Theriogenology 67 (2007) 824834

www.theriojournal.com

Reproductive performance of lactating dairy cows and heifers

resynchronized for a second insemination with an intravaginal
progesterone-releasing device for 7 or 8 d with estradiol benzoate
injected at the time of device insertion and 24 h after removal
J. Cavalieri a,*, G. Hepworth b, V.M. Smart c, M. Ryan c, K.L. Macmillan a
a

Department of Veterinary Science, The University of Melbourne, Werribee 3030, Australia

Statistical Consulting Centre, Department of Mathematics and Statistics, The University of Melbourne, Parkville 3010, Australia
c
Genetics Australia, P.O. Box 195, Bacchus Marsh 3340, Australia
Received 22 December 2005; accepted 10 October 2006

Abstract
One aim of this study was to compare the reproductive performance of cows and heifers when resynchronizing returns to estrus
for a second insemination by treating with an intravaginal progesterone-releasing device (IVD) for 7 or 8 d when estradiol benzoate
(EB) was administered at the start of treatment and again 24 h after device removal. An additional aim was to document the pattern
of onset and characteristics of estrus with each resynchrony treatment. Lactating cows in three herds were synchronized for a first
estrus and AI by treatment with an IVD for 8 d, starting on Day 0, cloprostenol (0.5 mg im) at device removal and EB at device
insertion (2.0 mg im) and 24 h after removal (1.0 mg im). Cows were resynchronized for a second estrus starting on Day 23 by
reinsertion of IVDs for 7 (IVD-7-EB; n = 449) or 8 d (IVD-8-EB; n = 445) with EB (1.0 mg im) administered at device insertion
and 24 h after removal. Cows were resynchronized for a third estrus by administration of EB (1.0 mg im) on Day 46, but subsequent
treatments (no further treatment, reinsertion of CIDR or administration of EB on Day 55) varied among herds as part of separate
studies. Maiden heifers (7-Day, n = 68; 8-Day, n = 69) were similarly treated as cows in a separate herd, but doses of EB were
always 1.0 mg im at device insertion and 0.75 mg im 24 h after removal. Heifers were not resynchronized for a third estrus. Cattle
were inseminated on detection of estrus at each synchronized estrus. Cumulative pregnancy rates 4 week (66.0%, 276/418 versus
59.1%, 247/418) and 7 week (72.7%, 304/418 versus 67.7%, 283/418) after the start of AI were greater (P < 0.05) in the IVD-7-EB
cows compared to the IVD-8-EB cows, respectively; this was associated with a 9% increase in conception rates at the second estrus
(P = 0.051) in the IVD-7-EB cows. Treatment did not significantly affect reproductive performance in heifers. Characteristics of
estrus measured with radiotelemetry did not differ significantly between the two treatment groups, but more cows were detected in
estrus 36 h after removal of IVDs in the IVD-8-EB cows compared to the IVD-7-EB cows (P < 0.05). We concluded that
reproductive performance in resynchronized dairy cows but not heifers was greater following resynchronization of estrous cycles
after AI with an IVD for 7 compared to 8 d when EB was injected at the start of treatment and 24 h after device removal.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Resynchronization; Estrus synchronization; CIDR; Radiotelemetry; Cattle

* Corresponding author. Present address: School of Veterinary and

Biomedical Sciences, James Cook University, Townsville, Qld 4811,
Australia. Tel.: +61 419882290; fax: +61 747791526.
E-mail address: john.cavalieri@jcu.edu.au (J. Cavalieri).
0093-691X/$ see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.10.016

1. Introduction
Reproductive performance in seasonally calving
dairy herds can be improved by maximizing the

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

percentage of cows that conceive within 6 week of

artificial insemination start date (AISD) [1]. To achieve
this, a high proportion of cows within a herd needs to be
inseminated within 3 week following AISD. In addition,
a high proportion of cows that do not conceive to the
first insemination needs to be reinseminated in the
second 3 week period. Synchronization of estrus in
cows for a first insemination and resynchronization of
estrous cycles for a second and even a third insemination have been used to condense the conception pattern
in seasonally calving dairy herds and shorten intervals
from the start of AI to conception [25].
By using a hormonal treatment strategy that involves
treatment with an intravaginal progesterone releasing
device (IVD) for 7 or 8 d, and estradiol benzoate (EB)
administered at the start of treatment and again 9 d later,
submission rates of over 90% have been achieved
within 4 d of removing devices [36]. Estrous cycles in
cows can be resynchronized for a second AI by
reinserting IVDs 12 to 14 d after a first insemination,
leaving devices in place for 7 or 8 d, and injection of EB
at the start of treatment and again 9 d later. Using this
method of resynchrony over 90% of cows that are
reinseminated within 4 d of removing devices can be
detected in estrus within a period of 24 h [2,5]. This
precise pattern of synchronizing and resynchronizing
estrus is used by some commercial dairy herds in
Australia and New Zealand to facilitate the use of labor
and restrict the amount of time needed for estrus
detection and AI. Bulls are often placed in the herd
between programmed windows of estrus detection and
AI to enable cows that return to estrus at other times the
opportunity to be bred.
Treatment with EB has been used for two purposes
with estrus synchronization systems that use IVDs.
Firstly, administration of EB at the start or during
treatment with an IVD synchronized emergence of a
new follicular wave containing a potential ovulatory
follicle about 3 to 5 d after administration [7]. Secondly,
injection of EB after removal of IVDs, during a
synchronized proestrus improved submission rates to
AI following estrus synchronization, improved the
synchrony of estrus, and induced estrus and ovulation in
cows that were anestrus at the commencement of the
synchronization treatment [810]. While this combined
use of EB has been shown to result in precise onset of
estrus and ovulation, fertility could be compromised by
using inappropriate durations of treatment with an IVD.
The optimum duration of treatment with an IVD and
intervals between concurrent treatments with EB for the
purpose of resynchronizing estrus is currently not
known.

825

If the duration of treatment with an IVD to

resynchronize returns to estrus is too long, a prolonged
interval between emergence of potential ovulatory
follicles and ovulation could occur, resulting in longer
durations of dominance of preovulatory follicles and
reduced fertility at a synchronized estrus [1113]. If
however, EB is used to induce ovulation too soon after
new wave emergence when potential ovulatory follicles
are still immature, then reduced fertility may also occur.
This has been demonstrated in studies where the use of
EB during a synchronized proestrus has induced
premature ovulation of preovulatory follicles and also
resulted in anovulatory estrus in some cows [14,15].
This can lead to lower concentrations of progesterone
following the induced estrus [16] and reduced fertility
[17]. We interpret these findings to suggest that when
designing synchrony and resynchrony treatments, it is
expected that an optimum duration of treatment with an
IVD and interval between treatments with EB should
exist, one that is both not too long but also not too short.
In this study, we examined the use of two different
protocols for resynchronizing estrus that involved
treatment with an IVD for either 7 or 8 d and
administration of EB at device insertion and 24 h after
removal. Our aim was to determine if this small
difference in treatment interval with an IVD would
affect fertility following a treatment designed to
resynchronize estrus in heifers and lactating dairy cows.
Radiotelemetry has been used to study characteristics of estrus in cattle experiencing spontaneous
[18,19] and synchronized estrous cycles [20]. Rumpmounted devices electronically record the time, duration, and frequency of mounts received by cows in
estrus. There have been few studies that have, however,
monitored the pattern of onset and characteristics of
estrus in pasture-based, lactating dairy cows which had
their estrus cycles resynchronized after a first synchronized estrus and AI. An additional aim in this study was
to document the pattern of onset and characteristics of
estrus in pasture-based dairy cows with each resynchrony treatment with the aid of radiotelemetry.
2. Materials and methods
2.1. Treatments
This study was undertaken in commercial dairy
herds located in the western district region of Victoria
(Herd A, latitude 38.3333, longitude 143.5833; Herd
B, latitude 38.1667, longitude 142.9500), southern
NSW (Herd C, latitude 36.0000, longitude, 146.3833)
and in the North Eastern region of Victoria (Kiewa

826

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

Valley; Herd D, latitude

36.4333, longitude
147.0667). Lactating cows were used in Herds A, B,
and C and nulliparous Holstein heifers were used in
Herd D. Studies were undertaken in Herds A, B, and D
during winter and Herd C during the spring. Herds A, B,
and D were seasonally calving herds in which cows
calved during a single time period each year, which is
usually less than 3 months [1]. Herd C was a splitcalving herd in which cows within the herd were mated
and calved in two distinct time periods each year [1].
The spring-bred, winter calving herd in Herd C was
used for the study. In all herds, owners attempted to
have as many cows conceive as possible on or as close
as possible after the AISD.
Lactating cows were maintained on ryegrass-clover
dominated pastures throughout the year and supplemented with 1 to 5 kg of cereal concentrate during
milking. Variable amounts of conserved forage were fed

out on pasture depending on the balance between the

nutritional requirements of the cows, and pasture
production and quality. Average annual milk production
per cow for herds enrolled in these studies was
estimated to range between 5000 and 6000 L. Heifers
in Herd D ranged in age from 13 to 15 months and were
maintained on pasture only. Holstein (Herds A, B, C)
and cross-breed Holstein and Jersey (Herd C) cows that
had calved a minimum of 18 d before the start of the
synchronization program were enrolled in the study in
Herds A, B, and C.
All treatments that were administered in lactating
cows were conducted during milking between 0530 and
0900 h. A diagrammatic representation of the treatment
protocol used is shown in Fig. 1. Cows were treated with
an IVD impregnated with 1.9 g of progesterone (CIDR
B, Genetics Australia, Bacchus Marsh, Vic.) for 8 d and
2.0 mg of EB im (Cidirol, Genetics Australia, Bacchus

Fig. 1. Diagrammatic representation of the treatment protocol in Herds A, B, and C. Estrous cycles were synchronized for a first estrus (Estrus 1) by
administering an IVD for 8 d starting on Day 0. Estradiol benzoate (EB) was administered on the day of insertion and 24 h after removal. An
analogue of PGF2a (PG) was injected at the time of removal of devices. Cows were inseminated on detection of estrus from Days 8 to 12. Devices
were reinserted on Day 23 and removed 7 (IVD-7) or 8 d (IVD-8) later. Again, EB was administered at device insertion and 24 h after removal. Cows
were inseminated on detection of estrus from Days 30 to 35 (Estrus 2). Cows detected in estrus at Estrus 2 were retreated to synchronize estrous
cycles for a third consecutive time. These cows were treated with EB on Day 46. In Herds A and B, cows that were not detected in estrus by Day 55
were alternatively treated or not treated with EB. Cows in Herd C were alternatively assigned to be either treated or not treated with an IVD from
Days 46 to 54. Cows were inseminated on detection of estrus from Days 53 to 58 (Estrus 3). Bulls were placed with the herd from Days 13 to 29, 36 to
53. Heifers in Herd D were given the same treatment for the first two consecutive estrous cycles except the dose of EB used on Days 0, 9, and 30 or 31
was less than in the cows. Heifers were not synchronized for a third estrous cycle.

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

827

Marsh, Vic.) at the start of treatment (Day 0). At the

time of device removal, 0.5 mg im of cloprostenol
(CIDRPG, Genetics Australia), an analogue of PGF2a,
was administered and 1.0 mg of EB im was injected
24 h later.
Devices that had been used to synchronize cows for
the first estrus were washed, disinfected in chlorhexidine solution by soaking for a minimum of 8 h. They
were then dried and reinserted on Day 23 of the program
with 1.0 mg of EB im being administered concurrently.
Cows were assigned by odd and even ear tag number to
have devices removed either 7 (Day 30; IVD-7-EB) or
8 d (Day 31; IVD-8-EB) later. Twenty-four hours after
devices had been removed from each cow, 1.0 mg of EB
im was administered. The experimental design and
synchronization treatment used in the heifers in Herd D
was identical to the cows except that the doses of EB
injected on Days 0, 9, 23, 30 or 31 were 1.0, 0.75, 1.0,
and 0.75 mg, respectively. Heifers in Herd D at birth
were allocated ear tag numbers sequentially in order of
birth and so the method used to allocate heifers to
groups (odd and even ear tag numbers) was expected to
balance each treatment for age. Only cows and heifers
that had been inseminated at the first synchronized
estrus were included in the results of the study to assess
the effect of differences in the resynchrony treatments
that were applied. The numbers of cattle in Herds A, B,
C, and D, respectively that were assigned to 7 or 8 d
treatment interval were, 7 d: n = 121, 96, 232, 68; and
8 d: n = 107, 95, 243, 69.
Cows detected in estrus between Days 31 to 35 of the
study had estrous cycles synchronized for a third estrus
as part of separate studies. In each herd, these cows were
treated with 1.0 mg of EB im on Day 46. Nine days later
(Day 55), in Herds A and B, these treated cows that had
not been detected in estrus were alternatively treated or
not treated with 1.0 mg of EB im to synchronize returns
to estrus. In Herd C on Day 46 of the program, devices
that had been used to synchronize cows for the first two
consecutive estrous cycles were reinserted or not
inserted alternatively into cows that had been inseminated between Days 31 and 35 and 1.0 mg of EB im was
administered. Eight days later (Day 54), devices were
removed. Heifers in Herd D were not synchronized for a
third estrus.

estrous cycles (Days 8, and 30 or 31). Heatmount

detectors (Kamar, Agrigene, Wangaratta) were also
used to detect estrus in Herds A and B, with detectors
applied 1 to 3 d before removal of devices. Cows were
monitored for estrus twice daily for 5 d (Days 30 to 35),
at the morning and evening milking, from the time of
first removal of devices. The system described by
Macmillan et al. [21] was used to record changes in tail
paint. A subset of cows in Herds A (n = 53) and B
(n = 50) were also fitted with a rump-mounted, radiotelemetric pressure-sensing system (Heatwatch, HW;
DDX Inc., Denver, CO, USA). Cows were located
within 300 m of repeaters that were located within
600 m of a signal receiver, a buffer that received and
stored information and a computer. Software (Heatwatch for Windows, DDX Inc.) processed the information into the identification of the cow and transmitter
being mounted and the time and duration of mounts
1 s. Characteristics of estrus recorded by the software
included the number and duration of mounts received
1 s. Cows were considered to be in estrus if three
mounts were recorded within a 4 h period. The
proportion of cows detected in estrus relative to the
time of removal of devices was derived from combining
the results of observations of estrous detection aids in
Herds A, B, and C that were conducted twice daily at the
time of milking.

2.2. Detection of estrus

2.4. Pregnancy diagnosis

Estrus was detected using tail paint (Herds A, B, and

C) and tail chalk (Herd D) that was applied to the base of
the tail of each cow on the day inserts were removed
following treatment to synchronize cows for successive

Pregnancy diagnosis was carried out 13 week after

the AISD by a single operator. Pregnancy diagnosis was
undertaken by ultrasonography (Aloka SSD500,
5.0 MHz transducer, Medtel Pty Ltd., Oakleigh, Vic.,

2.3. Artificial insemination

Inseminations were carried out from Days 8 to 12 (all
herds), 30 to 35 (all herds), and 53 to 57 (Herds A, B,
and C only) by professional technicians. Semen
processed in Caprogen [22] was used at the first estrus
in Herds A and B and thawed frozen semen was used in
Herds C and D. Thawed frozen semen was used in all
herds at the second estrus and third estrus where AI was
used. Bulls were placed with herds between Days 13 to
30 and 36 to 52, and removed during the days when
inseminations were performed. Previous studies have
indicated that less than 5% of pregnancies arise from
bulls mating cows between two consecutive synchronized estrous cycles [3]. Bulls were also placed with
each herd after completion of AI and the breeding of
cows continued for at least 7 weeks after the AISD.

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J. Cavalieri et al. / Theriogenology 67 (2007) 824834

Australia) with the transducer fixed to a flexible metal

extender. Conception dates were determined by the
visual estimation of fetal size [23] and insemination
dates.
2.5. Statistical analyses
Statistical analyses were conducted using the
statistical software package SPSS for Windows version
11.0 (SPSS Inc., Chicago, IL, USA). Logistic regression
[24] was used to assess the effect of relevant
explanatory variables on dependent variables. The
dependent variables that were included in analyses
included the cumulative pregnancy rates at the end of
the first, fourth, and seventh week of the breeding
program, the magnitude of the increase in pregnancy
rates between Weeks 1 to 4, and 4 to 7, the conception
rate of cows inseminated on Days 31 to 35 of the
program, and the submission rate of cows recorded on
Days 31 to 35 of the program. Age, BCS, calving to
AISD interval, breed, and the interaction of Herd with
treatment were included as explanatory variables. Ages
of cows were categorized as 2, 3, 4, 5 to 7 and more than
7 year; BCS was separated into 2.5, 2.75, 3.0, and
3.25; intervals from calving to AISD were categorized
as less than 6 week, 69 week, 912 week and more
than 12 week; breed was divided into cows that were
75% Holstein or greater than 75% Holstein. In the
analyses conducted for this study, Herd was considered
to be a random effect and while included in logistic
regression and ANOVA models, effects due to herd
were not reported.
Terms were considered for elimination from the
model using backwards step-wise logistic regression.
The test for elimination was a likelihood-ratio test [24]
using a significance level of P  0.10. If an interaction
was significant at P < 0.10, the associated main effects
were included in the model. Probability values for all
main effects remaining in models were determined
using the approximate chisquared distribution of the
likelihood ratio statistic. Odds ratios and 95%
confidence intervals were also calculated for all main
effects remaining in models and included in the results
listed in Tables 3 and 4. P-values <0.05 were
considered significant and 0.05 < P < 0.10 were
regarded as a tendency, or an indication of an effect.
Logistic regression was also used to examine the
potential effects of treatments that were used to
synchronize the third estrus on dependent variables
(conception rate of cows inseminated at the second
estrus and 7 week pregnancy rates). A model was
constructed for Herds A and B that contained the

explanatory variables listed above, but included the

effects of treatment that was used to synchronize cows
for the third estrus and the interaction of treatments that
were used to synchronize cows for the second estrus
with treatments used to synchronize the third estrus.
The interaction of treatments with Herd was also
included. The same model was used to examine
treatments conducted in Herd C, with the exception
that Herd was not included in the model.
Submission rate was defined as the proportion of
cows retrospectively diagnosed as nonpregnant during
the first 20 d of the breeding program (Days 10 to 30;
first round of AI) that were detected in estrus and
inseminated at the second round of insemination.
Conception rate was defined as the proportion of cows
retrospectively diagnosed as nonpregnant at the first
estrus that were presented for insemination from Days
31 to 35 of the study and that were classified as
conceiving to that insemination.
The synchrony of cows returning to the second estrus
(homogeneity of variance), relative to the time of
removal of inserts for cows monitored with radiotelemetry, was tested using Levines test [25]. The
proportion of cows first detected in estrus within 96 h of
removing devices was compared with a chi-square test.
3. Results
3.1. Omitted animals
Cows (6.5%; 58/894) or heifers (0.73%; 1/137) that
lost devices when resynchronizing estrous cycles for the
second estrus were omitted from all analyses as the
duration of treatment with devices was uncertain.
3.2. Reproductive performance
Significant interactions were not detected between
the treatment used at the second estrus and treatment
used at the third estrus in Herds A, B, and C. These
treatments were therefore considered to be acting
independently and effects of treatments used to
synchronize cows for the third estrus were omitted
from further analyses of effects due to treatments that
were used to synchronize the second estrus.
Reproductive performance of cows and heifers
enrolled in this study are summarized in Tables 1 and
2, respectively. Pregnancy rates after 1 week of mating
were not significantly affected by treatment. Four-week
pregnancy rates were 7% greater in cows that had
estrous cycles resynchronized with an IVD for 7 d
compared with cows that were treated with an IVD for


0.10
0.012
0.047
0.85
0.051

Values derived from the final statistical model. Herd by treatment interactions were not significant.
Percentage of cows retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI from Days 30 to 35 of the study.
Pregnancy rate of cows retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI from Days 30 to 35 of the study.
a

8
40.0
59.1
67.7
72.5
40.1
(180/418)
(276/418)
(304/418)
(180/238)
(89/180)
7
43.1
66.0
72.7
75.6
49.4
(100/224)
(147/224)
(163/224)
(91/124)
(40/91)
8
44.6
65.6
72.8
73.4
44.0
(94/215)
(145/215)
(158/215)
(90/121)
(46/90)
7
43.7
67.4
73.5
74.4
51.6
Treatment interval (d)
1-Week pregnancy rate (%)
4-Week pregnancy rate (%)
7-week pregnancy rate (%)
Submission rate - second estrusb (%)
Conception rate - second estrusc (%)

7
50.9
70.2
77.2
76.8
51.2

(58/114)
(80/114)
(88/114)
(43/56)
(22/43)

8
39.2
58.8
68.6
69.4
46.5

(40/102)
(60/102)
(70/102)
(43/62)
(20/43)

7
31.5
57.3
65.2
77.0
44.7

(28/89)
(51/89)
(58/89)
(47/61)
(21/47)

8
29.3
43.5
54.3
73.8
27.1

(27/92)
(40/92)
(50/92)
(48/65)
(13/48)

C
B
A

829

Table 2
Reproductive performance of maiden dairy heifers that had estrous
cycles resynchronized for a second estrus after AI with an intravaginal
progesterone releasing device for either 7 or 8 d, with estradiol
benzoate administered at device insertion and 24 h after removal

(167/418)
(247/418)
(283/418)
(182/251)
(73/182)

Pa
Total
Herd
Variable

Table 1
Reproductive performance of lactating dairy cows that had estrous cycles resynchronized for a second estrus after AI with an intravaginal progesterone releasing device for either 7 or 8 d with
estradiol benzoate administered at device insertion and 24 h after removal

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

Variable

Herd D

Treatment interval (d)

1-Week pregnancy rate (%)
4-Week pregnancy rate (%)
Submission rate - second estrusa
Conception rate - second estrus b

7
38.8
71.6
95.1
53.8

(26/67)
(48/67)
(39/41)
(21/39)

P
8
43.5
68.1
84.6
48.5

(30/69)
(47/69)
(33/39)
(16/33)

0.58
0.65
0.11
0.65

Percentage of heifers retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI from
Days 30 to 35 of the study.
b
Pregnancy rate of heifers retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI
from Days 30 to 35 of the study.

8 d (P = 0.012; Table 1). Submission rates at the second

estrus were not significantly affected by treatment.
Conception rates of cows inseminated at the second
estrus were 9% greater in cows that had been treated
with an IVD for 7 d compared to 8 d (P = 0.051;
Table 1). Seven-week pregnancy rates were greater by
5% in the cows treated with an IVD for 7 d compared to
those treated for 8 d (Table 1, P = 0.047). While both 4
and 7 week pregnancy rates were greater in the IVD-7EB compared to the IVD-8-EB treated cows, differences in the magnitude of the increase in pregnancy
rates between Weeks 1 and 4, and Weeks 4 and 7 for the
IVD-7-EB and IVD-8-EB treatments were only 3.8 and
1.9%, respectively (P > 0.10). Significant Herd by
treatment interactions was not detected for any of the
reproductive indices examined in the cows (P  0.55).
No significant differences in any of the reproductive
indices measured between a treatment period of 7 or 8 d
were detected in the heifers in Herd D (P > 0.10;
Table 2).
The interval from calving to AISD had a positive
effect on 1 (P = 0.004), 4 (P < 0.001), and 7 week
(P < 0.001; Table 3) pregnancy rates, and conception
rates at the second estrus (P = 0.001; Table 4).
Pregnancy rates after 1, 4, and 7 week of the breeding
season were greater in cows that had been calved from
nine to12 week compared to cows that had been calved
less than 9 week before the first insemination (Table 4).
Submission rates at the second estrus were greater in
cows that had calved less than 6 week at the time of the
first insemination compared to cows that had calved
from 6 to 12 week (P = 0.040; Table 4). Cows with a
BCS 2.5 had lower 4 (P = 0.038) and 7 week
pregnancy rates (P = 0.013; Table 3) and submission
rates (P < 0.001; Table 4) at the second estrus

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J. Cavalieri et al. / Theriogenology 67 (2007) 824834

Table 3
Factors affecting cumulative pregnancy rates after resynchronizing estrous cycles after AI with an intravaginal progesterone releasing device for 7
(IVD-7-EB) or 8 d (IVD-8-EB) with estradiol benzoate injected concurrently with device insertion and 24 h after removal
Dependant variable

Variable

d.f.

1-Week pregnancy rate

IVD-7-EB
Calving-AISD a
Calving-AISD (<6 week)
Calving-AISD (69 week)
Calving-AISD (>12 week)

1
3
1
1
1

0.10
0.004

IVD-7-EB
Calving-AISD
Calving-AISD (<6 week)
Calving-AISD (69 week)
Calving-AISD (>12 week)
BCSb
BCS (2.5)
BCS (2.75)
BCS (3.25)

1
3
1
1
1
3
1
1
1

0.012
<0.001

IVD-7-EB
Calving to AISD
Calving-AISD (<6 week)
Calving-AISD (69 week)
Calving-AISD (>12 week)
BCS
BCS (2.5)
BCS (2.75)
BCS (3.25)
Agec
Age (2)
Age (4)
Age (57)

1
1
1
1
1
3
1
1
1
4
1
1
1

0.047
<0.001

4-Week pregnancy rate

7-Week pregnancy rate

a
b
c

Odds ratio

Confidence interval

Reference group

1.29

0.951.77

IVD-8-EB

0.40
0.60
0.85

0.230.72
0.400.89
0.521.40

912 week
912 week
912 week

1.51

1.092.08

IVD-8-EB

0.30
0.59
1.43

0.180.50
0.400.86
0.812.51

912 week
912 week
912 week

0.60
0.82
1.29

0.380.95
0.541.26
0.702.37

BCS (3.0)
BCS (3.0)
BCS (3.0)

1.41

1.001.99

IVD-8-EB

0.41
0.69
1.66

0.240.68
0.461.04
0.883.13

912 week
912 week
912 week

0.55
0.75
1.39

0.340.89
0.471.18
0.712.71

BCS (3.0)
BCS (3.0)
BCS (3.0)

0.52
0.85
0.60

0.290.94
0.451.60
0.351.03

Age (3)
Age (3)
Age (3)

0.038

0.013

0.066

Calving to AI start date interval in days.

Body condition score.
Age (years).

compared to cows with a BCS = 3.0. Seven-week

pregnancy rates (P = 0.066) and submission rates at the
second estrus (P = 0.054; Table 4) tended to be less in
the 2 year old and 57 year old cows compared to the 3
year old cows.

detected in estrus 36 h after removal of inserts in the

cows treated with an IVD for 8 compared to 7 d
(P < 0.001). At 48 h after removal of inserts, there was
a tendency for a greater percentage of cows to be first
detected in estrus in the cows treated for 7 d compared
to the cows treated for 8 d (P = 0.068).

3.3. Characteristics of estrus

4. Discussion
Characteristics of estrus for cows treated with an
IVD for 7 or 8 d and monitored with radiotelemetry are
shown in Table 5. No significant effects of treatment, or
herd by treatment interactions were detected for any of
the characteristics of estrus examined. The synchrony of
returns to estrus relative to the time of removal of inserts
also did not differ significantly between treatments in
cows in which onset of estrus was monitored with
radiotelemetry (P = 0.86). The proportion of cows
detected in estrus relative to the time of removal of
inserts for cows treated with an IVD for 7 or 8 d is
shown in Fig. 2. A greater proportion of cows were first

Our interpretation of the results of this study are that

when using an IVD to resynchronize returns to estrus,
with EB administered at the start of treatment and 24 h
after removal of devices, that a treatment interval of 7 or
8 d is similarly effective in resynchronizing a return to
estrus in on average 74% of cows and 90% of heifers
that are not pregnant to the first insemination. We also
found that a treatment interval of 7 compared to 8 d with
EB injected at the start of treatment and 24 h after
removal can improve reproductive performance in
lactating dairy cows, although numerical differences in

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

831

Table 4
Factors affecting conception and submission rates after resynchronizing estrous cycles after AI with an intravaginal progesterone releasing device for
7 (IVD-7-EB) or 8 d (IVD-8-EB) with estradiol benzoate injected concurrently with device insertion and 24 h after removal
Dependant variable

Variable

d.f.

Conception rate - second estrus a

IVD-7-EB
Calving-AISD b
Calving-AISD (<6 week)
Calving-AISD (69 week)
Calving-AISD (>12 week)

1
1
1
1
1

0.051
0.001

IVD-7-EB
Calving-AISD
Calving-AISD (<6 week)
Calving-AISD (69 week)
Calving-AISD (>12 week)
BCSd
BCS (2.5)
BCS (2.75)
BCS (3.25)
Agee
Age (2)
Age (4)
Age (57)
Age (>7)

1
3
1
1
1
3
1
1
1
4
1
1
1
1

0.76
0.040

Submission rate - second estrusc

Odds ratio

Confidence interval

Reference group

1.58

1.002.52

IVD-8-EB

0.26
0.74
1.58

0.120.53
0.441.30
0.773.23

912 week
912 week
912 week

1.08

0.661.78

IVD-8-EB

3.08
1.02
1.67

1.257.55
0.571.84
0.674.18

912 week
912 week
912 week

0.25
0.85
1.98

0.120.53
0.431.68
0.616.41

BCS (3.0)
BCS (3.0)
BCS (3.0)

0.30
0.66
0.42
0.68

0.120.76
0.241.82
0.181.01
0.222.10

Age
Age
Age
Age

<0.001

0.054
(3)
(3)
(3)
(3)

a
Pregnancy rate of cows retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI from Days 30 to
35 of the study.
b
Calving to AI start date interval in days.
c
Percentage of cows retrospectively determined to be nonpregnant at the time of the second estrus that were presented for AI from Days 30 to 35 of
the study.
d
Body condition score.
e
Age (years).

cumulative pregnancy rates after 4 and 7 week of

breeding for each treatment were relatively small
(<7%). Greater conception rates at the second
synchronized estrus in the cows treated with the
IVD-7-EB treatment compared to those treated with
the IVD-8-EB treatment was one of the effects of
treatment that was detected, suggesting that treatment
did affect fertility at the second resynchronized estrus.
Reductions in fertility at a synchronized estrus have
been attributed to a prolonged duration of dominance of

preovulatory follicles and ovulation of aged oocytes

[26]. A progressive reduction in pregnancy rates from 0,
10 to 15, and 20 to 50% has been recorded with a
duration of dominance of 27, 48, and more than 10 d,
respectively [13]. Greater fertility in cows that ovulate
dominant follicles arising from a third compared to a
second follicular wave after estrus has also been
attributed to differences in the duration of dominance
and size of preovulatory follicles [27]. In the present
study, the interval from the start of treatment to estrus

Table 5
Characteristics of estrus (mean (S.E.M.) in cows after resynchronizing estrous cycles after AI with an intravaginal progesterone releasing device
for 7 (IVD-7-EB) or 8 d (IVD-8-EB) with estradiol benzoate injected concurrently with device insertion and 24 h after removal
Variable

n
Interval to estrusa (h)
Number of mounts received (log)
Duration of estrus (h)
Total duration of mounts received (s) (log)
Mounts received per hour
Duration per mount received (s)
a

Relative to time devices were removed.

Treatment
IVD-7-EB

IVD-8-EB

20
42.9  1.8
14.1  2.5
9.9  1.2
24.4  4.7
1.1  0.20
1.8  0.14

17
42.0  2.3
12.3  3.0
7.5  0.76
22.8  6.6
0.87  0.15
1.8  0.13

Total

37
42.5  1.4
13.2  1.9
8.8  0.76
23.7  3.9
0.98  0.13
1.8  0.10

0.89
0.55
0.10
0.66
0.44
0.96

832

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

Fig. 2. The percentage of cows detected in estrus in Herds A, B, and C

combined relative to the time of removal of devices for cows treated
with an intravaginal progesterone releasing device for 7 (&) or 8 d
(&), with estradiol benzoate administered at device insertion and 24 h
after removal. **P < 0.001.

was delayed by 1 d in the cows treated with an IVD for

8 d compared to cows treated for 7 d. A greater
proportion of cows were detected in estrus by 36 h after
removal of inserts in cows treated with an IVD for 8
compared to 7 d. Perhaps the preovulatory follicles
were more mature at the time of removal of devices in
the cows treated for 8 d. Intervals to estrus following the
induction of luteolysis are inversely related to the
maturity of preovulatory follicles at the time of inducing
luteolysis [28]. The interval to estrus is less if a mature
ovulatory follicle is present and is still functionally
dominant at the time of the induced luteolysis whereas
if a dominant follicle has become atretic or is only
emerging, the interval to estrus will be greater [29,30].
The longer interval from the start of treatment to estrus
and shorter interval from device removal to estrus in the
IVD-8-EB cows could imply that the duration of
dominance was greater in these cows compared to the
IVD-7-EB cows and contributed to the lower conception rates observed in the IVD-8-EB cows. Our failure
to detect a significant difference in the conception rates
at the second estrus in the heifers may have been due to
the small number of heifers enrolled in the study and the
inherently low statistical power. It could also be that
differences in location, dose rates of EB or physiological differences in factors such as follicle dynamics
differed in heifers compared to cows with the treatments
used. Further studies with greater numbers of heifers
will be needed to determine if significant differences in
pregnancy rates in heifers occur as a result of the
treatments that were applied.
Cumulative pregnancy rates after each resynchronized estrus are influenced by pregnancy rates at the
preceding synchronized estrus and submission and
conception rates at the resynchronized estrus. Although
differences in pregnancy rates were detected after 4 and

7 week of breeding in the cows of this study, differences

in the magnitude of the change in pregnancy rates
between Weeks 1 and 4, and 4 and 7 did not differ
between treatments. Thus, although treatments preceding the first synchronized estrus were identical, small
differences in the pregnancy rates achieved at the first
synchronized estrus would have contributed to some of
the differences in pregnancy rates by Weeks 4 and 7.
This could suggest that the potential impact of
treatments on overall reproductive performance was
smaller than suggested by the differences in cumulative
pregnancy rates at 4 and 7 week that were found.
One of our concerns with shorter intervals of
treatment such as the IVD-7-EB treatment used in this
study was that less time may have elapsed between
follicle emergence after treatment with EB at the time of
device insertion and administration of EB 24 h after
device removal compared with the IVD-8-EB treated
cows. Consequently, diameters of ovulatory follicles
could have been reduced along with subsequent CL
function in the IVD-7-EB treated cows compared with
cows treated with the IVD-8-EB protocol. The diameter
of ovulatory follicles following administration of EB
during a synchronized proestrus is proportional to the
period of time between follicle emergence and removal
of IVDs, although differences in CL function are mainly
observed following ovulation of follicles that are less
than 10 mm in diameter [16]. Since reproductive
performance was not compromised in this study by
using the 7 d compared to the 8 d treatment protocol, the
fertility of ovulating oocytes and CL function subsequent to ovulation was unlikely to be compromised by
the shorter treatment protocol.
Characteristics of estrus measured in this study with
radiotelemetry did not differ between the two treatment
groups and were within ranges of values reported in
other studies where estrus in dairy cows was monitored
with radiotelemetry [1820]. Submission rates of
retrospectively diagnosed nonpregnant cows at the
second synchronized estrus in the present study ranged
from 69 to 77% in cows, which was similar to previous
reports [3,4], and ranged from 85 to 95% in heifers.
Early embryonic loss, prolonged luteal phases of the
estrous cycle and failure to detect estrus have been
suggested as some of the causes of reduced submission
rates at a resynchronized estrus [3133]. There was
evidence in this study that significant differences in
submission rates will result from treatment with an IVD
for 7 or 8 d in both cows and heifers.
Increasing calving to AISD interval, lower body
condition score, and cows being aged 2, or 57 year
compared to 3 year old cows were associated with

J. Cavalieri et al. / Theriogenology 67 (2007) 824834

reduced submission rates in this study. Submission rates

in this study would be affected by the proportion of cows
that are in estrus at the time when estrus detection and AI
occurred, and the extent to which cows returning to estrus
exhibited behavioral signs of estrus. In a previous study,
cows classified as anestrus at the start of treatment with
an IVD and EB had lower submission rates at a
resynchronized estrus and lower body condition scores
compared with cows that were classified as undergoing
estrous cycles at the start of treatment [5]. Anestrus is also
more common among 2 year old cows compared to older
cows, due to partitioning of energy needs between
maintenance, growth, and lactation [34]. Negative energy
balance had an inhibitory effect on fertility and was
associated with greater body condition loss in dairy cows
[35]. Therefore, reduced submission rates among cows
with lower BCS and 2 year old cows may have been due
to greater negative energy balance in these cows and a
higher incidence of anestrus at the time treatment started.
A direct relationship between calving to mating
interval and the likelihood that nonpregnant cows would
be resubmitted for AI within 25 d of insemination has
been noted previously in studies conducted in dairy
cows with [4] and without [36] resynchronized estrus.
We are therefore unsure as to why there was a negative
relationship between calving to AISD interval and
submission rate in this study. It has been shown that
submission rates of cows resynchronized after a first
synchronized estrus is reduced following insemination
at the first synchronized estrus [33], suggesting that
conception and early embryonic loss can reduce
submission rates at a resynchronized estrus. In this
study, cows that had calved less than 6 week before the
AISD were most likely to be submitted for AI at the
resynchronized estrus, compared with those with a
longer postpartum interval before AISD. Perhaps in this
study, a more hostile uterine environment or other
factors increased the rate of conception failure or very
early embryonic loss prior to the normal timing of
maternal recognition of pregnancy in cows with shorter
intervals from calving to AISD. As a result, luteolysis
and a return to estrus were not delayed in cows with a
shorter calving to AISD, which contributed to the
improvement in submission rates at the second estrus
among cows with shorter calving to AISD intervals. A
longer calving to AISD interval was also observed in
this study to improve cumulative pregnancy rates after
1, 4, and 7 week of mating and pregnancy rates at the
second estrus. This is likely to reflect the improvement
in fertility in cows that has been reported previously
with increasing interval from calving to insemination
[1,37].

833

We concluded that a significant but numerically small

improvement in reproductive performance of lactating
dairy cows occurred following resynchronization of
estrous cycles after AI with an IVD for 7 compared to 8 d
with EB injected at the start of treatment and 24 h after
device removal. The improvement in reproductive
performance was associated with increased conception
rates at the resynchronized estrus following a 7 compared
to an 8 d period of treatment with an IVD. However, there
was no significant difference between treatment protocols for reproductive performance in maiden heifers. A
lack of difference in submission rates at the resynchronized estrus between cows treated with the two
resynchrony treatments supported the conclusion that
both treatments were equally effective in resynchronizing returns to estrus after AI. Relative to the time of
removing IVDs, the pattern of onset of estrus after
treatment was similar, apart from more cows being in
estrus earlier with the longer duration of treatment.
Characteristics of the resynchronized estrus, measured
with radiotelemetry were similar for both treatments.
Acknowledgments
We thank Glenormiston Agricultural College, J. and
A. Cirillo, Kunanadgee Station and S. and S. Crooke for
the generous provision of animals and technical
assistance for this study. We also thank K. Davis for
providing technical assistance. Financial support for
these studies was provided under the Strategic Partnerships with Industry Research and Training Scheme, of
the Australian Research Council and the Department of
Education, Training and Youth Affairs, by Genetics
Australia, Pharmacia-Upjohn and the Dairy Research
and Development Corporation. We thank DDX, Inc.
(Denver, CO) for supplying HeatWatch equipment used
in the study (http://www.heatwatch.com).
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