Theriogenology 64 (2005) 14571474

www.journals.elsevierhealth.com/periodicals/the

Progesterone (CIDR)-based timed AI protocols

using GnRH, porcine LH or estradiol cypionate
for dairy heifers: Ovarian and endocrine
responses and pregnancy rates
J.D. Ambrose a,*, J.P. Kastelic b, R. Rajamahendran c,
M. Aali c, N. Dinn c
a
Livestock Development Division, Alberta Agriculture Food and Rural Development,
Suite 205, 6903-116 Street, Edmonton, Alta., Canada T6H 5Z2
b
Agriculture and Agri-Food Canada, Lethbridge, Alta., Canada T1J 4B1
c
University of BC, Vancouver, British Columbia, Canada V6T 1Z4
Received 30 August 2004

Abstract

The overall objective was to compare the efficacy of GnRH, porcine LH (pLH) and estradiol
cypionate (ECP), in a modified Ovsynch/fixed-time AI (FTAI) protocol that included a controlled
internal drug [progesterone] release (CIDR) device. In Experiment 1, heifers received a CIDR on Day

10, and PGF (25 mg) on Day
3. At CIDR insertion, heifers received 100 mg of GnRH (n = 6),
0.5 mg of ECP (n = 6), 5.0 mg of pLH (n = 6) or 2 mL of saline (n = 7); these treatments were
repeated on Day
1, except for ECP, that was repeated on Day
2, concurrent with CIDR-removal.
The 5.0 mg pLH was the least effective with a longer interval to ovulation than the other groups
combined (102 versus 64 h; P < 0.05). Overall mean LH concentrations (1.6 ng/mL) and area under
the curve (AUC) did not differ among treatments, but mean peak LH concentration was lower in
heifers given 5 mg of pLH compared to all other groups (4.5 versus 10.3 ng/mL; P < 0.05). In
Experiment 2, heifers on CIDR-based Ovsynch protocols were given 12.5 mg pLH (n = 6; pLH-low),
25.0 mg pLH (n = 6, pLH-high), or 100 mg GnRH (n = 5; control). Heifers in the pLH-high group had
greater (P < 0.01) plasma LH concentrations (between 12 and 20 h) than GnRH-treated heifers, but
the pLH treatments did not differ (P > 0.10). Area under the curve for LH (ng/32 h) was at least 50%
greater (P < 0.01) in pLH-treated heifers compared to GnRH-treated heifers (mean, 41.3, 56.3 and

* Corresponding author. Tel.: +1 780 422 0807; fax: +1 780 427 1439.
E-mail address: divakar.ambrose@gov.ab.ca (J.D. Ambrose).

0093-691X/$ see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2005.03.010
1458 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

20.3 for pLH-low, pLH-high and GnRH, respectively). Ovulation occurred in 15 of 17 heifers.
Progesterone concentrations were higher on Days 9 and 14 in heifers given 25 mg of pLH, suggesting
enhanced CL function. In Experiment 3, 240 heifers were assigned to CIDR-based Ovsynch/FTAI
protocols. The first and second hormonal treatments (with an intervening PGF treatment on Day
3)
were GnRH/GnRH (100 mg), ECP/ECP (0.5 mg), pLH/pLH (12.5 mg) or GnRH/ECP, respectively;
pregnancy rates were 58.7, 66.1, 45.9 and 48.3%, respectively (ECP/ECP > both pLH/pLH and
GnRH/ECP; P  0.05). In conclusion, CIDR-based Ovsynch/FTAI protocols using either GnRH/
GnRH or ECP/ECP yielded pregnancy rates about 20% points higher than previously reported for
dairy heifers bred to Ovsynch/FTAI in the absence of a CIDR.
# 2005 Elsevier Inc. All rights reserved.

Keywords: Dairy heifers; Ovsynch; CIDR; Estradiol cypionate; LH; Pregnancy rate

1. Introduction

A controlled breeding program that allows fixed-timed AI (FTAI) without estrus

detection in dairy heifers is highly desirable. The Ovsynch protocol [1,2] that involves two
treatments of GnRH (given 9 days apart) and a single treatment of prostaglandin F2a (PGF)
given 7 days after the first GnRH treatment, followed by FTAI, is widely used for breeding
dairy cows. Even though FTAI following Ovsynch yields acceptable (3540%) conception
rates in dairy cows [13], conception rates in heifers are also in this range [2,46]. For
nulliparous heifers, a conception rate of 40% to FTAI is unacceptably low, considering
that conception rates >60% are achieved with synchronization protocols that include estrus
detection at AI [4,5]. Although reasons for low conception rates with the Ovsynch/FTAI
protocol in heifers are not well understood, failure of the first GnRH treatment to
consistently cause ovulation, and premature luteolysis and estrus prior to FTAI are believed
to contribute to the poor fertility. Premature estrus and ovulation could be prevented by the
administration of exogenous progesterone [7]. In that regard, an intravaginal progesterone-
releasing, controlled internal drug release (CIDR) device (Bioniche Animal Health,
London, Ont.) is available in Canada. In beef cattle, high pregnancy rates have been
reported with the use of CIDR-based protocols when AI was performed at detected estrus
[8,9]. The efficacy of a CIDR-device for synchronization of estrus in dairy cattle has been
reported [1012]. Although CIDR-based Ovsynch protocols have been used for FTAI in
beef cows [13] and heifers [1416], there is a paucity of information regarding the use of
CIDR-based Ovsynch protocols for FTAI in dairy heifers.
Porcine luteinizing hormone (pLH) and estradiol cypionate (ECP) can potentially be
used in lieu of GnRH in CIDR-based, Ovsynch protocols, and may have potential benefits
over GnRH in the formation of a robust CL. Treatment with as little as 5 mg of pLH
induced ovulation in beef heifers [17]. Similarly, low doses (0.5 or 1.0 mg) of ECP have
been used to synchronize ovulation in dairy cattle [18,19], and ECP has been used
successfully in FTAI protocols for lactating dairy cows [20] and beef heifers [15,16].
Although pLH and ECP have been used in Ovsynch-type FTAI protocols in beef and dairy
cattle, neither product has been tested in a CIDR-based FTAI protocol for dairy heifers.
The primary objective of this study was to compare the efficacy of pLH and ECP to that
of GnRH, in a CIDR-based, modified Ovsynch protocol. Three experiments were
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1459

conducted in Holstein heifers treated with a CIDR device for 8 days. Specific objectives
were to determine: (a) the effects of low doses of ECP (0.5 mg) or pLH (5.0 mg) to a
standard dose of GnRH (100 mg) on ovarian follicular dynamics, plasma concentrations of
LH and progesterone, and synchronization of ovulation (Experiment 1); (b) ovulation
response and plasma LH concentrations after treatment with 12.5 or 25 mg of pLH
(Experiment 2); and (c) pregnancy rates after FTAI protocols using GnRH or pLH or ECP
to initiate new follicular growth and to synchronize ovulation, or using GnRH to initiate
follicular growth, and ECP to synchronize ovulation (Experiment 3).

2. Materials and methods

2.1. Animals

Heifers used in all experiments were postpubertal, nulliparous, Holstein, 1315 months
of age, with a body weight that ranged from 350 to 420 kg. Heifers were group-housed and
managed in accordance with Canadian Council of Animal Care Guidelines [21].
Experimental procedures were approved by the Animal Policy and Welfare Committee,
University of Alberta (Protocol 2000-26C).

2.2. Experiment 1

Twenty-five heifers received a CIDR device intravaginally on Day
10 (without regard
to stage of the estrous cycle). Approximately 5 cm of the plastic cord attached to the CIDR
device was trimmed (to minimize the length of the cord that protruded from the vulva). All
heifers received 25 mg of PGF (Lutalyse, Pharmacia Animal Health, Orangeville, ON,
Canada) on Day
3 and the CIDR device was removed on Day
2. At CIDR insertion,
heifers were randomly allocated to receive one of the following treatments (all given im):
0.5 mg ECP (ECP, Pharmacia Animal Health, Orangeville, ON, Canada; n = 6); 5 mg pLH
(Lutropin-V, Bioniche Animal Health; Belleville, ON, Canada; n = 6); 100 mg GnRH
(Fertiline, Vetoquinol N.A. Inc., Lavaltrie, QC, Canada; n = 6); or 2 mL saline (n = 7).
Heifers that received ECP on Day
10 received a second treatment of ECP on Day
2,
concurrent with CIDR-removal, whereas heifers that received pLH, GnRH, or saline on
Day
10 were given a second treatment of the same product 48 h after PGF (Day
1;
Fig. 1).
Ovarian structures were monitored using a real-time ultrasound scanner (Aloka 500V,
Aloka Co., Tokyo, Japan) equipped with a 7.5 MHz transrectal transducer. Ultrasono-
graphy was performed once-daily from Days
10 to
2 (to determine ovarian follicular
changes) and twice-daily on Days
1, 0 and +1 (to confirm ovulation). At each
examination, the total number of Class 1 (<6 mm) follicles on each ovary was recorded.
The number, size and location of all Class 2 (69 mm) and Class 3 (>9 mm) follicles, and
CL diameter were also recorded. The day of establishment of the dominant follicle is
defined as the day on which the dominant follicle was first identified as a Class 2 follicle.
Sequential blood samples were collected from the jugular vein via an indwelling catheter
[22] over a 36-h period (starting 24 h after CIDR-removal), for determination of plasma LH
1460 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

Fig. 1. Modified Ovsynch protocols used in Experiment 1. Intramuscular treatments, 100 mg of GnRH, 5 mg of
pLH, 0.5 mg of ECP, 2 mL of saline, and 25 mg of PGF were given as shown. A CIDR-device was placed
intravaginally for 8 days in all heifers. Blood samples (for determination of plasma LH concentrations) were
collected over 36 h, beginning 24 h after CIDR-removal in all heifers.

concentrations. Samples were obtained 1 h prior to the second GnRH, pLH or saline
treatments (0 h), then every 15 min for 1 h, every hour for the next 5 h, and thereafter every
2 h until 32 h. In the ECP group, sampling frequency remained the same as above, but the
second ECP treatment was given concurrent with CIDR-removal (Fig. 1). Blood samples
were also collected every 4 h in ECP-treated heifers, during the 24 h from the time of
second ECP treatment (or CIDR-removal) to 0 h (corresponding to the second GnRH, pLH
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1461

or saline treatments of other heifers). These additional samples were obtained to determine
if any LH surges occurred in the first 24 h following ECP treatment. On Day
10, Days
3
to 7, and on Days 9, 11, 13, and 14, blood samples were collected (by venipuncture of a
coccygeal vessel) for determination of plasma progesterone concentrations. Blood samples
were immediately placed on ice, and plasma was separated within 4 h by centrifugation
(1500  g, 20 min) and stored at
20 8C until LH or progesterone concentrations were
determined.

2.3. Experiment 2

2.3.1. Experiment 2a
Without regard to the stage of the estrous cycle, heifers (n = 16) received an intravaginal
CIDR-device (Day
10), PGF 7 days later (Day
3), with CIDR-removal on Day
2 (i.e.,
8 days after insertion). Heifers were allocated to receive either 12.5 or 25 mg of pLH (low
and high dose, respectively) at the time of CIDR insertion, with the same dose repeated
48 h after CIDR-removal (Day 0). Ovarian status was determined by real-time
ultrasonography once each on Days
10 and
2, then once-daily until ovulation was
confirmed (for a maximum of 3 days after the second pLH treatment). At each
examination, the location and diameter of Class 3 (>9 mm) follicles were recorded. The
location of the largest and the second largest follicle(s) present on Day
2 was recorded
and these follicles were followed until ovulation was confirmed.

2.3.2. Experiment 2b
Seventeen heifers were assigned to CIDR-based Ovsynch protocols. Heifers received a
CIDR and either 12.5 mg (n = 6; pLH-low) or 25.0 mg (n = 6; pLH-high) of pLH, or GnRH
(100 mg, n = 5; control) on Day
10. On Day
3, PGF was given and CIDR-removed on
Day
2. A second treatment of pLH (same dose as given on Day
10) or GnRH was given
on Day
1. Ovarian status was monitored by ultrasonography, once-daily from Days
10
to 0, and then at 12-h intervals (up to 48 h after pLH or GnRH treatment) to determine the
synchrony of ovulation. Ovarian ultrasonography were performed on Day 9 to confirm the
presence of a CL. Blood samples (for determination of plasma LH concentrations) were
obtained from an indwelling jugular catheter from all heifers beginning 1 h prior to the
second pLH or GnRH treatment on Day
1, at 15-min intervals during the first hour, then
every hour until 6 h, and every 2 h thereafter for up to 32 h after pLH or GnRH treatment.
Blood samples (for determination of plasma progesterone concentrations) were also
collected by coccygeal venipuncture on Days
3 and 0, and then on Days 9, 12 and 14.

2.4. Experiment 3

Two hundred and forty postpubertal Holstein heifers, at random stages of the estrous cycle,
were assigned randomly to four different protocols (Fig. 2). The FTAI protocols were GnRH/
GnRH (n = 63), ECP/ECP (n = 56), GnRH/ECP (n = 60), pLH/pLH (n = 61). A CIDR-device
was inserted in all heifers on Day
10 and removed on Day
2, with PGF given on Day
3
(24 h before CIDR-removal). Heifers were given 100 mg of GnRH, 0.5 mg of ECP, or 12.5 mg
of pLH at CIDR insertion. The second treatment of GnRH (100 mg) or pLH (12.5 mg) was
1462 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

Fig. 2. The four modified Ovsynch protocols used in Experiment 3 for synchronization of ovulation and fixed-
time AI (FTAI). A CIDR device was placed intravaginally for 8 days in all heifers and PGF was given 24 h prior to
CIDR-removal. Intramuscular treatments of 100 mg GnRH, 0.5 mg ECP, 12.5 mg pLH, and 25 mg PGF were
given as shown.

given on Day
1, 24 h after CIDR-removal. In the ECP/ECP and GnRH/ECP groups, the
second treatment (ECP; 0.5 mg), was given on Day
2, concurrent with CIDR-removal.
Because heifers were group-housed in a pen near other breeding-age heifers, experimental
heifers were also monitored for estrous behavior whenever estrus detection occurred for non-
experimental heifers (though not on a consistent basis). Heifers were inseminated on Days 0,
40 to 44 h after CIDR-removal. After FTAI, these heifers were observed twice-daily for
estrous behavior. If a heifer was detected in estrus within 24 h after FTAI, no further action was
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1463

taken. However, any heifer detected in estrus >24 h after FTAI was re-inseminated and
considered nonpregnant to FTAI. Transrectal ultrasonography was conducted 3045 days
after FTAI to confirm pregnancy.

2.5. Hormone assays

Plasma concentrations of progesterone were determined by a solid-phase ELISA

validated for bovine plasma [23]. Plasma LH concentrations were determined by double-
antibody RIAs [24] validated for bovine plasma (Endocrine Lab Services, University of
Saskatchewan, Saskatoon, SK, Canada). The intra- and inter-assay CVs, respectively, were
5.3 and 8.0%, for progesterone and were 4.2 and 7.6%, for LH. The LH assay used is known
to cross-react with porcine LH (pLH). However, the standard curves for porcine and bovine
LH have very different slopes. Therefore, confounding effects were unavoidable (when
measuring a mixture of bovine and porcine LH) and LH data in pLH-treated heifers must
be interpreted cautiously.

2.6. Statistical analyses

Data were analyzed by ANOVA for a completely randomized design using either GLM
or MIXED procedures of SAS [25] as described. Repeated measures on plasma LH and
progesterone concentrations were analyzed using the MIXED procedure with the following
model:
Yik j m ai b j abi j ei jk

where m is the population mean; ai, a population parameter corresponding to treatment i;

bj, the fixed effect of time j; (ab)ij, the effect of treatment by time interaction; and eijk, the
residual error. The KenwardRoger procedure was used for approximating the degrees of
freedom. Animal nested in treatment was considered as the subject on which repeated
measures were taken and covariance structures modeled. Based on the smallest values of fit
statistics for Akaike information criteria (AIC), AIC corrected (AICC), and Bayesian
information criteria (BIC), the covariance structure of the unequally spaced repeated
measurements for LH and progesterone in both trials was modeled separately either as
compound symmetry, heterogenous compound symmetry, first-order antedependence, or
spatial power law [26]. For modeling the changes in follicle and luteal size with time, an
autoregressive order-1 matrix structure with random effect for heifer was used. The area
under the curve (AUC) for LH profiles was calculated using the trapezoidal rule. For
discrete variables, such as AUC, a model similar to the one described above was used,
except that time and interaction with time were not considered. Maximum size of dominant
follicle (Days
10 and 0), ovulation response, day of establishment of dominant follicle,
interval from CIDR-removal to ovulation, mean CL diameter, mean and peak LH
concentrations, and mean progesterone concentration were analyzed by GLM. Preplanned
treatment comparisons were made with the probability of difference (PDIFF) option and
declared significant at P < 0.05. Pregnancy data were analyzed by a Chi-square test. The
variance in the interval from CIDR-removal to ovulation (Experiment 1) was analyzed
using a Bartletts test.
1464 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

3. Results

3.1. Experiment 1

Following CIDR insertion and the first hormonal treatment, ovulation occurred in three
of six heifers given GnRH, but did not occur in any heifer given ECP, pLH or saline.
Following the second treatment and CIDR-removal, ovulation was detected in all heifers
(with the exception of one saline-treated heifer). The mean interval to ovulation after
CIDR-removal for saline-, ECP-, GnRH-, and pLH-treated heifers, were: 70.0, 66.0, 56.6,
and 102.0 h, respectively (Table 1), with pLH-treated heifers having the longest
(P < 0.001) interval. Even though the mean interval to ovulation after CIDR-removal did
not differ among Saline, ECP and GnRH groups, it was more variable (P < 0.05) in the
ECP group than in the Saline and GnRH groups (Table 1). Treatment and the treatment by
day interaction were not significant (P > 0.10) for follicle diameter. The maximum
diameter of the ovulatory follicle, non-ovulatory dominant follicle, and the day of
establishment of a new dominant follicle were similar among treatments (Table 1).
Although overall mean LH concentrations (ng/mL) did not differ (P > 0.10) among
treatments, the mean peak LH concentration was lowest (P < 0.05) in heifers treated with
Table 1
Ovarian responses, LH, and progesterone concentrations in heifers treated with estradiol cypionate (ECP, 0.5 mg),
GnRH (100 mg), porcine LH (pLH, 5 mg) or saline (2 mL) in a CIDR-based Ovsynch protocol (Experiment 1)
End point Saline ECP GnRH pLH S.E. P-value
No. of heifers 7 6 6 6
Mean diameter (mm) of largest 13.4 12.1 14.0 12.3 0.63 0.18
follicle on Day
10
Ovulation (%) after first treatment 0 0 50 0 0.11 0.02
Mean day of establishment of new 5.7 6.3 5.2 4.3 0.79 0.36
dominant follicle
Mean maximum diameter (mm) of 15.3 13.9 13.9 13.8 0.50 0.14
preovulatory dominant follicle
Ovulation (%) after second treatment 86 100 100 100 7.60 0.49
a a a b
Mean interval (h) from CIDR-removal 70 66 57 102 10.6 0.03
to ovulation
S.D. 4.9x 12.6y 5.9x 51.9z 0.03
Range in interval (h) from CIDR-removal 6072 4884 4860 48168
to ovulation
Mean interval (h) to LH peak after 20.0a 38.1c 1.6b 8.2b 2.83 <0.01
second treatment
Range in interval (h) to LH peak 1032 24.550 0.753 122
after second treatment
Mean peak LH concentration (ng/mL) 10.3a 9.3a 11.3a 4.5b 1.53 0.03
Range in peak LH concentration (ng/mL) 5.016.9 4.611.8 6.917.2 1.310.6
Mean LH concentration (ng/mL) 1.37 1.62 2.11 1.26 0.34 0.29
Area under the curve, LH (ng/32 h) 67.72 67.77 52.65 53.76 5.81 0.29
Mean maximum diameter of CL (mm) 18.19 18.65 16.57 16.61 1.89 0.65
Mean progesterone concentration (ng/mL) 3.23 3.25 3.19 2.01 0.38 0.07
Means within row, with different superscripts (a, b, c) differ. Variances with different superscripts (x, y, z) differ.
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1465

Fig. 3. Least-square means for plasma LH concentrations of heifers given a CIDR and allocated to receive saline
(SAL; control), GnRH, estradiol cypionate (ECP) or porcine LH (pLH) in Experiment 1. Heifers in the saline,
GnRH and pLH groups received their treatments at 0 h; heifers in the ECP group received the treatment at
24 h.
There were effects of treatment and a treatment by time interaction (P < 0.001; pooled S.E.M. = 0.34 ng/mL).

5 mg pLH (Table 1). The mean interval to LH peak after the second injection of ECP,
GnRH, pLH or saline differed (P < 0.01) among treatment groups and there was a
treatment by time interaction (P < 0.001) for plasma LH concentrations (Fig. 3); GnRH-
treated heifers had the most acute, synchronous increase in plasma LH concentrations. The
rise in LH occurred (P < 0.05) 15 min after giving GnRH, reached peak concentrations in
2 h, and returned to pre-treatment concentrations (P > 0.10) within 6 h (Figs. 3 and 4a).
Heifers treated with saline (control) or ECP had a much more variable LH pattern;
spontaneous LH surges in saline-treated heifers occurred between 8 and 30 h after
treatment (Fig. 4c) and mean concentrations were greater (P < 0.05) at 20 h (Fig. 3)
compared to other treatments. None of the ECP-treated heifers had an LH surge during the
24-h period following the second ECP treatment. In one heifer (#E1D; Fig. 4b) LH began to
rise 16 h after second ECP treatment and peaked at 24.5 h. In another heifer (#E1B;
Fig. 4b), the LH increase began at 24 h, with a surge 810 h later. In other ECP-treated
heifers, occurrence of the LH peaks ranged from 32 to 50 h after the second ECP treatment.
Approximately a three-fold increase (P < 0.05) in LH concentrations (over pre-treatment
concentrations) was detected in all pLH-treated heifers by 15 min after treatment; however,
the increase was minimal in three of six heifers, with peak LH concentrations that ranged
from 1.3 to 2.1 ng/mL. A characteristic LH peak (810 ng/mL) was measured only in two
of six heifers, with a peak of 3.3 ng/mL in another heifer (Fig. 4d). It is noteworthy, that
only these latter three heifers ovulated within the first 36 h after treatment. The average
interval to ovulation in the other three heifers (peak LH concentrations < 2.1 ng/mL), was
124 h. All saline-treated controls had spontaneous LH peaks >5 ng/mL. On average, LH
surges lasted for 10, 6.5, 11.7, and 20.7 h in heifers treated with saline, GnRH, ECP, and
pLH, respectively. Mean area under the curve for the LH profile did not differ (P > 0.10)
among treatments (Table 1).
There was a treatment by time interaction (P = 0.03) for plasma progesterone
concentrations (Fig. 5); progesterone concentrations were similar (P > 0.10) among
1466 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

Fig. 4. Individual LH profiles in heifers treated with 100 mg GnRH (a); 0.5 mg ECP (b); 2.0 mL saline (c); 5.0 mg
pLH (d); 12.5 mg pLH (e); and 25.0 mg pLH (f) (Experiments 1 and 2).

treatments from Days 0 to 2, but were generally lower in pLH-treated heifers after Day 3.
Furthermore, overall mean progesterone concentrations tended (P = 0.07) to be lower for
pLH-treated heifers relative to other treatments (Table 1). There was no (P > 0.10)
treatment effect or treatment by time interaction for CL diameter (Table 1).

3.2. Experiment 2

3.2.1. Experiment 2a
Except for one, all low-dose heifers had a large (>9 mm) follicle at the second treatment
of pLH. Ovulation occurred within 48 h after the second pLH treatment in 6/8 and 8/8 low-
and high-dose heifers, respectively. One of the two remaining low-dose heifers ovulated
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1467

Fig. 5. Least-square means for postovulatory (Days 014) progesterone concentrations in heifers given a CIDR
and saline (SAL), GnRH, estradiol cypionate (ECP) or porcine LH (pLH) in Experiment 1. There were effects of
treatment (P = 0.03), time (P < 0.01) and a treatment by time interaction (P = 0.04). Progesterone concentrations
were lower (P < 0.05) in pLH-treated heifers (compared to all other groups) on Days 37, 11 and 14 (pooled
S.E.M. = 0.38 ng/mL).

within the next 24 h and in the remaining low-dose heifer, ovulation had not occurred by
96 h after the second pLH treatment.

3.2.2. Experiment 2b
Mean (S.E.M.) plasma LH concentrations in heifers in the GnRH, pLH-low, and pLH-
high groups were 1.28  0.4, 1.34  0.37 and 1.94  0.37 ng/mL (no difference among
treatments, P > 0.10). However, there was a treatment by time interaction (P = 0.03;
Fig. 6). Heifers given GnRH had an acute response, with LH surging from basal
concentrations (<0.5 ng/mL) to a mean peak of 6.2 ng/mL by 2 h. One heifer (#G2A;
Fig. 4a) had a minimal LH surge (peak, 1.3 ng/mL), yet ovulated within 32 h.
Concentrations of LH also increased immediately after the second treatment in pLH-
treated heifers, regardless of the dose (Fig. 6). Whereas LH returned to basal
concentrations (<1.0 ng/mL) within 5 h in GnRH-treated heifers, LH concentrations
remained elevated for a much longer period in pLH-treated heifers (before ultimately
reaching basal concentrations). Consequently, heifers in the pLH-high group had greater
(P < 0.01) plasma LH concentrations between 12 and 20 h (Fig. 6) compared to GnRH-
treated heifers. Furthermore, the area under the curve for the LH profile was about 50%
greater (P < 0.01) in heifers receiving pLH compared to GnRH-treated heifers (GnRH:
20.3  8.1; pLH-low 41.3  7.4; pLH-high 56.3  7.4). Individual LH profiles of pLH-
low and pLH-high heifers are presented (Fig. 4e and f).
Although ultrasonographic examinations were not conducted frequently enough to
definitively determine the synchrony of ovulation, ovulation response (occurrence or non-
occurrence of ovulation) was determined based on plasma progesterone concentrations of
Days
3, 0 and 9 and by ultrasonography on Day 9. With the exception of two heifers (one
each from the high- and low-pLH groups), all other heifers ovulated in response to the
second GnRH or pLH treatment. Progesterone concentrations differed at 9 and 14 days
1468 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

Fig. 6. Least-square means for plasma LH concentrations in heifers given GnRH, a low dose of pLH (12.5 mg;
pLH-L) or a high dose of pLH (25 mg; pLH-H) in Experiment 2b (pooled S.E.M. = 0.28 ng/mL). Heifers were
placed on a CIDR-based Ovsynch protocol and blood samples were collected starting 1 h prior to the second pLH
or GnRH treatment (Day
1) up to 32 h after treatment. There was a treatment by time interaction (P = 0.03).
Compared to GnRH-treated heifers, those treated with 25 mg of pLH had higher (P < 0.01) concentrations of LH
between 12 and 20 h, but the two pLH treatments did not differ (P > 0.10).

after treatment (Table 2). On Day 9, heifers treated with either GnRH or pLH-low had
lower (P < 0.02) progesterone concentrations compared to heifers treated with pLH-high.
Even though the progesterone concentrations were not different between pLH- and GnRH-
treated heifers on Days 12 and 14, the two pLH doses remained separated at Days 12
(P = 0.06) and 14 (P = 0.02).

3.3. Experiment 3

Within the limited estrus detection performed, no premature estrous behavior was
reported prior to 12 h preceding FTAI. Furthermore, only eleven heifers were detected in
estrus (between FTAI and the scheduled day of pregnancy diagnosis) and re-inseminated.
Pregnancy rate to FTAI after the ECP/ECP protocol (66.1%) was greater than that of
GnRH/ECP (48.3%; P = 0.05) and pLH/pLH (45.9%; P = 0.03) protocols, but was not
different from that following the GnRH/GnRH protocol (58.7%; P = 0.41).

Table 2
Plasma concentrations of progesterone in dairy heifers 9, 12 and 14 days after im treatment with GnRH (100 mg)
or porcine LH (pLH, 12.5 or 25.0 mg) to synchronize ovulation (Experiment 2b)
Treatment group Progesterone (ng/mL  S.E.M.)
Day 9 Day 12 Day 14
a a,b
GnRH 3.1  0.7 5.1  0.7 4.8  0.7a,b
pLH (12.5 mg) 3.2  0.6a 4.4  0.6a 3.9  0.6a
pLH (25.0 mg) 5.5  0.7b 6.2  0.7b 6.4  0.7b
Probability 0.02 0.06 0.02
Within a column, data with different superscripts (a, b) differ (P < 0.05).
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1469

In all three experiments, the intravaginal placement of a CIDR-device was easily

accomplished and none of the heifers prematurely lost its CIDR device (100% retention
rate).

4. Discussion

4.1. Experiment 1

Ovulation in response to the first treatment occurred only in three GnRH-treated heifers,
whereas none ovulated from the other treatment groups. Poor ovulatory responses to GnRH
in randomly-cycling heifers is well documented [4,2729]; induction of ovulation of an
existing dominant follicle in randomly-cycling heifers is dependent on the stage of the
estrous cycle [29]. It was expected that at least some of the pLH-treated heifers would
ovulate; in a previous study [28], 78% of pLH-treated heifers ovulated. However, in that
study, 25 mg pLH was used; presumably 5 mg of pLH is not a high enough dose to
consistently cause ovulation. The absence of ovulation after the first ECP treatment was not
unexpected, as exogenous estradiol rarely stimulated an LH surge in the presence of
elevated plasma progesterone concentrations [30]. Instead, estradiol induced the regression
of the dominant follicle present at the time of treatment and hastened the emergence of the
next follicular wave [30].
Regardless of whether or not ovulation occurred in response to the first treatment, a
large dominant follicle was present in all heifers (of all groups) at the time of PGF
administration (Day
3), that is, 7 days after the first treatment. Although one study in beef
heifers [28] reported that only heifers ovulating in response to the first injection of GnRH
had a synchronized emergence of a new follicular wave, in dairy heifers, with the exception
of those that received their first injection of GnRH on Day 2 of the cycle (metestrous phase)
when a dominant follicle was absent, synchronized emergence of a follicular wave
occurred at all other stages of the cycle, regardless of ovulation [29]. Synchronized wave-
emergence occurred in the present study; the dominant follicle was identifiable after a
mean interval of 5.4 days after treatment and all heifers (with the sole exception of one
saline-treated heifer) ovulated the dominant follicle in response to the second treatment.
A dominant follicle was present 7 days after the first GnRH treatment (Ovsynch
protocol) in 100% of heifers [29] and 19 of 24 (79%), ovulated in response to the second
GnRH treatment. Heifers started on an Ovsynch/FTAI protocol during the metestrous
phase, although likely to have synchronous ovulation, could ovulate an aged follicle with
compromised oocyte competence [29] and poor fertility. In the present study, heifers were
at random (and unknown) stages of the estrous cycle at CIDR insertion. Regardless, in
heifers of all groups, a new dominant follicle was identified by 6 days after CIDR insertion.
Based on previous reports on the efficacy of 5.0 mg of pLH [17] and 0.5 mg of ECP [19]
in synchronizing ovulation, we expected that these low doses of both pLH and ECP would
be as effective as a standard dose (100 mg) of GnRH for synchronizing ovulation in dairy
heifers (in a CIDR-based Ovsynch protocol). Although there were no significant
differences among groups in the interval to establishment of the new dominant follicle, its
diameter, or the percentage of heifers ovulating in response to the second treatment, there
1470 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

was a notable lack of synchrony in ovulation (range 48168 h following CIDR-removal) in

heifers given 5.0 mg pLH. Furthermore, mean peak LH concentration and mean
progesterone concentrations were also lower in heifers that received 5.0 mg pLH. Even
though the variance in the interval from CIDR-removal to ovulation was greater in ECP-
treated heifers than in GnRH- or saline-treated heifers, 0.5 mg ECP was comparable to
100 mg GnRH in synchronizing ovulation, and in peak LH and postovulatory progesterone
concentrations. Although low (0.5 or 1.0 mg) doses of ECP given at random stages of the
estrous cycle to lactating dairy cows had limited efficacy in synchronizing ovulation in one
study [18], ECP effectively synchronized ovulation in dairy heifers [19,31] and in lactating
dairy cows [20,32]. It is noteworthy that label directions recommend giving 35 mg of ECP
for induction of estrus in anestrous cattle. However, these doses resulted in high estradiol
concentrations that persisted for extended intervals [33] and could potentially hinder
ovulation and/or interfere with gamete transport.

4.2. Experiment 2

Because 5.0 mg of pLH did not consistently synchronize ovulation, the second
experiment compared the efficacy of higher doses (12.5 and 25.0 mg) of pLH to that of
GnRH (100 mg). The protocol used in Experiment 2a differed slightly from that of
Experiment 1; the second dose of pLH was given (inadvertently) on Day 0 instead of Day

1. Notwithstanding, we were still able to determine the synchrony of ovulation. In
Experiment 2b, although one heifer from each treatment group did not ovulate for at least
96 h after the second treatment, ovulation was relatively synchronous in the remaining
heifers. With the exception of one heifer that had a concentration of 7.6 ng/mL (#L25F,
Fig. 4f), in all pLH-treated heifers the LH surge was apparently blunted (peak
concentrations ranged from 1.4 to 4.3 ng/mL). Furthermore, four of the six heifers given
5 mg pLH (Experiment 1) also had low LH peaks. We inferred that pLH treatment
(regardless of dose) suppressed endogenous LH surges in a majority of heifers. Even
though some heifers (e.g., #L12B, L25A, L25C, L25E, Fig. 4e and f) apparently had a rise
in LH (with peaks occurring at about 16 h), the shape of their LH curves differed from that
of typical endogenous LH surges. Nevertheless, typical LH surges, with peaks >5 ng/mL,
were uncommon in pLH-treated heifers. As mentioned elsewhere, even though this LH
assay cross reacts with porcine LH (e.g., Lutropin), the standard curves (for porcine and
bovine LH) have very different slopes. Therefore, while very low concentrations of LH of
porcine and bovine origin might calculate somewhat similarly, higher concentrations (e.g.,
>0.5 ng/mL) could be very different. While absolute concentrations differ, the trends
would remain similar (i.e., high concentrations will be measured high and low
concentrations measured low).
Plasma progesterone concentrations on Days 9, 12 and 14 were higher in heifers given
25 mg of pLH than in heifers given 12.5 mg pLH. Therefore, although 100 mg of GnRH
and 12.5 mg of pLH were apparently equally efficacious in synchronizing ovulation in
dairy heifers, 25 mg of pLH induced a CL that produced more progesterone. Although we
did not determine progesterone concentrations before Day 9, it seems likely that heifers
given 25 mg pLH had increased plasma progesterone concentrations early in diestrus; this
could enhance embryonic development [34] and fertility.
 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474 1471

4.3. Experiment 3

It is well established that FTAI following Ovsynch results in poor pregnancy rates in
dairy heifers [2,46], perhaps due to a more rapid turnover of ovarian follicular waves in
heifers than in lactating cows [35] and the premature onset of estrus [2,27,29]. Our long-
term objective includes developing an FTAI protocol that can yield good fertility for a
modest cost. Since treatment with 0.5 mg of ECP or 12.5 mg of pLH was as effective as
100 mg GnRH in synchronizing ovulation, we evaluated pregnancy rates after CIDR-based
modified Ovsynch/FTAI protocols in a field trial. Three of the four protocols were based on
our findings of Experiments 1 and 2; the fourth protocol (GnRH/ECP) was a modification
of reports from the University of Florida [19,20] that used GnRH to initiate new follicular
growth and ECP to synchronize ovulation, either with [19] or without [20] the addition of
an oral progestin (melengesterol acetate). In the absence of rigorous estrus detection, the
incidence of premature estrus (if any) could not be determined accurately in the present
study. However, in lactating dairy cows, a CIDR-based FTAI protocol effectively prevented
the onset of premature estrus [7] and increased (P < 0.05) pregnancy rates (41.2%)
compared to standard Ovsynch/FTAI (20.6%) or AI at detected estrus (20.0%). Likewise,
the occurrence of premature estrus was 0% in dairy heifers assigned to an Ovsynch-type
protocol that included a CIDR, compared to 24% in the absence of a CIDR [6].
In our study, the numerically highest pregnancy rate (66%) was attained in ECP/ECP
heifers, but it was not different from that (59%) of GnRH/GnRH heifers. Since ECP is less
expensive than GnRH and pLH, the ECP/ECP protocol was also the most cost-effective.
Furthermore, the pLH/pLH protocol had the lowest pregnancy rate and was also the most
expensive protocol, making it the least cost-effective. A higher (56%) pregnancy rate to
FTAI has been reported [36] in beef heifers when 12.5 mg pLH was used in a CIDR-based
Ovsynch protocol. Although a 25 mg dose of pLH is likely to improve pregnancy rates,
potentially through greater ovulation rates and higher postovulatory progesterone
concentrations, there is little economic advantage in it. At 59%, the pregnancy rate
achieved in the present study with the GnRH/GnRH protocol seemed greatly superior to
the 32% reported [6] after a modified Ovsynch protocol that included a CIDR for only 6
days, with GnRH given on Days 0 and 8, PGF on Day 6, and FTAI on Day 8, performed
concurrent with the second GnRH treatment. It must be noted, however, that inseminator
effects reportedly influenced the poor pregnancy rates attained in that [6] study. It is not
clear why the pregnancy rate in heifers treated with the GnRH/ECP protocol was lower
than that of heifers treated with the ECP/ECP protocol. In an earlier study [19] FTAI
following a GnRH/ECP protocol that included melengesterol acetate (instead of CIDR) as
the progestin resulted in only a 39.2% pregnancy rate. The reasons for the poor pregnancy
rates with the GnRH/ECP protocol warrants further investigation. It is likely that by
delaying the insemination 812 h, FTAI would occur that many hours closer to ovulation
time, potentially increasing the chances for conception. This remains to be tested.
In terms of animal-handling, the ECP/ECP protocol required no more handling than the
standard Ovsynch protocol (four times including AI), whereas the pLH/pLH and GnRH/
GnRH protocols required one additional handling. The increased handling in these
protocols was due to our design, deliberately leaving the CIDR for 8 days (i.e., 1 day
beyond PGF treatment), with the expectation that this would, further, reduce precocious
1472 J.D. Ambrose et al. / Theriogenology 64 (2005) 14571474

estrus and enhance the synchrony of ovulation following FTAI. In a recently concluded
study, we [37] did not detect a significant difference in the interval from CIDR-removal to
standing estrus or ovulation in dairy heifers when a CIDR was removed after 7 days,
concurrent with PGF treatment, or after 8 days (as in the present study) with PGF given 1
day earlier. Therefore, a CIDR-based GnRH/GnRH protocol with four-times animal
handling should be possible without a high incidence of precocious estrus. However,
pregnancy rates to FTAI in a GnRH/GnRH protocol with CIDR-removed after 7 days (i.e.,
concurrent with PGF treatment) are yet to be determined.
In conclusion, CIDR-based, Ovsynch/FTAI protocols using either GnRH/GnRH or
ECP/ECP yielded pregnancy rates about 20% points higher than previously reported for
Ovsynch/FTAI [46]. Although synchronization of ovulation was satisfactory with
12.5 mg of pLH, pregnancy rates after Ovsynch/FTAI using 12.5 mg of pLH remained
lower than those achieved with Ovsynch/FTAI using ECP.

Acknowledgements

This research was supported by grants from Alberta Milk, Alberta Agricultural
Research Institute and Alberta Agriculture Food and Rural Development, and in-kind
contributions from and Bioniche Animal Health (CIDR), Pharmacia Animal Health
(Lutalyse) and Vetoquinol NA Inc. (Fertiline). The assistance of Prasanth Chelikani with
statistical analyses, and the technical assistance of Pavol Zalkovic, Melissa Currie, Dr.
Leonardo Brito, and Randy Wilde are gratefully acknowledged. We thank Dr. Julie Small,
Agriculture and Agri-Food Canada Research Centre, Brandon, Manitoba, for facilitating
the progesterone assays. Portions of these data were presented at the 8th International
Congress of Biotechnology in Animal Reproduction (ICBAR), Bernburg, Germany, 2426
September 2001, and at the 9th ICBAR, Chennai, India, 24 December 2002.

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