7 after reinsemination, pregnant from natu-
the study, and pregnancy on d 65 was used for first ral service, pregnant after the first AI (reinsemination
AI. Survival analyses were performed to calculate the and natural service combined), and pregnant at the end
rate of pregnancy throughout the breeding season, and of the breeding period. Adjusted proportions and 95%
pregnancy on d 65 was used for first AI. Cows that CI are presented.
were sold, died, or remained as nonpregnant at the end Reproductive responses were analyzed with AMH as
of the 100-d breeding season were censored when the an independent variable categorized as low, intermedi-
respective event occurred. ate, or high in a logistic regression model using the
GLIMMIX procedure of SAS. Univariable analyses
Repeatability of AMH Concentrations in Plasma were initially performed to identify factors that were
in 2 Distinct Phases of the Estrous Cycle associated (P ≤ 0.10) with the reproductive response of
interest. Factors considered were farm, breed of the cow,
A group of 60 lactating cows in 1 of the 2 farms parity, DIM at first AI, and progesterone ≥1 ng/mL
in the study had their estrous cycle synchronized, as on d −8. Sire and technician of AI were also included
Journal of Dairy Science Vol. 97 No. 11, 2014
6892 RIBEIRO ET AL.

in models evaluating P/AI. Multivariable models were

then built including AMH.
The interval to pregnancy was analyzed by the Cox’s
proportional hazard model using the PHREG procedure
of SAS. Multivariable analyses were performed and the
initial explanatory variables included farm, breed of the
cow, parity, DIM at first AI, and progesterone ≥1 ng/
mL on d −8. Those that remained with P ≤ 0.10 were
included in the final multivariable models concurrent
with AMH concentrations. The adjusted hazard ratio
and the respective CI were calculated for the response
evaluated. The time variable used in the model was the
interval in days between study d 0 and pregnancy or the
day when the cow was censored because she was sold,
died, or remained open on study d 100. A cow was con-
sidered pregnant based on gestation d 65 after breeding.
Proportionality was assessed by evaluating the Kaplan-
Meier curves. The median and mean days to pregnancy
were obtained from the LIFETEST procedure of SAS
9.3. Survival plots were generated with MedCalc ver-
sion 12.7.2 (MedCalc Software, Mariakerke, Belgium).
Differences with P ≤ 0.05 were considered significant Figure 2. Regression plot of anti-Müllerian hormone (AMH) con-
centrations between d 7 and 15 of the estrous cycle in 60 lactating
and 0.05 < P ≤ 0.10 were considered tendencies. grazing dairy cows. Linear regression equation: y = 0.951x + 28.51.
The coefficient of determination and the average CV between samples
from d 7 and 15 of the estrous cycle were 0.903 and 0.111, respectively.
RESULTS

AMH Concentrations in Plasma (Figure 4B). Farm and DIM at first AI had no statisti-
cal effect on concentrations of AMH in plasma.
Samples collected from the 60 cows on d 7 and 15 Differences in AMH concentrations in plasma accord-
of the estrous cycle were highly correlated within cow ing to binary reproductive responses were evident (P
(r2 = 0.903), indicating repeatability of values despite < 0.03) for detection of estrus at timed AI, pregnancy
the day of the cycle. The CV between the 2 samples from breeding after spontaneous estrus, and pregnancy
measured within each cow averaged 11.1% (Figure 2).
The AMH concentrations in plasma from 1,237
cows ranged from 10 to 3,198 pg/mL (Figure 3). The
mean (±SD) and median concentrations of AMH were
320.3 ± 251.1 and 266 pg/mL. Samples classified as
low AMH, comprising the 20% lowest concentrations,
averaged 85 pg/mL and ranged from 10 to 140 pg/
mL; those classified as intermediate AMH averaged 263
pg/mL and ranged from 141 to 450 pg/mL; and those
classified as high, comprising the 20% highest concen-
trations of AMH, averaged 631 and ranged from 451 to
3,198 pg/mL.
Factors identified to be associated with AMH concen-
trations in plasma were lactation number (P < 0.01)
and breed of the cow (P < 0.01; Figure 4). Lactation
number had a quadratic effect (P < 0.01) on AMH
concentration, which was greater for cows on second
and third lactations compared with those on the first
and fourth or greater lactations (Figure 4A). Concen-
trations of AMH differed (P < 0.05) among all breeds,
Figure 3. Histogram of frequency distribution of anti-Müllerian
and Jersey cows had the greatest average concentration hormone (AMH) concentrations in plasma of lactating dairy cows (n
of AMH, followed by crossbreds and then Holsteins = 1,237).

Journal of Dairy Science Vol. 97 No. 11, 2014

 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6893

reinsemination and natural service periods were evalu-

ated separately, cows that became pregnant from rein-
semination using AI tended (P = 0.06) to have greater
concentrations of AMH than those that did not became
pregnant, and those that became pregnant during the
natural service period had greater (P = 0.02) concen-
trations of AMH than those that failed to become preg-
nant. Finally, cows that were pregnant at the end of
breeding season had greater (P = 0.02) concentrations
of AMH than those that failed to become pregnant.
The concentrations of AMH did not differ for cows ac-
cording to progesterone above or below 1 ng/mL on d
−8, return to estrus after first AI, and pregnancy or
pregnancy loss from timed AI.

AMH as Predictor of Reproductive Responses

Concentration of AMH in plasma was associated (P

< 0.05) with the proportion of cows that had progester-
one ≥1 ng/mL, expressed estrus at timed AI, became
pregnant from breeding after spontaneous estrus, and
experienced pregnancy loss from gestational d 30 to
65 (Table 2). Cows with high AMH were less likely
(P < 0.05) to have progesterone ≥1 ng/mL compared
with cows with low or intermediate AMH. Detection of
estrus was greater (P < 0.02) in cows with low AMH
compared with those with intermediate or high AMH.
A greater (P = 0.01) proportion of cows with high AMH
became pregnant from breeding after spontaneous es-
trus than cows with low AMH, mostly because high
AMH resulted in an increased proportion of pregnancy
during the natural service period (Table 2). Although
category of AMH was not associated with overall P/AI
from reinsemination, pairwise comparisons resulted in
a tendency (P = 0.09) for those with low AMH to have
less P/AI than cows with intermediate AMH (Table
2). Pregnancy loss was greater (P < 0.04) for cows
with low AMH than for those with intermediate or high
AMH. In general, as category of concentration of AMH
increased, a tendency (P = 0.09) for a linear positive
Figure 4. Factors associated with the concentration of anti-Mülle-
rian hormone (AMH) in plasma of lactating dairy cows: lactation num-
association was observed with the proportion of preg-
ber (P < 0.01; panel A) and breed (P < 0.01; panel B). A quadratic nant cows at the end of the breeding season (Table 2).
relationship (P < 0.01) was observed between lactation number and Nevertheless, category of AMH was not associated with
AMH concentration in plasma. Different lowercase (a,b) and upper-
case (A,B) letters among bars denote statistical difference (P ≤ 0.05
P/AI at the AI or the proportion of cows returning to
and P < 0.10, respectively). estrus after first AI (Table 2).
Detection of estrus at first insemination was greater
at the end of the breeding period (Table 1). Cows de- (P < 0.01) for cows initiating the timed AI protocol
tected in estrus at timed AI had a smaller (P < 0.01) with progesterone <1 ng/mL compared with those with
concentration of AMH than those that were not de- progesterone ≥1 ng/mL (48.1 vs. 34.3%). No interac-
tected in estrus. Moreover, cows that became pregnant tion was observed between category of progesterone
from breeding after spontaneous estrus, including rein- concentration on d −8 and AMH category for detection
semination and natural service periods, had greater (P of estrus (Figure 5A). An interaction (P = 0.03), how-
< 0.01) concentrations of AMH in plasma than those ever, was observed between progesterone concentration
that failed to become pregnant. When pregnancy from on d −8 and category of AMH for P/AI (Figure 5B).
Journal of Dairy Science Vol. 97 No. 11, 2014
6894 RIBEIRO ET AL.

Table 1. Concentrations of anti-Müllerian hormone (AMH) in plasma according to reproductive responses (LSM of AMH concentration in pg/
mL with 95% CI in parentheses)

Incidence of the event

Event No Yes P-value

Progesterone ≥1 ng/mL on d −8 277.1 (261.2–293.6) 267.1 (257.9–276.4) 0.57
Estrus at timed AI 289.3 (279.1–299.7) 240.9 (228.2–254.0) <0.01
Return to estrus of cows nonpregnant from first AI 260.4 (242.0–279.4) 280.3 (266.8–294.1) 0.39
Timed AI
Pregnant on d 65 271.4 (261.3–281.7) 268.0 (255.6–280.6) 0.83
Pregnancy loss between gestation d 30 and 65 277.2 (269.2–285.4) 250.0 (228.0–272.9) 0.26
Pregnant after spontaneous estrus
Reinsemination 255.0 (237.6–272.9) 305.9 (285.3–327.2) 0.06
Natural service 222.7 (204.8–241.4) 280.6 (263.2–298.5) 0.02
Reinsemination and natural service 228.3 (210.7–246.5) 292.0 (278.6–305.8) <0.01
Pregnant at the end of the breeding season 228.3 (210.9–246.4) 278.4 (269.4–287.6) 0.02

Overall, cows with progesterone ≥1 ng/mL had greater Pregnancy Rate Throughout the Breeding Season
(P < 0.01) P/AI on d 65 after timed AI than cows with
progesterone <1 ng/mL (45.2 vs. 29.9%). Nevertheless, The rate of pregnancy throughout the breeding sea-
the negative effect of initiating the timed AI protocol son was not affected by category of AMH (Figure 6A;
with progesterone <1 ng/mL on pregnancy was only Table 3). When cows that became pregnant from timed
observed in cows with intermediate or high AMH (Fig- AI were excluded from the analysis, then those with
ure 5B). low AMH had a smaller (P < 0.05) rate of pregnancy
Cows detected in estrus at timed AI had greater (P than cows with intermediate or high AMH (Figure 6B;
< 0.01) P/AI on d 65 than those not in estrus (49.2 Table 3). The rate of pregnancy was 29% greater for
vs. 30.2%). Interestingly, the negative effect of initiat- cows with intermediate AMH and 40% greater for cows
ing the timed AI protocol with low progesterone on P/ with high AMH compared with those with low AMH
AI was not observed when cows expressed estrus on (Table 3). No differences were seen in pregnancy rate
the day of AI (Figure 5C). Pregnancy losses were also between intermediate and high AMH.
affected by detection of estrus at timed AI, and those
detected in estrus had less (P = 0.03) pregnancy loss DISCUSSION
from gestation d 30 to 65 than those not detected in
estrus (6.0 vs. 14.1%). The association between expres- Concentration of AMH in plasma has been used as
sion of estrus and pregnancy loss was more pronounced a marker to estimate the size of the ovarian reserve,
in cows initiating the timed AI protocol with low pro- and the AFC, a proxy for the ovarian reserve, has been
gesterone (Figure 5D). associated positively with fertility traits (Ireland et al.,

Table 2. Association between concentrations of anti-Müllerian hormone (AMH) in plasma and fertility responses in dairy cows

Categorized AMH Contrast1

Event Low Intermediate High P-value L Q

Number of cows 245 746 246 — — —
AMH concentration on d −8, pg/mL [mean (range)] 85 (10–140) 263 (141–450) 631 (451–3,198) — — —
Progesterone ≥1 ng/mL on d −8, adjusted % 77.1a 78.5a 68.4b 0.02 0.05 0.04
Estrus at timed AI, adjusted % 49.3a 39.4b 34.5b <0.01 <0.01 0.45
Return to estrus in nonpregnant cows, adjusted % 69.8 69.1 69.1 0.99 0.92 0.91
Timed AI, adjusted %
Pregnant on d 65 37.4 38.6 34.8 0.58 0.56 0.39
Pregnancy loss between gestation d 30 and 65 16.7a 8.5b 8.0b 0.03 0.05 0.21
Pregnant after spontaneous estrus, adjusted %
Reinsemination 44.7A 57.3B 55.6 0.17 0.17 0.16
Natural service 62.6b 66.9b 78.9a 0.04 0.02 0.30
Reinsemination and natural service 68.9b 75.4b,B 82.3a,A 0.04 0.01 0.81
Pregnant at the end of the breeding season, adjusted % 83.3A 85.0 88.7B 0.23 0.09 0.56
a,b
Least squares means in the same row with different superscripts differ (P ≤ 0.05).
A,B
Least squares means in the same row with different superscripts differ (P < 0.10).
1
Orthogonal polynomial contrasts with linear (L) and quadratic (Q) effects of concentration of AMH based on categorized values.

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 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6895

Figure 5. Relationships among categorical anti-Müllerian hormone (AMH), progesterone concentrations on d −8, and detection of estrus at
timed AI with fertility responses. In panels A and B, the number of cows, adjusted mean and range of AMH concentrations in plasma (pg/mL)
in each category were low (n = 245; mean = 85; range = 10 to 140), intermediate (n = 746; mean = 263; range = 141 to 450), and high (n =
246; mean = 631; range = 451 to 3,198). Different letters (a–c) among bars denote statistical difference (P ≤ 0.05).

2011). Our results support a positive association be- AI was observed when cows had ovulation synchronized
tween AMH and fertility in lactating dairy cows. Cows for timed AI, and a negative association was detected
with low AMH concentration had smaller pregnancy between AMH and detection of estrus at timed AI.
rates after first service and greater incidence of preg- Lack of an association between AMH and P/AI at
nancy loss between d 30 and 65 of gestation. Moreover, first AI may be attributed to the reproductive man-
cows that failed to become pregnant by the end of the agement implemented. Reproduction is controlled by
breeding season had smaller concentration of AMH in a multitude of factors, and perhaps, at first AI, the
plasma than cows that became pregnant. Nevertheless, association of AMH with fertility was masked by the
no association between concentration of AMH and P/ application of a timed AI system that optimized follicle

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6896 RIBEIRO ET AL.

Figure 6. Survival probability of pregnancy throughout the breeding season according to anti-Müllerian hormone (AMH) concentrations in
plasma categorized as low, intermediate, or high. Panel A includes all cows enrolled in the study (n = 1,237); panel B includes only cows that
failed to become pregnant from timed AI on the first day of breeding season (n = 684). In panel A, the number of cows, adjusted mean and
range of AMH concentration in plasma (pg/mL) in each category were low (n = 245; mean = 85; range = 10 to 140), intermediate (n = 746;
mean = 263; range = 141 to 450), and high (n = 246; mean = 631; range = 451 to 3,198). In panel B, the number of cows, adjusted mean and
range of AMH concentration in plasma (pg/mL) in each category were low (n = 140; mean = 85; range = 10 to 140), intermediate (n = 404;
mean = 267; range = 141 to 450), and high (n = 140; mean = 631; range = 451 to 2,591).

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 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6897
Table 3. Cox’s proportional hazard model for daily pregnancy rate according to categorized anti-Müllerian hormone (AMH) concentration in
plasma

Mean day to Median day to

pregnancy pregnancy Adjusted hazard
Item (±SEM) (95% CI) ratio (95% CI) P-value
1
AMH in all cows (n = 1,237) 0.29
Low 39.0 ± 2.6 24 (20–36) 1.00 —
Intermediate 33.3 ± 1.4 21 (20–23) 1.14 (0.97–1.34) 0.12
High 32.6 ± 2.3 22 (20–25) 1.11 (0.91–1.35) 0.31
AMH in cows not pregnant from timed AI (n = 684)2 0.05
Low 68.2 ± 2.6 71 (61–85) 1.00 —
Intermediate 60.8 ± 1.6 58 (50–65) 1.29 (1.01–1.65) 0.04
High 56.5 ± 2.6 50 (42–59) 1.40 (1.05–1.87) 0.02
1
Number of cows, means, and ranges of AMH concentration in plasma (pg/mL) in each category were low (n = 245; mean = 85; range = 10 to
140); intermediate (n = 746; mean = 263; range = 141 to 450); and high (n = 246; mean = 631; range = 451 to 3,198).
2
Number of cows, means, and ranges of AMH concentration in plasma (pg/mL) in each category were low (n = 140; mean = 85; range = 10 to
140); intermediate (n = 404; mean = 267; range = 141 to 450); and high (n = 140; mean = 631; range = 451 to 2,591).

development, luteal regression, and ovulation such that fertility of dairy cows (Bisinotto et al., 2010; Santos et
variation in AMH concentrations was not associated al., 2010; Wiltbank et al., 2011) and might have been
with pregnancy response to the first insemination. The particularly important for cows with low AFC or low
association between AMH and fertility was apparent AMH. In addition, increased proportion of high AMH
in subsequent inseminations to cows detected in estrus cows had low progesterone at the initiation of the timed
and mated with bulls via natural service, whereby AI protocol and did not show estrus at timed AI, 2
sequential ovarian or uterine effects possibly associ- factors associated negatively with fertility (Bisinotto et
ated with AMH concentrations and fertility were more al., 2010, 2013), which also could have contributed to
apparent. Others have shown a positive association the lack of positive association between AMH and P/
between AFC and P/AI when cows were inseminated AI for first AI.
following detected estrus, which was evident at the first Pregnancy maintenance was affected by AMH. Cows
AI postpartum (Mossa et al., 2012). Thus, hormonal with low AMH had greater risk of pregnancy loss be-
synchronization for follicle development and ovulation tween d 30 and 65 of gestation than cows with inter-
may have optimized fertility of cows with low AMH, mediate or high AMH. Based on the low-AFC model
which might have disrupted the association between (Jimenez-Krassel et al., 2009), cows with low AMH are
fertility and AMH concentrations at the first synchro- expected to have low concentrations of progesterone
nized timed AI. throughout the estrous cycle. Progesterone is essential
Cows with low AFC, which are expected to have for establishment of uterine receptivity to gestation and
low AMH, have compromised follicle development and maintenance of pregnancy (Bazer et al., 2008). In fact,
oocyte quality (Ireland et al., 2011). Cows with low low progesterone concentrations before and after breed-
AFC have heightened secretion of gonadotropins and ing have been associated with a greater incidence of
reduced concentrations of progesterone (Jimenez-Kras- pregnancy loss (Wiltbank et al., 2011) and, therefore,
sel et al., 2009). The latter 2 factors can compromise may have contributed to the increased incidence of loss
fertility because of premature resumption of meiosis observed in cows with low AMH. In addition, cows
and oocyte maturation, especially when associated with low AFC did not present normal dynamic changes
with extended follicle dominance (Revah and Butler, in endometrial thickness through the estrous cycle
1996; Cerri et al., 2009). The synchronization program (Jimenez-Krassel et al., 2009), which suggest that the
used in the present study not only reduces follicle domi- endometria of such cows are less capable of adequate
nance but also optimizes progesterone concentrations remodeling needed for proper conceptus implantation
during ovulatory follicle development (Santos et al., and pregnancy maintenance.
2010). The majority of cows in our study were expected An interesting finding in the current study was the
to be in early diestrus at the initiation of timed AI inverse relationship between detection of estrus at timed
program and bear a dominant follicle that, in case of AI and concentrations of AMH in plasma. This result
ovulation induced by the first GnRH injection, would is likely related with potential effects of AMH on fol-
form an accessory corpus luteum and further enhance licular steroidogenesis. Estrous behavior in dairy cows
concentrations of progesterone during the development occurs after increases in estradiol concentrations from
of the ovulatory follicle. Those features are critical for the preovulatory follicle triggers behavioral centers in

Journal of Dairy Science Vol. 97 No. 11, 2014

6898 RIBEIRO ET AL.

the hypothalamus (Allrich, 1994). In several species, granulosa cells. A good example of genetic differences
the expression of AMH is inversely related with the ex- in the regulation of folliculogenesis is the comparison
pression of aromatase CYP19A1 in granulosa cells that between Bos taurus and Bos indicus breeds. Bos indicus
converts androgens to estrogens and, consequently, with have greater number of antral follicles in their ovaries
concentrations of estradiol in follicular fluid (Ireland et compared with Bos taurus breeds, which does not seem
al., 2009; Eilsø Nielsen et al., 2010; Monniaux et al., to be related to the size of ovarian reserve (Silva-Santos
2011, 2013; Campbell et al., 2012). Although expression et al., 2011).
of AMH decreases as follicles develop to larger sizes, The quadratic effect of lactation on concentrations of
AMH concentrations are maintained relatively constant AMH is likely related to the number of antral follicles.
by the production of preantral and small antral fol- Similar response has been reported for AFC in beef
licles (Monniaux et al., 2013), which may have para- heifers (Cushman et al., 2009) and dairy cows (Mossa
crine actions in the preovulatory follicle. In addition, et al., 2012). These results suggest that the number of
cumulus cells of preovulatory follicles still express AMH antral follicles recruited may change with age, reaching
(Monniaux et al., 2013) and may also play a role in a maximal number at 4 to 5 yr of age and reducing
the steroidogenesis near ovulation. Nonetheless, a dif- thereafter. The increase in AFC and AMH might be
ference in estrous expression associated with AMH in related with somatic growth, maturation, and the endo-
plasma was not observed later in the breeding season, crine regulation involved with body development that
as similar proportions of nonpregnant cows returned to probably also affects the gonads. The reduction in AFC
estrus after first service regardless of category of AMH and AMH with age is likely related with reduction in
concentration. Thus, the difference in estrous expres- the size of ovarian reserve by gradual depletion of pri-
sion in cows with different AMH concentrations may mordial follicles over time. Nevertheless, the association
be restricted to the 72 h of induced proestrus allowed between age and AMH concentrations in plasma cannot
by the timed AI program, but AMH does not seem to be addressed directly in the present study because of
affect expression of spontaneous estrus. Supporting this the inevitable culling of cows in commercial farms. The
finding, others have shown similar detection of sponta- relationships listed above were measureable, detected
neous estrus in cows with different AFC (Mossa et al., associations, but to clearly evaluate the relationships
2012). between cow age and concentrations of AMH would
Detection of estrus at timed AI resulted in increased require measurements of AMH in the same cows at dif-
P/AI. Interestingly, this increment was greater in cows ferent ages such that longevity in the herd or culling
with low progesterone at the initiation of the synchroni- policies could not potentially influence the interpreta-
zation protocol. The interaction between progesterone tion of the results. Nevertheless, others using the same
concentration during follicle development and detection approach as cross-sectional studies observed associa-
of estrus at AI on P/AI observed in the current study tions between age and parity and AFC (Mossa et al.,
has also been demonstrated by others in previous stud- 2012), which might explain the observations from the
ies with dairy cows in grazing (Ribeiro et al., 2012a,c) current study between AMH and parity of cows.
or confinement systems (Bisinotto et al., 2013). The Finally, concentrations of AMH in plasma were highly
marked depression in P/AI in cows developing the variable among individual cows and support the results
follicle under low concentrations of progesterone that by others demonstrating high variation of AMH con-
do not display estrus might be related to inadequate centrations among dairy heifers (Ireland et al., 2011),
synchrony of follicle growth with luteal regression or and those reporting high variation in the size of ovarian
altered endocrine milieu during proestrus that affect reserve based on follicle numbers at birth (Erickson,
expression of estrus and fertility. As AMH concentra- 1966) and in adults (Ireland et al., 2008). The fact that
tion was inversely related with detection of estrus, it the variation in AMH concentrations within individual
is reasonable to speculate that low AMH benefited cows is small but large among cows in the same popula-
fertility of cows with low progesterone by improving tion allows the use of a single AMH measurement for
folliculogenesis and steroidogenesis of the dominant fol- phenotyping cows, as previously reported (Ireland et
licle as previously discussed. al., 2008; Rico et al., 2009).
Two important factors were found to be associated
with concentration of AMH in plasma: breed of the CONCLUSIONS
cow and lactation number. The association with breed
of the cow may be related to distinct genetic control Even though AMH was not associated with P/AI at
in several events of folliculogenesis, including ovarian the first postpartum insemination when cows were sub-
reserve formation, activation and recruitment of grow- jected to timed AI, positive associations were observed
ing follicles, apoptosis of follicles, or AMH secretion by between AMH and measures of fertility in lactating
Journal of Dairy Science Vol. 97 No. 11, 2014
 ANTI-MÜLLERIAN HORMONE AND FERTILITY IN DAIRY COWS 6899

dairy cows during the breeding season. Cows with a the high variation in the ovarian reserve on ovarian function and
fertility, utility of anti-Müllerian hormone as a diagnostic marker
low AMH concentration had greater pregnancy loss be- for fertility and causes of variation in the ovarian reserve in cattle.
tween 30 and 65 d of gestation and smaller pregnancy Reprod. Fertil. Dev. 23:1–14.
rate during breeding following spontaneous estrus. Ireland, J. J., F. Ward, F. Jimenez-Krassel, J. L. Ireland, G. W.
Smith, P. Lonergan, and A. C. Evans. 2007. Follicle numbers are
Moreover, cows that failed to become pregnant by the highly repeatable within individual animals but are inversely cor-
end of the breeding season had smaller concentrations related with FSH concentrations and the proportion of good qual-
of AMH in plasma than cows that became pregnant. ity embryos after ovarian stimulation in cattle. Hum. Reprod.
22:1687–1695.
Nevertheless, a negative association between AMH and Ireland, J. J., A. E. Zielak-Steciwko, F. Jimenez-Krassel, J. Folger, A.
detection of estrus at timed AI was noted, which might Bettegowda, D. Scheetz, S. Walsh, F. Mossa, P. G. Knight, G. W.
indicate differences in steroidogenesis in dominant Smith, P. Lonergan, and A. C. O. Evans. 2009. Variation in the
ovarian reserve is linked to alterations in intrafollicular estradiol
follicles of cows with distinct AMH concentrations. production and ovarian biomarkers of follicular differentiation and
Synchronization of follicle development and ovulation oocyte quality in cattle. Biol. Reprod. 80:954–964.
seemed to override positive associations between AMH Ireland, J. L. H., D. Sheetz, F. Jimenez-Krassel, A. P. N. Themmen, F.
Ward, P. Lonergan, G. W. Smith, G. I. Perez, A. C. O. Evans, and
and fertility, and AMH might play a role on fertility of J. J. Ireland. 2008. Antral follicle count reliably predicts number
dairy cows inseminated on estrus. of morphologically healthy oocytes and follicles in ovaries of young
adult cattle. Biol. Reprod. 79:1219–1225.
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