Theriogenology 79 (2013) 528533

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Theriogenology
journal homepage: www.theriojournal.com

Circadian inuence on the preovulatory LH surge, ovulation, and

prolactin concentrations in heifers
O.J. Ginther a, b, *, F.L.V. Pinaf a, F.A. Khan a, L.F. Duarte a, M.A. Beg b
a
b

Eutheria Foundation, Cross Plains, Wisconsin, USA

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 September 2012
Received in revised form 3 November 2012
Accepted 5 November 2012

A novel circadian study of the effect of clock hours on the preovulatory LH surge, ovulation,
and maximal PRL concentration was done in 13 nontreated Holstein heifers. Hourly blood
sampling and hourly ultrasound examinations to detect the hour of ovulation began at 8
and 48 hours, respectively, after CL area (cm2) had decreased 15% from the area at 15 days
postovulation. The resulting experimental period began at the beginning of postluteolysis
(progesterone, <1 ng/mL) and encompassed a mean of 3.5 days until ovulation. The
frequency of the peak of the preovulatory LH surge for the three 8-hour periods of
a 24-hour day was different (P < 0.02) between 2:00 AM to 9:00 AM (N 9), 10:00 AM to
5:00 PM (N 3), and 6:00 PM to 1:00 AM (N 1). The median was 6:00 AM. The
frequency of ovulations for 8-hour periods was different (P < 0.02) between 3:00 AM to
10:00 AM (N 9), 11:00 AM to 6:00 PM (N 3), and 7:00 PM to 2:00 AM (N 1). The
median was 7:30 AM. Two or three clusters of PRL pulses occurred during the 3.5 days.
Based on all available PRL pulse clusters (N 36), the clock hours of the maximal
concentration/cluster was greater (P < 0.0001) for 9:00 AM to 2:00 PM (N 33 clusters)
than for each of the three other 6-hour periods (N 0, 1, or 2 per period). The median was
11:30 AM. The hypothesis was supported that the peak of the preovulatory LH surge,
ovulation, and maximal PRL concentration during pulse clusters occur with greater
frequency during certain clock hours in heifers.
2013 Elsevier Inc. All rights reserved.

Keywords:
Circadian rhythms
Diurnal variation
LH surge
Ovulation
Prolactin

1. Introduction
Daily rhythms in reproductive events have been
described as diurnal [14] or circadian [57]. Circadian
regulation of reproductive events has been described in
detail in rats and hamsters [57]. Administration of
a barbiturate during a few critical clock hours delays the LH
surge for 24 hours. The LH surge is timed by a clock in the
suprachiasmatic nucleus of the hypothalamus. The importance of the hypothalamic nucleus has been demonstrated
by lesion experiments [8]. Estradiol acts permissibly to
prime the hypothalamic release of gonadotropin-releasing

* Corresponding author. Tel.: 1 608 798 3777; fax: 1 608 798 3722.
E-mail address: ginther@vetmed.wisc.edu (O.J. Ginther).
0093-691X/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2012.11.003

hormone [3,8]. The effect of environmental light on the

retina is the predominant external cue for the endogenous
timing system that regulates the rhythmicity of seasonal
and daily reproductive mechanisms [7,8]. The light information is processed by the suprachiasmatic nucleus which
then signals the pineal gland to begin nocturnal production
of melatonin. In addition to the response to light, the
hypothalamic nucleus is also a self-sustained oscillator.
Therefore, in the absence of a rhythmic environmental
factor (e.g., continuous darkness) an endocrinological
rhythm might be only partially altered.
The incredibly complex endogenous circadian timekeeping system has been studied in rodents in detail [58].
At the other extreme in farm animals, even descriptions of
circadian patterns of reproductive hormones and events
are limited. In ewes at 10 days postmating, progesterone

O.J. Ginther et al. / Theriogenology 79 (2013) 528533

concentration is lesser and LH concentration is greater

during the day (10:00 AM to 6:00 PM; 10:00 AM to 4:00
PM) than at night (10:00 PM to 4:00 AM) [4]. In mares,
based on blood samples collected each day at 1:00 AM, 7:00
AM, 1:00 PM, and 7:00 PM throughout the estrous cycle,
a circadian pattern occurs on Days 4 to 12 (Day 0
ovulation) in progesterone and on Days 4 to 5 in LH [1]; the
lowest concentration for both hormones was at 1:00 AM. In
women, the preovulatory LH surge occurs between
midnight and 8:00 AM, but there have been few additional
studies on circadian rhythms in human reproduction [7]. In
two cows, diurnal variation in progesterone and LH was not
detected in blood samples collected hourly during estrus
and until ovulation [9]. Blood sampling in heifers every 6
hours indicated that the concentration of LH on Days 5 to 9,
but not on Days 10 to 14, is lower at 7:00 AM than at each of
the other three time periods [2]. The effect of hour of the
24-hour day on the hour of the preovulatory surge and
ovulation in cattle apparently has not been reported.
Rhythmic pulses of PRL have been described in heifers
for 12 hours before the beginning of the luteolytic period to
36 hours after the end of the luteolytic period [10]. The
interval between the peaks of adjacent pulses is 4 or 5
hours. In lactating cows, serum PRL is greatest at 4:00 PM;
however, the stage of the estrous cycle and time of year
were not reported [11]. Based on a preliminary study in
July, multiple pulses of PRL form clusters during the
follicular phase or during 3.5 days before ovulation in
heifers. Using PRL means that were normalized to ovulation, the maximum concentration of a cluster was
approximately 24 hours from the maximum of an adjacent
cluster. The 24-hour cycle indicated that PRL clustering was
inuenced by environmental factors, but the clock hours
associated with clustering and with ovulation were not
determined.
The effect of daylength in cattle has been studied more
for its role in seasonal or circannual hormonal rhythms
than for its role in daily or circadian rhythms. Circannual
rhythms occur in circulating concentrations of LH [12] and
PRL [13,14]. The circannual rhythm in LH has been
demonstrated in ovariectomized heifers, and the PRL
rhythm occurs in intact cattle. Prolactin increases fourfold
in prepubertal heifers exposed to photoperiods of 16 hours
light:8 hours dark compared with daily photoperiods of
<12 hours [15].
The current study in heifers used hourly blood sampling
and hourly ovulation detection to test the hypothesis that
the peak of the preovulatory LH surge, ovulation, and
maximal PRL concentration during pulse clusters occur
with greater frequency during certain clock hours in
heifers.
2. Materials and methods
2.1. Heifers and clock hours
Holstein dairy heifers (N 13) aged 18 to 24 months and
weighing 450 to 580 kg were used during July 2011 in the
northern temperate zone (latitude: 43120 N). Only natural
estrous cycles were used without induced luteolysis,
induced ovulation, or synchronization of estrus or

529

ovulation. The heifers were kept under natural light in an

open shelter and were maintained by ad libitum access to
water, trace mineralized salt, and primarily grass hay.
Heifers were selected that had docile temperament and
were acclimated to the chute system. The day of ovulation
was detected by transrectal ultrasonography, and the rst
day of the experiment (Day 15 postovulation) occurred for
the rst of the 13 heifers on June 26. The last ovulation at
the end of the experiment occurred on July 20. The
experiment therefore was initiated shortly after the
summer solstice of June 20. Sunrise and sunset for the
summer solstice are 5:19 AM and 8:42 PM (16 hours light:8
hours dark).

2.2. Protocol
Cross-sectional area (cm2) of the CL was determined
every 8 hours by transrectal ultrasound using the
maximum CL image, beginning on Day 15 (Day 0 ovulation). Hourly blood sampling and hourly ultrasound
examinations to detect the hour of ovulation began 8 hours
and 48 hours, respectively, after the CL area had decreased
15% from the area on Day 15. Progesterone concentrations
for these samples indicated that the hourly sampling began
at the beginning of postluteolysis (progesterone, <1 ng/mL)
and encompassed a mean of 3.5 days until ovulation. Mean
day and diameter of the future ovulatory follicle at the
beginning of the hourly examinations for ovulation were
Day 19.7  0.6 and 14.6  0.6 mm, respectively. To minimize animal stress, hourly examinations for ovulation
detection were made quickly (<5 minutes) with a simple
search for disappearance of the preovulatory follicle. Blood
was collected from an indwelling catheter that was inserted 8 hours before the beginning of the hourly blood
sampling. Heifers were released from the holding chute
after every blood sampling or ultrasound examination and
given access to feed and water.
Clusters of PRL pulses were recognized as illustrated
(Fig. 1). A cluster consists of 2 to 6 pulses of PRL. The
maximum value in a cluster was represented by the pulse
peak with the greatest concentration. The clock hour when
ovulation occurred was determined from the recorded
hourly ultrasound records. The clock hour for concentrations of LH and PRL were determined from the recorded
clock hour of ovulation and the known number of hours
from ovulation retroactively to each hourly sample. For
example, if ovulation in an individual occurred at 7:00 AM
and the peak of the preovulatory LH surge occurred 25
hours before ovulation, the LH peak was assigned to 6:00
AM of the previous day. A day encompassed midnight
11:00 PM. The frequency of an event was compared among
sets of hours that divided evenly into 24 hours (2, 3, 4, 6, 8,
and 12 hours). The hour-set that appeared to best encompass the hours of an event was then used for comparisons
over the 24 hours. For the peak of the LH surge and
ovulation, the duration of the selected hour set was 8
hours. For maximal concentration in a PRL cluster, the
duration of the selected set was 6 hours. In addition, the LH
and PRL concentrations for the two complete days
(midnight to 11:00 PM for each day) that preceded the day

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O.J. Ginther et al. / Theriogenology 79 (2013) 528533

determine the hour effect of sequential hourly samples. A

least signicant difference test or Student paired t tests
were used to locate the differences among hours. A probability of P  0.05 indicated that a difference was signicant, and probabilities of P > 0.05 to 0.1 indicated that
a difference was approached. Data are represented as the
mean  SEM unless otherwise indicated.

Individual heifer
= Pulse peak

PRL

Day 3

= Cluster maximum

Day 2

Day 1

140
120
100

3. Results

80

The frequency of the peak of the LH preovulatory surge

for 8-hour periods was different (P < 0.02) among 2:00 AM
to 9:00 AM (N 9 peaks), 10:00 AM to 5:00 PM (N 3
peaks), and 6:00 PM to 1:00 AM (N 1 peak; Table 1). The
median clock hour of the peak of the LH surge was 6:00 AM.
The hourly LH concentrations for the nearest complete day
preceding ovulation differed (P < 0.0001) among clock
hours (Fig. 2); the maximum mean LH concentration
occurred at 7:00 AM.
Ovulation occurred more frequently (P < 0.01) during
the 12 hours after 3:00 AM (N 11) than for the 12 hours
after 3:00 PM (N 2). The frequency of ovulations for
8-hour periods was different (P < 0.02) among 3:00 AM to
10:00 AM (N 9), 11:00 AM to 6:00 AM (N 3), and 7:00
PM to 2:00 AM (N 1). The median clock hour of ovulation
was 7:30 AM.

Concentration (ng/mL)

60
40
20
0
70

PRL

Means (N = 13)

60
50
40
30
20
-80 -72 -64 -56 -48 -40 -32 -24 -16 -8

Hours from ovulation

Table 1
Frequency distributions of the peak of the preovulatory LH surge, ovulation, and maximum concentration for clusters of PRL pulses during the 24
h of a day in 13 heifers.

Fig. 1. Concentrations of PRL for an individual heifer showing CV-identied

pulse peaks (single ring) and the maximal concentration (double rings) in
clusters of multiple pulses (upper panel). Mean  SEM for three clusters of
PRL pulses in 13 heifers (lower panel).

12-h
clock

24-h
clock

Number of events
Peak of
LH surgea

Ovulation

Maximum
value in a PRL
clusterb

of ovulation were analyzed for each hormone for a difference among the 24 hours in each day.

12 AMc
1 AM
2 AM
3 AM
4 AM
5 AM
6 AM
7 AM
8 AM
9 AM
10 AM
11 AM
12 PM
1 PM
2 PM
3 PM
4 PM
5 PM
6 PM
7 PM
8 PM
9 PM
10 PM
11 PM

0000c
0100
0200
0300
0400
0500
0600
0700
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300

1
0
2d
0d
0d
1d
2d
2d
1d
1d
0
0
1
0
0
0
0
0
2
0
0
0
0
0

0
0
0
1d
2d
0d
0d
3d
1d
1d
1d
0
0
0
2
0
0
0
1
0
1
0
0
0

0
0
1
0
0
0
0
0
0
5d
1d
11d
6d
9d
1d
0
0
0
0
1
1
0
0
0

2.3. Hormone assays

Blood samples were collected into heparinized tubes
and immediately placed in ice water for 10 minutes before
centrifuging (2000 g for 10 minutes). The plasma was
decanted and stored (20  C) until assay. The assays have
been validated and described for bovine plasma in our
laboratory and used RIA for LH [2,16] and PRL [17]. The
intra- and interassay CV and sensitivity, respectively, were
11.8%, 12.8%, and 0.04 ng/mL for LH and 9.2%, 7.4%, and
0.25 ng/mL for PRL.
2.4. Statistics
Data that were not normally distributed were transformed to natural logarithms or ranks. The statistical
analyses were done by using SAS PROC MIXED (Version 9.2;
SAS Institute Inc., Cary, NC, USA). Because of repeated
measures in each heifer, a REPEATED statement was used to
minimize autocorrelation by modeling AR(1) covariance
structure between sequential measurements. One-way
ANOVA was used for hormone concentrations to

Preovulatory LH surge occurred on the day before ovulation.

Two or three clusters were available per heifer at intervals between
the maximum of adjacent clusters of 24.1  0.6 h.
c
Also called midnight or 0000 h.
d
Number of events for each hour of an 8-h period that had a greater
(P < 0.02) total number of events per period than for any other 8-h period,
except a 6-h period was used for number of PRL clusters.
b

O.J. Ginther et al. / Theriogenology 79 (2013) 528533

531

6
LH
5
Day 2

Day 1

Day 0

3
Concentration (ng/mL)

2
1
0
70

PRL

60
50
40

Median
clock
hour of
ovulation

30
20

0000 0400 0800 1200 1600 2000 0000 0400 0800 1200 1600 2000 0000 0400 0800
Clock hours on Days 2, 1, and 0 (Day 0 = ovulation)
Fig. 2. Mean  SEM for concentrations of LH and PRL during the last two complete days (midnight to 11:00 PM on Days 2 and 1) preceding the day of
ovulation (Day 0; N 13). The median clock hour of ovulation is shown. Concentrations of both LH and PRL differed (P < 0.0001) among hours.

Two or three clusters of PRL pulses occurred during the

3.5 days and the maximum PRL concentration per cluster
occurred at means of 66, 44, and 22 hours before ovulation.
Using all available clusters (N 36), the frequency of the
maximum concentration per cluster for 6-hour periods was
greater (P < 0.0001) for 9:00 AM to 2:00 PM (33 clusters)
than for each of the three other 6-hour periods (N 0, 1, or
2). The median clock hour of maximum PRL concentration
was 11:30 AM. The hourly PRL concentrations for each of
the two nearest complete days preceding ovulation differed
(P < 0.0001) among clock hours (Fig. 2). The greatest mean
concentration occurred at 12:00 noon or 1:00 PM for the 2
days.
4. Discussion
The hypothesis was supported that the peak of the
preovulatory LH surge, ovulation, and maximal PRL
concentration during pulse clusters occur with greater
frequency during certain clock hours in heifers. These are
novel ndings in heifers. The hours of the greatest
frequency of each of these events are illustrated (Fig. 3).
Although it is not known if the length of the photoperiod
affected the results, the approximate light:dark cycle
during the experiment is included in the illustration. The
light-dark cycle determines the clock hour of the preovulatory LH surge in rats [18]. In Holstein heifers, the number
of hours of light (8 hours light:16 hours dark vs. 16 hours
light:8 hours dark) did not affect the timing, amplitude, or
pattern of preovulatory surges of LH or FSH, the rise in PRL
associated with estrus, or the length of the estrous cycles
[19]. However, in another study, the light-dark cycle
determined the magnitude of the PRL concentration in

prepubertal heifers [15]. Environmental light is the

predominant cue for circadian rhythms in other species [8]
and should be considered as a potential factor in the
current study. Ambient temperature and photoperiod in
cattle affect several endocrinological events, especially the
concentration of PRL [13]. For example, cold ambient

24-hour clock for July

Midnight
12 AM
2300 0000 0100
2200

0200
0300

2100
SUNSET
2000

0400
Peak of
LH surge

1900

0500

Light:dark
cycle

1800

SUNRISE
0600

Ovulation

1700

0700

Maximum value
in a PRL cluster

1600

0800

1500

0900
1400

1000
1300

1200 1100
12 PM
Noon

Fig. 3. Illustration of day:night hours at the time of the experiment and

hours of the greatest frequency of the peak of the LH surge, hour of ovulation the next day, and maximum PRL concentration per cluster during the
follicular phase.

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O.J. Ginther et al. / Theriogenology 79 (2013) 528533

temperatures markedly suppress the ability of a light:dark

hour ratio of 16 hours light:8 hours dark to stimulate PRL
secretion in Holstein heifers [20].
The current experiment was not intended to control the
periods of light and dark. Darkness was interrupted by
articial light (50-W light bulb) at the restraining chute for
<5 minutes each hour during handling of a heifer. In this
regard, endocrinological rhythms are only partially altered
by the articial interruption of day or night, because of
a self-sustained oscillator in the hypothalamic nucleus that
partially allows for exibility in the exposure to an environmental rhythm [8]. Further study in cattle with
controlled periods of light and dark are indicated.
In the current study, the peak of the preovulatory LH
surge, the most salient event of the estrous cycle (ovulation), and the maximum concentration in a cluster of PRL
pulses were most likely to occur from 2:00 AM to 2:00 PM
in heifers. The interval of 2:00 AM to 9:00 AM for the peak
of the LH surge in the current study in heifers seems similar
to the reports in which the LH surge in women occurs
between midnight and 8:00 AM [7]. The peak of the LH
surge at 2:00 AM to 9:00 AM and ovulation at 3:00 AM to
10:00 AM the next day each involved 69% of heifers. The LH
surge and ovulation were inuenced by the clock hour but
were not daily rhythms in that they occur only once during
the 21-day estrous cycle. However, the PRL clusters
involved an approximate 24-hour rhythm during the 3.5day study of the preovulatory period. The maximum
value in a PRL cluster involved 9:00 AM to 2:00 PM in 94%
of clusters and 82% occurred in a 3-hour span (11:00 AM to
1:00 PM). These comparisons indicate that the maximal
value of PRL clusters was more tightly controlled by the
environmental factors associated with clock hours than the
peak of the LH surge and the resulting LH-induced
ovulation.
The mean interval from the peak of the LH surge to
ovulation for these samples was 25.9  0.5 hours (range
23 to 29 hours) based on both blood sampling and
ovulation detection every hour. In the current study, the
mean interval from the peak of the LH surge to ovulation
approximated a 1-hour earlier clock hour for the peak of
the LH surge than for ovulation on the following day; the
8-hour interval with the greatest frequency began at 2:00
AM for the peak of the LH surge and 3:00 AM for ovulation. The mean interval between the LH surge and ovulation also approximated the interval between the hour of
the maximum LH concentration per 24 hours and the
interval to the median hour of ovulation the following day
(24.5 hours). These results indicate that research consideration should be given to circadian regulation in the
control of the LH surge and ovulation in cattle, even
though the range of clock hours was much broader than in
rodents [5]. The median of 11:30 AM for maximal PRL
concentration per cluster was close to the noon hour for
the greatest mean PRL concentration in the day preceding
ovulation without regard to the location of clusters.
In comparison, PRL was reported to be greatest at 4:00 PM
in lactating cows at an unknown stage of the estrous
cycle [11].
Daily cycles in reproductive events, including changes in
hormone concentrations, have received limited attention in

heifers/cows. A circadian inuence on ovarian steroids and

LH was not detected with hourly sampling during estrus in
two cows [9]. Another study involved 10 heifers on Days
0 to 16, and blood samples were available only at 6-hour
intervals (1:00 AM, 7:00 AM, 1:00 PM, and 7:00 PM).
Concentration of LH was lower at 7:00 AM than at each of
the other three-hour periods but only on Days 5 to 9. The
results of the previous LH report cannot be readily assimilated with the present results, because of differences in
month, stage of the cycle, and frequency of blood sampling.
Further comprehensive studies of circadian inuence on
the LH surge, ovulation, and PRL concentrations should also
use hourly sampling and should include differences among
months, because of the potential effects of day length and
ambient temperature. Studies on PRL concentrations and
clustering should consider also other stages of the estrous
cycle.
4.1. Conclusions
Twentyfour-hour or circadian rhythms were detected
during the follicular phase in heifers. The peak of the
preovulatory LH surge was more likely to occur at 2:00 AM
to 9:00 AM than for either of the other two 8-hour periods
of the 24-hour day. Ovulation occurred the next day, and
was more likely to occur at 3:00 AM to 10:00 AM than for
the other 8-hour periods. Pulses of PRL occurred in a cluster
each day for the approximate 3 days of the study, and the
maximal concentration of a cluster usually (82%) occurred
at 11:00 AM to 1:00 PM.
Acknowledgments
This research was supported by the Eutheria Foundation, Cross Plains, WI, USA (Project B2-OJ-11). The authors
thank M. Hoffman for assistance in data handling, manuscript processing, and gure preparation.
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