Pregnancy Rates Per Artificial Insemination for Cows

and Heifers Inseminated at a Synchronized

Ovulation or Synchronized Estrus1
J. R. PURSLEY,*,2 M. C. WILTBANK,*,3 J. S. STEVENSON,†
J. S. OTTOBRE,‡ H. A. GARVERICK,§ and L. L. ANDERSON||
*Department of Dairy Science, University of Wisconsin, Madison 53706
†Department of Animal Sciences, Kansas State University, Manhattan 66506
‡Department of Animal Sciences, The Ohio State University, Columbus 43210
§Department of Animal Science, University of Missouri, Columbia 65211
||Department of Animal Science, Iowa State University, Ames 50011

ABSTRACT time AI at a synchronized ovulation provided similar

Two synchronization protocols were tested for lac-
tating dairy cows and heifers. Nulliparous dairy heif-
ers (13 to 23 mo; n = 155) and primiparous and
multiparous dairy cows (60 to 289 d postpartum; n =
310) were assigned randomly to two treatments. Con-
trols received 25 mg of PGF2a and were artificially
inseminated according to the a.m.–p.m. rule following
detected estrus. All controls that were not detected in
estrus were injected with 25 mg of PGF2a at
14-d intervals until artificial insemination ( A I ) at a
detected estrus or until timed AI at 72 to 80 h after a
third sequential injection of PGF2a. Treated cows and
heifers received a protocol that used GnRH and
PGF2a to synchronize ovulation (Ovsynch). Cows and
heifers that were treated with Ovsynch were injected
i.m. with 100 mg of GnRH at a random stage of the
estrous cycle. Seven days later, cows and heifers in
this group received 25 mg of PGF2a, followed by a
second injection of 100 mg of GnRH 30 to 36 h later.
Subsequently, the treated cows and heifers received
AI 16 to 20 h after the second injection of GnRH.
Pregnancy rates per AI were similar (38.9% vs.
37.8%) for control cows and cows treated with the
Ovsynch protocol, respectively. However, pregnancy
rate per AI was greater for control heifers (74.4%)
than for heifers treated with Ovsynch (35.1%).
Evaluation of serum progesterone concentrations at
each hormonal injection indicated that the first injec-
tion of GnRH synchronized luteal function of lactating
dairy cows but not of heifers. In summary, one fixed-
Received October 19, 1995.
Accepted June 27, 1996.
1Partial funding for this project was provided by the Regional
Project NC-113 of the Cooperative State Research Service, USDA.
2Current address: Department of Animal Science, Michigan
State University, East Lansing 48824.
3Send reprint requests to M. C. Wiltbank, Department of Dairy
Science, 1675 Observatory Drive, Madison 53706.
pregnancy rates per AI as did AI following the a.m.–
p.m. rule after estrus had been induced by PGF2a in
lactating cows, but the fixed-time AI was not effective
for heifers because of the lack of synchronization.
( Key words: dairy cattle, synchronization of ovula-
tion, gonadotropin-releasing hormone, prostaglandin
F2a)
Abbreviation key: CL = corpus luteum.
INTRODUCTION
Methods to synchronize estrus have been developed
and used in many dairy herds to manage reproduction
more efficiently. Management of AI is more efficient
when synchronization schemes are used to reduce the
variation to first and subsequent AI ( 5 ) . Synchroni-
zation protocols using approved hormones for lactat-
ing dairy cows in the US have been limited to the use
of PGF2a (1, 4, 7, 9, 16, 17). Pregnancy rates per AI
were similar when dairy cows were bred at a detected
estrus after synchronization of estrus with PGF2a or
after a spontaneous estrus (16, 17). However, estrus
is not synchronized precisely with PGF2a because this
treatment does not synchronize growth of follicles but
only regulates the lifespan of the corpus luteum
( CL) . Thus, detection of estrus is needed over a
7-d period after two doses of PGF2a are administered
11 to 14 d apart (4, 6, 9, 14). Consequently, when
cows received a fixed-time AI at 72 to 80 h after a
second injection of PGF2a, pregnancy rates per AI
were considerably lower than those for cows receiving
AI at a detected estrus (15, 16, 17).
Recently, a novel protocol, Ovsynch, was developed
to synchronize ovulation in lactating dairy cows using
GnRH and PGF2a (11). This protocol synchronized
ovulation within an 8-h period from 24 to 32 h after
the second injection of GnRH. This precise synchrony
may allow for successful AI without the detection of
estrus (11). The current study was designed to evalu-

1997 J Dairy Sci 80:295–300 295

296 PURSLEY ET AL.
ate pregnancy rates obtained by timed AI after syn-
chronization of follicular growth and ovulation follow-
ing the Ovsynch protocol compared with those after a
traditional synchronization program using PGF2a and
detection of estrus. In addition, a previous study ( 1 1 )
indicated that follicular changes during the Ovsynch
protocol were different for lactating cows than for
heifers. Thus, a second objective of this study was to
compare pregnancy rates per AI for lactating cows
and nulliparous heifers in response to these two syn-
chronization strategies.
MATERIALS AND METHODS
This study was a collaborative project among sta-
tions of the NC-113 Project of the Cooperative States
Research Service of the USDA entitled “Methods for
Improvement of Fertility in Cows Postpartum”. Lac-
tating dairy cows from three experiment stations
(total of four dairy farms) and heifers from four ex-
periment stations (total of five dairy farms) were
used in this trial. In this experiment, 310 lactating
dairy cows, 60 to 289 d postpartum, and 155 dairy
heifers, 13 to 23 mo old, were assigned randomly by
location to the two treatments described in Figure 1.
Lactating cows were randomly assigned by lactation
number (1, 2, and ≥3 ) and stage of lactation (60 to
99, 100 to 149, and ≥150 d postpartum). The mean
number of days postpartum ( ±SEM) at the start of
treatment was 100.4 ± 4.3 and 96.0 ± 4.7 for the
control and Ovsynch groups, respectively.
Cows in the control group received up to three i.m.
injections of 25 mg of PGF2a 14 d apart. Only cows
and heifers that were not detected in estrus were
given subsequent injections. All control cows detected
in estrus after each PGF2a injection received AI ac-
cording to the a.m.–p.m. rule. All controls that did not
exhibit estrus after the third injection of PGF2a
received one fixed-time AI at 72 to 80 h after PGF2a.
Detection of estrus was performed twice daily by herd
personnel at each location for 7 d following the first
two injections of PGF2a and for 3 d following the third
injection (Figure 1).
Cows and heifers treated with the Ovsynch pro-
tocol were administered 100 mg of GnRH i.m. Seven
days later, cows and heifers received 25 mg of PGF2a.
This group of cows and heifers then received a second
injection of 100 mg of GnRH 30 to 36 h later and timed
AI 16 to 20 h later (Figure 1). Cows and heifers in
the Ovsynch group were not observed for estrus.
The semen that was used for AI was chosen by the
herd manager as part of routine management of the
herd. All injections were performed by or under the
supervision of NC-113 collaborators. All AI were per-
formed by the farm personnel that were normally in
charge of AI. Pregnancy diagnosis was determined by
Journal of Dairy Science Vol. 80, No. 2, 1997
Figure 1. Description of the timing of injections for the two
synchronization methods used in this experiment. Control cows
received AI in relation to estrus unless they were not detected in
estrus after any of the three PGF2a injections. Ovsynch cows had
timed AI.
ultrasound at 25 to 30 d postpartum on approxi-
mately 80% of the cows and heifers and by rectal
palpation between 35 to 49 d following AI on approxi-
mately 20% of the cows and heifers. The pregnancy
rate per AI was defined as the percentage of cows or
heifers that were confirmed pregnant at the single
pregnancy diagnosis (ultrasound or palpation) after
one AI.
In a subset of cows and heifers, blood was collected
to evaluate the progesterone status at the time of
injections, to determine the effect of PGF2a on CL
regression, and to determine the estrus detection rate
of controls. For control cows and heifers, blood was
collected at the time of each PGF2a injection and at
24 to 48 h after PGF2a. Blood from cows and heifers
in the Ovsynch group was collected at each hormone
injection. Serum was evaluated for progesterone con-
centration using the validated ELISA or radio-
immunoassay procedure at each station. Progesterone
concentrations were determined to be high at ≥1 ng/
ml or low at <1 ng/ml.
Effect of treatment on pregnancy rate per AI was
evaluated by chi-square analysis. The interactions of
treatment and stage of lactation (60 to 75 d, 76 to
100, and >100 d), treatment and parity (1, 2, and
≥3), and treatment and location were analyzed by
logistic regression using the CATMOD procedure of
SAS (12). No interactions were detected between
treatment and parity or treatment and location.
However, a trend ( P = 0.14) for an interaction be-
tween treatment and stage of lactation was observed.
Therefore, stage of lactation (60 to 75 and >75 d ) was
reanalyzed using chi-square analysis. In addition, in-
teractions of progesterone status and treatment and
 PREGNANCY RATES USING SYNCHRONIZATION OF OVULATION
interactions of pregnancy rate, progesterone status,
and treatment were analyzed by chi-square analysis.
RESULTS
The time from the start of treatment until AI was
13 ± 11 d ( x ± SD) for cows treated with PGF2a and 9
± 0 d for cows treated with Ovsynch. As shown in
Table 1, lactating cows had a similar pregnancy rate
per AI following the traditional PGF2a protocol (con-
trol) or the Ovsynch protocol. In contrast, control
heifers had a greater ( P < 0.01) pregnancy rate per
AI than did heifers treated with Ovsynch. Also, preg-
nancy rate per AI was greater ( P < 0.01) for control
heifers than for control cows. Cows in the Ovsynch
group that were >76 d postpartum had a greater ( P <
0.05) pregnancy rate per AI than did cows that were
60 to 75 d postpartum (Table 2).
Analysis of progesterone concentrations in control
cows and heifers allowed calculation of an estrus
detection rate [percentage of cows and heifers de-
tected in estrus after high ( ≥1 ng/ml) followed by low
(<1 ng/ml) progesterone concentrations] of 66% in
cows ( n = 160) and 60% in heifers ( n = 77). Control
cows with high concentrations of progesterone ( ≥1 ng/
ml) had a greater ( P < 0.05) pregnancy rate per AI
(49.3%; n = 83) than did control cows with low con-
centrations of progesterone (21.2%; n = 19) on the
day of PGF2a injection; however, progesterone status
had no effect on pregnancy rate per AI in heifers
(high = 82.7%, n = 52; low = 71.4%, n = 7; P > 0.1).
Most cows and heifers received AI after the first or
second PGF2a injection; only 18% (lactating cows)
and 23% (heifers) received the third PGF2a injection
(Table 3). Of the lactating cows that received the
third PGF2a injection, almost all were bred by timed
AI (92%), and the pregnancy rate from this AI was
very low (Table 3). In contrast, more than half of the
heifers that received the third PGF2a injection were
bred to a detected estrus (Table 3). Control cows that
were inseminated at an observed estrus had a greater
( P < 0.01) pregnancy rate per AI than did control
TABLE 1. Pregnancy rates in cows and heifers after treatment with
PGF2a (control) or synchronization of ovulation (Ovsynch).
Pregnancy rate
Control Ovsynch
(%)
Cows 38.9 ( n = 154) 37.8 ( n = 156)
Heifers 74.4 ( n = 78) 35.1 ( n = 77)
P <0.01 >0.10
297
TABLE 2. Influence of stage of lactation on pregnancy rates in
lactating cows after treatment with PGF2a (control) or synchroni-
zation of ovulation (Ovsynch). 1
Pregnancy rate
Stage
of lactation Control Ovsynch Total
(%)
60 to 75 d 39.4 ( n = 33) 26.0 ( n = 50) 31.3 ( n = 83)
≥76 d 38.8 ( n = 121) 43.4 ( n = 106) 41.0 ( n = 227)
P 0.88 0.04 0.12
1No differences between treatments.
cows that received timed AI (Table 3). However,
pregnancy rate per AI was similar ( P > 0.10) for
control cows inseminated only at estrus and for cows
in the Ovsynch group.
For cows and heifers with functional CL on the day
of PGF2a injection, CL regression occurred (high
progesterone on day of injection followed by low
progesterone 24 to 48 h later) in all control cows and
heifers (100%) and in 109 of 116 cows (94%) and 74
of 77 heifers (96%) in the Ovsynch group. Analysis of
progesterone concentrations for cows and heifers in
the Ovsynch group demonstrated that a similar per-
centage of heifers and cows had high progesterone
concentrations on the day of the first GnRH injection
( ∼60%; Table 4). At the PGF2a injection for cows in
the Ovsynch group, the percentage of lactating cows
with high concentrations of progesterone increased to
86% ( P < 0.05) but remained at 60% in heifers. The
pregnancy rate per AI was similar for cows, regard-
less of whether concentrations of progesterone were
high or low at the time of PGF2a, but heifers with low
progesterone concentrations at the time of PGF2a in-
jection had a lower pregnancy rate per AI than did
heifers with high progesterone concentrations at the
time of PGF2a injection (Table 4).
DISCUSSION
This study compared pregnancy rates per AI for
lactating dairy cows and dairy heifers after two differ-
ent synchronization strategies. Although both pro-
tocols allowed all dairy cows to be bred, the Ovsynch
protocol provided a very predictable time of AI after
the start of treatment. Ovsynch could benefit dairy
operations because it allows for timed AI of lactating
cows without detection of estrus. For lactating dairy
cows, synchronization of ovulation with GnRH and
P PGF2a resulted in a pregnancy rate per AI that was
similar to the rate for cows receiving PGF2a every
>0.10 other week. For those cows, 83% had AI after a de-
<0.01 tected estrus, and 17% had timed AI. In another
study with 333 lactating dairy cows, we also found
Journal of Dairy Science Vol. 80, No. 2, 1997
298 PURSLEY ET AL.

TABLE 3. Percentage of cows bred and pregnancy rates per AI after each treatment with PGF2a.
Lactating cows Heifers
( % bred ( % pregnant ( % bred ( % pregnant
of total) of bred) of total) of bred)
First PGF2a 48.5 46.3 39.7 71.0
Second PGF2a 33.3 45.7 37.2 82.8
Third PGF2a
AI to Estrus 1.4 0 12.8 70.0
Timed AI 16.7 4.3 10.3 50.0
1Percentage of pregnant cows was lower ( P < 0.01) for cows bred after third PGF
2a injection than
that after first or second PGF2a injection.

similar pregnancy rates for first, second, and third AI
using the Ovsynch protocol compared with breeding
at a detected estrus (10). In contrast to the 38%
pregnancy rate per AI after Ovsynch, only 8.3% of
cows conceived to the timed AI after the third PGF2a
injection in the control group. The control cows that
received timed AI were probably from a less fertile
population, because the other control cows had previ-
ously shown behavioral estrus, indicating estrous cy-
clicity. A similar number of this less fertile population
was probably included in the Ovsynch group, which
might have reduced pregnancy rate per AI for cows in
the Ovsynch group. Thus, pregnancy rates per AI
after Ovsynch were similar to breeding after a de-
tected estrus in lactating dairy cows, but Ovsynch did
not yield acceptable pregnancy rates per AI in heifers.
Pregnancy rates per AI at an observed estrus were
almost twice as great for heifers as for lactating dairy
cows in the current study. This difference has been
apparent in most dairy herds and has been reported
previously ( 8 ) . The reason for this large discrepancy
in fertility between cows and heifers has not been
well characterized. The obvious differences between
cows and heifers include lactation, parity, age, and
nutrition ( 2 ) . These factors may produce differences
in oocyte quality and uterine environment that reduce
fertility. Second, the response to the Ovsynch protocol
by heifers was dramatically different from the
response by lactating cows. In contrast to the normal
pregnancy rate per AI that was produced by Ovsynch
in lactating cows, heifers had a lower pregnancy rate
per AI after Ovsynch than after PGF2a and detected
estrus. Ovsynch may be the first synchronization pro-
tocol that has performed well in lactating cows but
not in heifers. An understanding of the differences
in follicular dynamics may explain differences in
response to Ovsynch.
Timed AI after PGF2a is ineffective in lactating
dairy cows because of anestrous cows ( 1 7 ) and be-
cause of the large variation in time from regression of
the CL to expression of estrus (15, 16, 17). This
variation in time to estrus is due to differences in the
developmental stage of the preovulatory follicle at the
time of PGF2a injection ( 3 ) and is not related to the
rate of progesterone decrease to basal concentrations
( 5 ) . The developmental stage of the preovulatory
follicle can be controlled by exogenous hormones, such
as GnRH (19), which is the basis of the Ovsynch
protocol. We previously reported that 85% of lactating
dairy cows ovulate after an injection of GnRH at a

TABLE 4. Pregnancy rates and progesterone ( P 4) status in a subset of cows and heifers treated with the Ovsynch protocol.
First GnRH injection PGF2a Injection
>1 ng of P4/ml <1 ng of P4/ml >1 ng of P4/ml <1 ng of P4/ml
(%) (no.) (%) (no.) (%) (no.) (%) (no.)
Cows
Pregnancy rate 40.9 29/71 33.3 15/45 37.0 37/100 43.8 7/16
% of Cows1 61.2 71/116 38.8 45/116 86.2 100/116 13.8 16/116
Heifers
Pregnancy rate 40.4 19/47 29.6 8/27 47.72 21/44 20.0 6/30
% of Heifers 63.5 47/74 36.5 27/74 59.5 44/74 40.5 30/74
1Percentage of cows with high P4 was different ( P < 0.05) at first GnRH versus PGF2a injection.
2Pregnancy rate was greater ( P < 0.05) for heifers with high ( ≥1 ng/ml) versus low (<1 ng/ml) P4.

Journal of Dairy Science Vol. 80, No. 2, 1997

 PREGNANCY RATES USING SYNCHRONIZATION OF OVULATION
random stage of the estrous cycle, and a new follicular
wave begins in all cows (11). One follicle from this
new follicular wave is functionally dominant by d 7
(time of PGF2a) and can be induced to ovulate with a
second injection of GnRH.
Heifers are poorly synchronized by Ovsynch. As
discussed previously, the success of the Ovsynch pro-
tocol is dependent on synchrony of both the ovulatory
follicle and the CL. Greater synchrony of CL function
was evident in lactating cows because high serum
progesterone concentrations were detected at the time
of PGF2a injection in 86.2% of cows compared with
only 59.5% of heifers (Table 4). This result agrees
with that of our previous study in which we found
that the first injection of GnRH stimulated ovulation
of a follicle in only 54% of heifers compared with 85%
of lactating dairy cows (11). Therefore, in contrast to
heifers, cows appear to have a functionally dominant
follicle that is responsive to LH for a greater propor-
tion of the estrous cycle. The percentage of heifers
with elevated serum progesterone concentrations was
not different 7 d following the first injection of GnRH
as was found at a random stage of the estrous cycle.
Approximately 60% of heifers had high progesterone
concentrations at the first injection of GnRH and at
PGF2a injection. Obviously, luteal development was
not synchronized by the first injection of GnRH in
heifers. The low ovulatory response in heifers, as well
as other differences in follicular dynamics, might also
decrease Ovsynch success because of a lack of follicu-
lar synchrony in heifers.
To achieve successful synchronization with the Ov-
synch protocol it is particularly critical to ovulate a
follicle when the first injection of GnRH is given
during the late luteal phase. If a follicle does not
ovulate during this period, the cow or heifer may cycle
normally and would most likely be in estrus before
the second injection of GnRH. If ovulation occurs from
the initial injection of GnRH during the late luteal
phase, the newly induced CL appears to be unrespon-
sive to the spontaneous pulses of PGF2a, and, even
though the spontaneous CL may regress, the new CL
continues to grow until the day of PGF2a injection
(Pursley and Wiltbank, 1996, unpublished data).
Lactating dairy cows primarily have two waves of
follicular growth during an estrous cycle (18), which
makes it likely that there would be a follicle that is
responsive to LH during the late luteal phase. Thus,
differences in numbers of follicular waves or length of
follicular waves may be crucial for determining the
success of synchronization with Ovsynch.
Follicle growth seems to be more rapid for heifers
than for lactating cows (11), and the length of a
299
follicular wave seems to be longer (Pursley and Wilt-
bank, 1996, unpublished data). Thus, newly emerg-
ing follicles of heifers that are not ovulated by the
first injection of GnRH may lose dominance status
(turnover). Specifically, the heifers that do not
respond to the first injection of GnRH would most
likely be in the first 3 d of a new follicular wave.
Seven days later, at the time of PGF2a, some of these
heifers would be at d 9 or 10 after the initiation of a
follicular wave, a time when the old follicle loses
dominance and a new follicular wave emerges. Thus,
the newly emerging dominant follicle would not be
sufficiently mature to respond to the second injection
of GnRH. Unlike our previous study ( 1 1 ) in which
heifers and cows received the second injection of
GnRH at 24 and 48 h after PGF2a, respectively, both
heifers and lactating cows in this study were given
the second injection of GnRH at 30 to 36 h after
PGF2a injection. The design of this study did not
allow us to evaluate the degree of follicular synchrony
in cows versus heifers because sequential ultrasound
evaluations of the ovaries were not performed. Ex-
trapolation from previous ultrasound evaluations
(11), the present results, and results of others ( 1 3 )
indicate that Ovsynch synchronizes follicular and lu-
teal development in lactating cows, but not in heifers.
Practical Implications
The ability to achieve acceptable fertility after a
timed AI could have a major impact on reproductive
management of lactating dairy cows. Breeding to a
synchronized ovulation would allow more control in
AI programs and would remove dependence on estrus
detection before AI. This achievement opens the door
to various ways of managing AI and the calving inter-
val. For example, traditional selection of a voluntary
waiting period might be replaced by selection of a day
of first AI. Managers could eliminate first AI that
occur too early or too late in lactation. The data from
the present study indicate that this day of first AI
should be after 75 d to achieve maximal pregnancy
rates per AI (Table 2). Use of this protocol at the
University of Wisconsin has allowed all first AI to
occur at about 70 d of lactation, and all AI are per-
formed on 1 d of the week. The timeliness of subse-
quent breedings can also be ensured because all open
cows could receive an effective timed AI at 9 to 10 d
after pregnancy diagnosis. Potentially, the need for
detection of estrus could be eliminated from a
reproductive management program. The effectiveness
of such a program is improved if early pregnancy
diagnosis is performed, for example with transrectal
Journal of Dairy Science Vol. 80, No. 2, 1997
300 PURSLEY ET AL.
ultrasonography (10). Thus, synchronization of ovu-
lation with GnRH and PGF2a provides an effective
way to manage reproduction in lactating dairy cows.
However, this protocol was not an effective synchroni-
zation method for heifers.
ACKNOWLEDGMENTS
The authors thank Myron Brown of Sanofi Animal
Health, Inc., Overland Park, Kansas, for providing
GnRH (Cystorelin) and John Chenault of The Up-
john Company, Kalamazoo, Michigan, for providing
PGF2a (Lutalyse) . Support was also provided by the
Wisconsin, Kansas, Ohio, Missouri, and Iowa Cooper-
ative Experiment Stations and Ohio Dairy Farmers
Cooperative. We thank Crave Bros. Dairy, Waterloo,
Wisconsin; Pfeifer Farms, Bucyrus, Ohio; and Stoll
Farms, Marshallville, Ohio, for providing cows and
heifers. In addition, we thank Jerry Guenther, Uni-
versity of Wisconsin, Madison; Ann Ottobre, Daniel
Grooms, and David Smith, The Ohio State Univer-
sity; Yasuhiro Kobayashi and Betty Hensley, Kansas
State University; and Brent Salfen, University of Mis-
souri, for providing technical assistance.
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