Domestic Animal Endocrinology 33 (2007) 7790

Effects of estradiol on gonadotrophin release, estrus

and ovulation in CIDR-treated beef cattle
M.F. Martnez a,1 , J.P. Kastelic b, , M.G. Colazo a,2 , R.J. Mapletoft a
a Western College of Veterinary Medicine, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada S7N 5B4
b Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1

Received 6 December 2005; received in revised form 13 April 2006; accepted 14 April 2006

Abstract

The effects of estradiol-17 (E-17) or estradiol benzoate (EB) on gonadotrophin release, estrus
and ovulation in beef cattle were evaluated in two experiments. In experiment 1, 16 ovariectomized
cows received a previously used CIDR insert from days 0 to 7 and 1 mg of EB on day 8; they also
received 5 mg of E-17 on days 0 or 1, or 5 mg of E-17 + 100 mg of progesterone on day 0. There
was only an effect of time (P < 0.0001) on plasma concentrations of progesterone, estradiol, FSH, and
LH. Following treatment with E-17, plasma FSH concentrations were suppressed for approximately
36 h, whereas plasma LH concentrations were reduced (P < 0.05) for 6 h, but surged within 24 h.
Injecting 1 mg of EB 24 h after CIDR removal decreased (P < 0.02) plasma LH concentrations for
6 h, followed by an LH surge at 18 h. In experiment 2, ovary-intact heifers (n = 40) received a used
CIDR and 5 mg of E-17 + 100 mg of progesterone on day 0. On day 7, CIDR were removed, PGF
given, and heifers received nothing (control) or 1 mg of EB 12, 24, or 36 h later. In these groups,
plasma LH peaked (mean SEM) 78.0 23.0, 37.8 8.5, 44.4 10.3, and 51.0 5.1 h after CIDR
removal (means, P < 0.001; variances, P < 0.001) and intervals from CIDR removal to ovulation were
102.0 6.7, 63.6 3.6, 81.6 3.5, and 78.0 4.1 h (P < 0.05). The interval from CIDR removal to
ovulation was shorter and less variable in EB-treated groups; the interval from EB to ovulation was
shortest (P < 0.05) in the 12-h group. In summary, E-17 or EB decreased both FSH and LH, but LH
increased after 6 h (despite elevated progesterone concentrations). Following CIDR removal, 1 mg of

Corresponding author. Tel.: +1 403 317 2236; fax: +1 403 382 3156.
E-mail address: kastelicj@agr.gc.ca (J.P. Kastelic).
1 Present address: Instituto de Reproducci on Animal Cordoba (IRAC), J.L. de Cabrera 106 X5000GVD,
Cordoba, Argentina.
2 Present address: Alberta Agriculture, Food, and Rural Development, Edmonton, AB, Canada T6H 5Z2.

0739-7240/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.domaniend.2006.04.009
78 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

EB effectively synchronized LH release, and ovulation (in intact cattle), but the interval from CIDR
removal to EB treatment affected the time of ovulation.
2006 Elsevier Inc. All rights reserved.

Keywords: Cattle; CIDR; Estradiol; Gonadotrophins; Progesterone

1. Introduction

Estradiol has become an important part of progestin-based estrus synchronization pro-

grams in cattle [1]. The administration of 5 mg of estradiol-17 (E-17) 1 day [2] after
insertion of a controlled internal drug release (CIDR) insert, or 5 mg of E-17 and 100 mg of
progesterone on the day of CIDR insertion [3], resulted in synchronous emergence of a new
follicular wave 34 days after treatment in beef heifers. The mechanism appeared to involve
suppression of plasma FSH concentrations, resulting in suppression of FSH-dependent fol-
licles, followed by the resurgence of circulating FSH concentrations and emergence of
a new follicular wave approximately 1 day later [4,5]. However, the effects of estradiol
and progesterone on gonadotrophin release in the absence of endogenous ovarian steroid
hormones have not been critically studied in cattle.
When follicular wave emergence is synchronized with an injection of estradiol at the
beginning of a progestin-based protocol in cattle, the newly recruited dominant follicle
is expected to ovulate following progestin removal 7 or 8 days later. Ovulation occurred
7284 h after CIDR removal in 75% of heifers [4]; in another study, all heifers ovulated
from 72 to 108 h after CIDR removal [3]. Although these treatments resulted in moderately
synchronous ovulation, no treatment was given to synchronize an LH surge and ovulation.
Conversely, estradiol has been given to induce estrus, LH release and ovulation in cattle. In
lactating beef cows, estradiol benzoate (EB) was given 48 h after prostaglandin F2 (PGF)
treatment; estrus occurred approximately 16 h later, nearly concurrent with LH release
(66 1.0 h after PGF treatment) [6]. In another study, 0.38 or 1.0 mg of EB given 2430 h
after CIDR removal resulted in the detection of estrus in 86 and 100% of heifers and cows,
respectively [7]. These doses of EB also induced an LH surge between 16 and 20 h after
treatment, and significantly improved pregnancy rates to AI compared to non-treated cattle
[7]. In a field study, giving EB at CIDR insertion (to synchronize follicular wave emergence)
and 24 h after CIDR removal (to synchronize ovulation), followed by fixed-time AI 28 h
later, resulted in highly acceptable pregnancy rates in heifers and lactating cows [3]. A
similar protocol, with new or once-used CIDR inserts, also resulted in high pregnancy rates
[8]. The hypothesis that administration of 1 mg EB at the time of progestin removal would
be as efficacious as 1 mg EB 24 h later in inducing a synchronous ovulation was tested in
a study involving serial ultrasound examinations [9]; the administration of EB 24 h after
progestin removal resulted in a more synchronous ovulation. However, the effect of the
interval from CIDR removal to EB treatment on the timing and synchrony of ovulation has
not been critically studied.
Two experiments were conducted to evaluate the effects of E-17 at CIDR insertion
and EB following CIDR removal on gonadotrophin release, estrus and ovulation in
cattle. The objectives of experiment 1 were to determine the effects of exogenously
 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790 79

administered E-17 at the initiation of CIDR treatment in ovariectomized cows on

circulating gonadotrophin concentrations when plasma progesterone concentrations were
low or elevated, and to characterize LH release following CIDR removal and treatment
with EB. The objective of experiment 2 was to determine intervals to LH release and
ovulation after treatment with EB at 12, 24, or 36 h after CIDR removal in ovary-intact
heifers, in which follicle wave emergence had been synchronized with E-17.

2. Materials and methods

2.1. Animals

Crossbred beef cows (Simmental, Charolais, and Hereford; 38 years old, and weighing
from 500 to 750 kg) that had been ovariectomized 1112 months earlier, were used in
experiment 1. Pubertal beef heifers (same breed crosses as the cows) were used in experiment
2. Cows and heifers were housed outdoors in feedlot pens at the Goodale Research Farm,
University of Saskatchewan, and fed barley silage, with free access to water, salt and a
mineral mix. The protocols for these experiments were approved by the University of
Saskatchewan Animal Care Committee.

2.2. Experiment 1

Ovariectomized beef cows (n = 16) received a previously used CIDR insert (Bioniche
Animal Health Inc., Belleville, ON, Canada) on day 0 (beginning of the experiment). The
CIDR inserts originally contained 1.9 g progesterone, but had been previously used for 7
days. Prior to insertion, the CIDR inserts were scrubbed with 1% iodine detergent (Providine
Detergent, Rougier Pharma Inc., Mirabel, QC, Canada) and disinfected by immersion in
a 0.1% iodine solution (Providine Solution, Rougier Pharma Inc.). Cows were allocated
randomly to three groups and given im injections of 5 mg of E-17 on day 0 (Group
E0; n = 6), 5 mg of E-17 on day 1 (Group E1; n = 5), or 5 mg of E-17 plus 100 mg of
progesterone on day 0 (Group EP; estradiol and progesterone, SigmaAldrich Canada Ltd.,
Oakville, ON, Canada; n = 5) in 2 mL of canola oil. The CIDR inserts were removed on
day 7 and all cows received 1 mg im of EB (SigmaAldrich) in 2 mL of canola oil on day
8. Although neither E-17 nor EB are licensed for use in food animals in the USA, they
can be compounded and given to beef cattle in Canada under the authority of a licensed
veterinarian. Blood samples were collected from the jugular vein every 6 h for 72 h after
CIDR insertion, then every 12 h until 24 h after CIDR removal (day 8), and then every 6 h
for an additional 48 h. These samples were used for determination of plasma concentrations
of progesterone, estradiol, LH, and FSH (Fig. 1).

2.3. Experiment 2

Beef heifers (n = 40) received a previously used CIDR insert (as described in experiment
1) and 5 mg of E-17 plus 100 mg of progesterone in 2 mL of canola oil im on day 0
(beginning of the experiment). On day 7, CIDR were removed, heifers were given an im
80 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

Fig. 1. Treatment schedules for ovariectomized beef cows (n = 16) given a once-used CIDR insert on day 0, and
given 5 mg of estradiol-17 (E-17) on day 1 (E1) or on day 0 (E0), or 5 mg of E-17 and 100 mg of progesterone
on day 0 (EP). The CIDR were removed on day 7, followed 24 h later by an injection of 1 mg of estradiol benzoate
(EB). Blood samples were taken every 6 h for 72 h after CIDR insertion, then every 12 h until 24 h after CIDR
removal, and then, every 6 h for 48 h (experiment 1).

injection of 500 g of cloprostenol (PGF; Estrumate, Schering-Plough Animal Health

Canada Inc., Pointe-Claire, QC, Canada), and were randomly allocated to four groups to
receive no treatment (control), or an im injection of 1 mg of EB in 2 mL of canola oil 12, 24,
or 36 h after CIDR removal (Fig. 2). Transrectal ultrasonographic examinations were done
once daily from days 0 to 7 (to monitor ovarian follicular development) and then twice
daily from EB treatment to ovulation (all heifers ovulated), using a real-time, B-mode
scanner (Model SSD-500; Aloka, Tokyo, Japan) with a 7.5 MHz linear-array transducer. In
the control group, examinations were performed as in the group given EB 24 h after CIDR
removal. Blood samples were collected daily for determination of plasma progesterone

Fig. 2. Treatment schedules for ovary-intact, beef heifers given a once-used CIDR and injected with estradiol-17
(E-17) and progesterone on day 0. The CIDR were removed on day 7 and 1 mg of estradiol benzoate (EB)
was injected 12, 24, or 36 h after CIDR removal. Blood samples were collected every 6 h from EB treatment to
ovulation (experiment 2).
 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790 81

concentrations, and every 6 for 48 h after administration of EB for determination of plasma

concentrations of estradiol and LH (three animals were randomly selected from each
EB-treated group).

2.4. Blood sample processing and assays

Blood samples were collected in heparinized tubes (Becton Dickinson, Franklin Lakes,
NJ, USA), maintained at approximately 4 C for up to 4 h, and then centrifuged (400 g
for 25 min). Plasma was collected and stored at 20 C until assayed. The progesterone
assay was a solid-phase, enzyme-linked immunoassay, as described [10]. Progesterone was
extracted with 2.5 mL of ether added to 250 L aliquots of plasma. The intra- and inter-assay
coefficients of variation were 5.4 and 10.6%, respectively, and the sensitivity of the assay
was 1.6 pg/well. Estradiol concentrations were determined by a modified human, double-
antibody radioimmunoassay kit (DPC Coat-a-Count; Diagnostic Products Corporation, Los
Angeles, CA, USA) [11], using a procedure previously described [12]. Plasma samples were
charcoal-stripped before processing and standards were prepared with dilution of plasma
from castrated animals. The sensitivity of the estradiol assay was 0.5 pg/mL and the intra-
assay coefficients of variation were 5.3 and 9.1% for reference sera with mean estradiol-
17 concentrations of 31.7 and 208.6 pg/mL, respectively. Plasma LH concentrations were
determined by a double antibody radioimmunoassay [13] and expressed in terms of NIDDK-
bLH4. The sensitivity of the assay was 0.06 ng/mL, assessed as the lowest concentration of
LH capable of displacing labeled LH from the antibody. Intra-assay coefficients of variation
were 7.9 and 5.5% for reference sera with mean LH concentrations of 0.4 and 1.0 ng/mL,
respectively. Plasma concentrations of FSH were determined using a liquid-phase double
antibody radioimmunoassay [14,15]. The first antibody used was NIDDK anti-oFSH-l and
FSH concentrations were expressed in terms of USDA-bFSH-1. The sensitivity of the assay
was 0.13 ng/mL. Intra-assay coefficients of variation were 6.8 and 6.8% for reference sera
with mean FSH concentrations of 1.7 and 3.6 ng/mL, respectively.

2.5. Statistical analyses

Time-series hormone data were analyzed using the MIXED procedure for repeated mea-
sures (Statistical Analysis Systems; SAS Institute, Cary, NC, USA); the main effects of
treatment, time, and the treatment-by-time interaction were determined [16], and the main
effects were compared by the protected LSD test. The mean and standard error of the mean
(SEM) were used to describe hormone concentrations. An LH peak was considered as the
highest mean concentration of plasma LH at a given time. One-way analysis of variance
(ANOVA) was used to compare the intervals from treatment to LH surge or FSH resur-
gence (statistically significant increases of circulating hormone concentrations >1 ng/mL).
In experiment 1, plasma FSH and LH concentrations for the first 48 h after estradiol treatment
were normalized to E-17 treatment; the effects of treatment, time, and treatment-by-time
interaction were determined. The effect of treatments on plasma estradiol, progesterone,
FSH, and LH concentrations after the single injection of EB after CIDR removal was also
analyzed. Although a probability of 5% (P < 0.05) was established a priori as the level for
statistical significance in all analyses, probability levels obtained in the statistical tests are
82 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

reported. In experiment 2, the diameters of the dominant follicle at 12, 24, or 36 h after CIDR
removal were analyzed by one-way ANOVA and means were compared by the protected
LSD test. Bartletts test was used to compare variances among groups. If data were not
normally distributed or their variances were unequal, the non-parametrical KruskalWallis
test was used. With the exception of the time-series hormone data, all of the remaining
statistical analyses were conducted with commercial software (Statistix Student Version,
Version 2.0; Analytical Software, Tallahassee, FL, USA).

3. Results

3.1. Experiment 1

Hormone concentrations following treatment with a used CIDR and E-17, with or
without progesterone, are shown in Fig. 3. Plasma progesterone concentrations increased
2 ng/mL (P < 0.0001) by 6 h after CIDR insertion and were higher (P < 0.0001) in the
EP group at 6 h than in the other groups. In all groups, plasma progesterone concentra-
tions decreased (P < 0.0001) by 84 h after CIDR insertion and remained relatively constant
thereafter. By 12 h after CIDR removal on day 7, progesterone concentrations had declined
(P < 0.0001) to <0.2 ng/mL.
In all groups, estradiol concentrations reached a peak (P < 0.0001) 6 h after treatment
with 5 mg of E-17, and subsequently decreased to baseline (P = 0.2) by 30, 24, and 30 h
after treatment in the E0, EP, and E1 groups, respectively. When LH data for the first 48 h
were normalized to the time of E-17 treatment, there was no effect of treatment (P = 0.80),
but there was an effect of time (P < 0.0001) and treatment-by-time interaction (P < 0.007).
Mean (SEM) LH concentrations decreased (P < 0.05) by 6 h after E-17 was given in
all treatment groups, and then increased (P < 0.05) and reached a peak 18 h after estradiol
was given in the E1 group (3.1 0.4 ng/mL) and at 24 h in the E0 (1.6 0.4 ng/mL) and
EP (2.6 0.4 ng/mL) groups. Thereafter, plasma LH concentrations declined to <1 ng/mL
where they remained.
When FSH data for the first 48 h were normalized to the time of E-17 treatment, there
were effects of treatment (P = 0.007) and time (P < 0.0001) and a treatment-by-time inter-
action (P < 0.0001). Mean (SEM) plasma FSH concentrations were reduced (P < 0.0001)
from 2.0 0.1 ng/mL at the time of treatment to 1.3 0.1 ng/mL by 6 h in all groups
(Table 1). By 30 h after treatment, plasma FSH concentrations reached a nadir in all groups
(average, 0.9 0.1 ng/mL), and began to increase thereafter (Fig. 3).

3.1.1. Hormone concentrations after EB treatment

There was no effect of treatment at CIDR insertion on plasma concentrations of proges-
terone, estradiol or LH at the time of EB treatment (24 h after CIDR removal), but there
was an effect on plasma FSH concentrations (P < 0.0025). Plasma estradiol concentrations
increased (P < 0.0001) from 1.0 0.4 pg/mL at the time of EB treatment to 15.0 2.1 pg/mL
by 6 h, continued to increase until 24 h (32.6 2.4 pg/mL), and then decreased (P < 0.01);
by 48 h, estradiol concentrations (9.9 1.6 pg/mL) were still higher (P < 0.05) than at the
time of treatment with EB (Fig. 4). Plasma LH concentrations decreased (P < 0.02) for
 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790 83

Fig. 3. Mean (SEM) plasma concentrations of progesterone, LH, estradiol and FSH in ovariectomized cows
(n = 16) treated with a once-used CIDR on day 0, and injections of 5 mg of estradiol-17 (E) on day 1 or on day
0, or 5 mg of E and 100 mg of progesterone on day 0 (EP), in experiment 1. The CIDR were removed on day 7,
followed 24 h later by an injection of 1 mg of estradiol benzoate (EB).
84 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

Table 1
Mean (SEM) interval from CIDR removal to estradiol and LH peaks, estrus and ovulation, and interval from
LH peak to ovulation, in control beef heifers and those given 1 mg of estradiol benzoate (EB) 12, 24, or 36 h after
CIDR removal (experiment 2; n = 10 heifers/group)
Control Interval from CIDR removal to EB (h)
12 24 36
Interval (h) from CIDR removal to
Estradiol peak* 56.4 3.6a 24.0 0.0b 42.0 3.5c 52.0 4.0ac
Range 2424 2436 4860
LH peak 78.0 7.27a 37.8 2.7b 44.4 3.3bc 51.0 1.6c
Estrus 63.6 7.0ax 33.3 1.4by 45.0 3.3by 52.0 2.7aby
Ovulation 102.0 6.7cy 63.6 3.6ax 81.6 3.5bx 78.0 4.1bxy
Median 96 60 72 84
Range 84144 4884 72108 4896
Interval (h) from LH peak to ovulation 24.0 5.4d 25.8 5.0d 37.2 2.0e 28.5 3.9de
ac Means and xy variances without a common superscript are different (interval to estrus, P < 0.05 and P < 0.01,

respectively; interval to ovulation, P < 0.05 and P < 0.07, respectively). de Means without a common superscript
differ (P < 0.06).
* Estradiol concentrations were determined for only three heifers in each EB-treated group.

6 h after EB treatment, then increased significantly, peaking at 18 h, and then declining

to basal concentrations by 30 h after treatment. Plasma FSH concentrations decreased by
6 h (P < 0.01), reaching a nadir by 30 h and then increased (P < 0.04) at 48 h after EB
treatment.

Fig. 4. Mean (SEM) plasma concentrations of progesterone, estradiol, FSH and LH after treatment with 1 mg
of estradiol benzoate (EB) 24 h after CIDR removal in ovariectomized beef cows (n = 16) in experiment 1. Blood
samples were collected from the jugular vein every 6 h for 48 h after CIDR removal for determination of plasma
concentrations of progesterone, estradiol, LH, and FSH.
 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790 85

3.2. Experiment 2

The overall mean (SEM) interval from treatment with E-17 to emergence of the new
follicular wave was 4.1 0.2 days. The diameter of the dominant follicle at the time of EB
treatment at 12, 24 or 36 h after CIDR removal was 9.4 0.7, 9.8 0.2, and 10.7 0.4 mm,
respectively (P = 0.3). The mean interval from CIDR removal to ovulation was shorter
(P < 0.05) and less variable (P < 0.05) in all EB-treated groups than in the control group.
There was an effect of time of EB treatment on the mean and variance of the interval from
CIDR removal to estrus (P = 0.001 and P = 0.004, respectively) and ovulation (P < 0.001 and
P < 0.07, respectively; Table 1). The interval from CIDR removal to ovulation was shorter
(P < 0.05) in heifers that received EB at 12 h than in those that received EB at 24 or 36 h.
Mean (SEM) plasma progesterone concentrations did not differ among groups
(P = 0.9). Combined for all groups, progesterone concentrations at CIDR insertion, CIDR
removal, and 24 h after CIDR removal were 3.0 0.3, 4.3 0.3, and 0.7 0.2 ng/mL,
respectively. Plasma estradiol concentrations increased (P < 0.001) within 6 h after treatment
with EB, and differed from those in the control group at that time (P = 0.0002). Maximal
mean concentrations of estradiol in heifers following treatment with EB at 12, 24, or 36 h
after CIDR removal were detected at 24 h (35.9 3.8 pg/mL), 42 h (39.6 3.8 pg/mL), and
52 h (42.8 3.8 pg/mL), respectively, and all were higher (P < 0.0001) than in the control
group (5.0 2.1 pg/mL) which peaked 48 h after CIDR removal. Maximal mean (SEM)
plasma LH concentrations after CIDR removal (Table 1) did not differ (P < 0.14) among the
control group and EB-treated groups (2.8 0.3 ng/mL). There was an effect of time of treat-
ment on the interval to LH peak (P = 0.02); the interval from EB treatment to the LH peak
was shorter (P = 0.007) in the group treated 36 h after CIDR removal (15.0 1.6 h) than
in the group treated at 12 h (25.8 2.7 h), whereas the group treated at 24 h (20.4 3.2 h)
was not different from either. The interval from the estradiol peak to the LH surge was less
variable (P = 0.02) in groups treated with EB than in the control group.

4. Discussion

Treatment with estradiol, after CIDR insertion or after CIDR removal, resulted in an
LH surge. It is noteworthy that estradiol treatment was followed initially by a decrease
and then an increase in plasma LH concentrations, and an LH surge occurred even when
progesterone was injected with the E-17 at CIDR insertion (further increasing progesterone
concentrations by >2 ng/mL). However, following the LH surge, there was a subsequent
suppression of LH in all groups, apparently due to the synergistic effects of estradiol and
progesterone. At the same time, there was also a suppression of plasma FSH. Treatment
with a low dose of EB after CIDR removal also induced an LH surge in both ovariectomized
and intact cattle. The LH surge was followed by earlier and more synchronous ovulation of
the selected dominant follicle in EB-treated versus untreated (control) ovary-intact heifers.
Although progesterone-releasing devices have been devised to inhibit estrus, LH surges,
and subsequent ovulation in intact animals [17], elevated plasma progesterone concentra-
tions did not prevent an estradiol-induced LH surge in ovariectomized cows in experiment 1.
The reason for this is not known, but progesterone released from the CIDR may have been
86 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

too low, even after the injection of progesterone, to block estradiol-induced LH release.
Following intravaginal insertion of a new CIDR in ovariectomized cows, progesterone
concentrations increased significantly by 6 h [18], and high plasma progesterone concentra-
tions suppressed LH pulse frequency [19]. Furthermore, the administration of progesterone
decreased the size and growth rate of the dominant follicle in a dose-dependent manner
[20], presumably by suppressing circulating LH concentrations [19,21]. However, it has
also been suggested that E-17-induced LH release may be due to the very high blood
estradiol concentrations following intramuscular injection; mean plasma estradiol concen-
trations in beef heifers were as high as 3250 pg/mL 2 h after an injection of 5 mg E-17
[22]. However, an estradiol-induced LH surge was not observed in CIDR-treated, intact
heifers given 5 mg of EB or estradiol valerate (EV) [23]; the injection of estradiol esters
(EB or EV) resulted in slower increases, and lower and more prolonged circulating estra-
diol concentrations than an injection of E-17 [23]. Furthermore, an estradiol-induced LH
surge was not observed when implants containing E-17 and progesterone were inserted
into ovary-intact [2426] or ovariectomized [2729] cattle. The absorption of E-17 from
an implant would be slower than absorption from an injection site. A subcutaneous implant
containing 6 mg norgestomet, placed 1 day before treatment with E-17, also blocked the
estradiol-induced LH release in ovary-intact [30] or ovariectomized [31] heifers, suggesting
that norgestomet may suppress estradiol-induced LH release more effectively than natural
progesterone. However, there have been no reports comparing the effects of CIDR inserts
and norgestomet implants on gonadotrophin release. When these two progestins were com-
pared in estrus synchronization programs in Holstein heifers using E-17 to synchronize
follicular wave emergence, no differences in ovarian dynamics were detected [32]. More-
over, the effect of body size and class of cattle used in our study cannot be excluded as
potential causes of the failure of progesterone to prevent an estradiol-induced LH surge.
Plasma FSH concentrations were suppressed by elevated estradiol concentrations, but
were unaffected by progesterone concentrations. In ovariectomized cows in the present
study, 5 mg of E-17 reduced plasma FSH concentrations for approximately 36 h. Similarly,
plasma FSH concentrations declined after treatment with 5 mg of E-17 in cyclic heifers
[30], or 10 mg of E-17 (with or without a norgestomet implant) in ovariectomized heifers
[31]. In experiment 1, plasma FSH concentrations did not reach pre-treatment concentrations
until 60 h after treatment, and in experiment 2, a new follicular wave emerged 4.1 days after
heifers were given 5 mg of E-17. A follicular wave was consistently preceded by a rise
in plasma FSH concentrations [33], similar to that reported previously in estradiol-treated
cattle [4,5,30].
In both experiments, administration of a small dose of EB following CIDR removal
induced an LH surge. Circulating estradiol concentrations peaked between 18 and 24 h
after EB treatment in ovariectomized cows (experiment 1), and at approximately 15 h in
ovary-intact heifers (experiment 2). Similar results were obtained in a CIDR-based estrus
synchronization protocol, when a small dose of EB (0.75 mg) given 24 h after CIDR removal
induced an LH peak (average, 50 pg/mL) 16 h after EB treatment [7]. In both ovariectomized
and intact cattle in the present study, estradiol concentrations of 15 and 20 pg/mL were
sufficient to induce increased plasma LH concentrations; this may be of importance with
the use of longer-lasting estradiol esters (e.g., estradiol cypionate), for the synchronization
of ovulation. Administration of estradiol cypionate resulted in a small and slow increase in
 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790 87

circulating estradiol concentrations due to its slow absorption and hydrolysis of estradiol-
17 from the parent molecule [34]. However, this small increase in circulating estradiol
concentrations, in addition to that produced by the preovulatory follicle, has resulted in
synchronous ovulation and highly acceptable pregnancy rates following fixed-time AI [35].
Plasma LH concentrations in heifers given EB 12 or 24 h after CIDR removal declined
slightly by 6 h and then increased, reaching maximal values between 20 and 25 h after estra-
diol treatment. Although there were no significant differences in the mean concentrations of
plasma LH at 6 h, there was a tendency for lower LH concentrations in the group treated with
estradiol (with or without progesterone) on day 0 than in the group treated with estradiol
on day 1 (that received only a CIDR insert on day 0). Similar hormonal patterns following
estradiol treatments have been attributed to a biphasic effect of estradiol on plasma LH
concentrations [31]. In that study, plasma LH concentrations in ovariectomized beef heifers
were lower between 4 and 12 h after treatment with 10 mg EB. However, mean plasma
LH concentrations were higher after 20 h in treated versus control animals. In the present
study, a low dose of EB was used; therefore, the progesterone decline may have influenced
LH release. When EB was given 12 h after CIDR removal in experiment 2, the interval
from EB treatment to the LH surge (25 h) was longer than when EB treatment was given at
36 h (15 h) after CIDR removal. Although plasma progesterone concentrations decreased
to <1.0 ng/mL by 12 h after CIDR removal in ovariectomized cows in experiment 1, blood
sampling was not frequent enough in experiment 2 to determine when progesterone concen-
trations reached baseline. In addition, other factors (e.g., stage of dominant follicle growth
and proximity to the selection of the dominant follicle at the time of EB treatment) may have
affected intervals to LH release and ovulation. In this regard, there was no effect of maturity
of the dominant follicle on the ovulatory response to EB treatment in CIDR-synchronized
heifers, and follicles of various sizes ovulated [36]. Follicles that were less mature (smaller
diameter) have resulted in a smaller CL, lower blood estradiol [37] and progesterone con-
centrations [3638], and reduced fertility [37,38]. Therefore, EB treatment 12 h after CIDR
removal may not allow sufficient time for follicle development to optimize preovulatory
diameter, CL size, progesterone production, and fertility.
Highly synchronous ovulation and appropriately timed AI are necessary for high preg-
nancy rates following fixed-time AI. In the present study, the interval from CIDR removal
to ovulation was shorter in heifers treated with EB 12 h after CIDR removal (64 h) than
in heifers treated with EB at 24 (82 h) or 36 h (78 h); the latter intervals were comparable
to those after treatment with 1 mg E-17 or EB 24 h after CIDR removal (75.8 3.0 and
77.3 1.9 h, respectively) [23]. Similarly, in another study [9], 1 mg of EB was given 24 h
after removal of a progesterone-releasing insert, and the intervals from insert removal to
ovulation ranged from 66 to 72 h. If ovulation is expected approximately 7584 h after CIDR
removal, fixed-time AI should be done just prior to the earliest ovulations (approximately
36 h after estradiol treatment, when estradiol is given 24 h after CIDR removal). The interval
from the LH surge to ovulation was numerically different between the control group and the
group treated with EB 24 h after CIDR removal (24 and 37 h, respectively). However, this
difference may not be real and was attributed to the ultrasonographic examinations, which
were performed at 12-h intervals.
In conclusion, estradiol induced an LH release in ovariectomized cows, even though cows
received a used-CIDR 24 h before or concurrent with estradiol treatment, or a used CIDR
88 M.F. Martnez et al. / Domestic Animal Endocrinology 33 (2007) 7790

and an injection of progesterone concurrent with estradiol. However, estradiol and proges-
terone treatment of intact heifers (given a used CIDR) resulted in synchronous emergence
of a new follicular wave 4.1 days later, presumably due to the increased FSH concentrations
that occurred approximately 48 h after treatment in ovariectomized cows. A low dose (1 mg)
of EB following CIDR removal in ovariectomized cows suppressed plasma FSH concen-
trations for at least 36 h, and plasma LH for 6 h, followed by an LH surge in 18 h; when
administered to intact heifers following CIDR removal, ovulation was more synchronous
than in controls. As early ovulations would decrease fertility in fixed-time AI protocols,
EB should be given 24 h after CIDR removal; this should minimize the incidence of cattle
ovulating prior to AI, and concurrently provide adequate opportunity for preovulatory folli-
cle growth and development. Furthermore, from a management perspective, 24-h intervals
between treatments would enable them to be given at a consistent time during the day,
minimizing errors and interference with other farm activities.

Acknowledgements

We thank the University of Saskatchewan, the Canada-Alberta Beef Industry Develop-

ment Fund, the Saskatchewan Agriculture Development Fund, Strategic Research Program,
Agriculture and Agri-Food Canada, and the Matching Investment Initiative, for financial
support. Bioniche Animal Health provided CIDR inserts and Schering-Plough Animal
Health provided Estrumate. Portions of these data were presented at the Annual Meetings
of the International Embryo Transfer Society (Foz do Iguassu, Brazil, 2002 and Auckland,
New Zealand, 2003).

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