J. Dairy Sci. 89:4694–4702
© American Dairy Science Association, 2006.
Endocrine Alterations Associated with Extended Time Interval
Between Estrus and Ovulation in High-Yield Dairy Cows
A. Bloch,* Y. Folman,† M. Kaim,† Z. Roth,* R. Braw-Tal,† and D. Wolfenson*
*Department of Animal Science, Faculty of Agriculture, The Hebrew University, Rehovot 76100, Israel
†Institute of Animal Science, Agricultural Research Organization, Bet-Dagan 50250, Israel
Short fertile half-lives of the male and female ga-
metes in the female tract necessitate accurate timing
of artiﬁcial insemination. We examined the possible
association between extension of the estrus to ovulation
(E-O) interval and alterations in concentrations of es-
tradiol, progesterone, and the preovulatory LH surge
before estrus and ovulation. High-yielding Holstein
cows (n = 74 from a total of 106) were synchronized
and were examined around the time of the subsequent
estrus. They were observed continuously for estrual
behavior. Blood samples were collected before and after
estrus, and ultrasound checks for ovulation were made
every 4 h. About three-quarters of the cows exhibited
short (but normal) E-O intervals of 22 to 25 h (25%)
or normal intervals of 25 to 30 h (47%); 17% of them
displayed a long (but normal) E-O interval of 31 to 35
h, and about 10% exhibited a very long E-O interval of
35 to 50 h. Extended E-O interval comprised estrus-
to-LH surge and LH surge-to-ovulation intervals that
were both longer than normal. Pronounced changes in
hormonal concentrations were noted before ovulation
in the very long E-O interval group of cows: progester-
one and estradiol concentrations were reduced, and the
preovulatory LH peak surge was markedly less than
in the other 3 groups. Postovulation progesterone con-
centrations during the midluteal phase were lesser in
the very long and the long E-O interval groups com-
pared with those in the short and normal interval
groups. Season, parity, milk yield, and body condition
did not affect the estrus to LH surge, LH surge to ovula-
tion, and E-O intervals. The results indicate an associa-
tion between preovulatory-reduced estradiol concentra-
tions and a small preovulatory LH surge, on the one
hand, and an extended E-O interval, on the other hand.
Delayed ovulation could cause nonoptimal timing of AI,
a less than normal preovulatory LH surge that may be
associated with suboptimal maturation of the oocyte
Received March 30, 2006.
Accepted June 26, 2006.
Corresponding author: email@example.com
before ovulation, or reduced progesterone concentra-
tions before and after ovulation. All may be factors
associated with poor fertility in cows with a very long
Key words: estrus, ovulation, luteinizing hormone
surge, dairy cow
Delayed ovulation in cows following estrus minimizes
the chances of successful fertilization. The short fertile
half-life of bovine gametes restricts the period during
which fertilization can occur (Dransﬁeld et al., 1998;
Nebel et al., 2000). Fertilization rate of the oocyte de-
creases signiﬁcantly 8 to 12 h postovulation, and insem-
ination 25 to 40 h before ovulation is associated with
a signiﬁcant decrease in conception rates (Hunter,
1994). In most studies, mean intervals between onset
of estrus and ovulation (E-O) in beef and dairy cattle
ranged from 23 to 33 h (Renger et al., 1978; Mikeska
and Williams, 1988; Lemaster et al., 1999), and the
mean was reported to be about 27 h in lactating dairy
cows (Walker et al., 1996).
Wide variation among E-O intervals of individual
cows or heifers in older studies was attributed to inade-
quate frequencies of detecting estrus and palpation of
ovaries to determine ovulation. Even in more recent
studies, however, in which modern electronic devices
were used to detect the onset of estrus, and in which
the ovulation time was more frequently monitored by
ultrasonography, a wide variation in E-O intervals was
reported. For example, Walker et al. (1996) reported
that 78% of lactating cows ovulated within 40 h of the
onset of estrus, but that 22% had not ovulated by 40
h. In another recent study of lactating Holstein cows,
a mean E-O interval of 30 h, with a range from 18.5 to
48.5 h was reported (Roelofs et al., 2005). A mean inter-
val of 28 h (ranging from 12 to 36 h) from visual estrus
to ovulation was noted for Bos indicus cows (Cavalieri
et al., 1997). Notably, mean E-O intervals and ranges
were similar for cows exhibiting spontaneous estrus
and for those in which estrus was induced by PGF
(Walker et al., 1996). A wide variation among E-O inter-
vals was recorded for heifers as well; 92% of Holstein
ENDOCRINE CHANGES ASSOCIATED WITH DELAYED OVULATION 4695
heifers ovulated within 36 h of the onset of estrus and
8% between 36 and 48 h after estrus (Hernandez-Ceron
et al., 1993). Lemaster et al. (1999) reported a mean
interval of 26 h and a range from 11 to 71 h in crossbred
Despite extensive documentation over the years that
conﬁrms large variation among E-O intervals of individ-
ual cows, the possible associations between extended
E-O intervals and endocrine traits are poorly docu-
mented. A single study by Saumande and Humblot
(2005) reported a negative correlation between the pre-
ovulatory peak in estradiol concentrations and duration
of the estrus-LH peak and E-O intervals. They hypothe-
sized an ovarian control of those intervals that was
related to a negative correlation between the size of the
preovulatory follicle and the E-O interval. The endo-
crine milieu, however, that is associated with delayed
ovulation and extended E-O interval has not been char-
acterized. The present study characterized groups of
cows that exhibited short, normal, long, or very long E-
O intervals. We focused on the exceptional group of
cows that exhibited a very long E-O interval because a
long E-O interval minimizes the chances of those cows
conceiving. We examined the possibility that the abnor-
mally large E-O interval in a group of high-yielding
cows was associated with alterations in steroid and
gonadotropin concentrations; namely, progesterone
concentrations during the preceding luteal phase, es-
tradiol concentrations before estrus, and LH concentra-
tions at the preovulatory peak.
MATERIALS AND METHODS
Holstein cows in their ﬁrst to ﬁfth lactations were
used. The experiment, which was designed with 9 clus-
ters of cows grouped according to their calving dates,
was performed during spring (March to May), summer
(July to August), and fall (October to November). Cows
were kept in an open shed with access to an adjacent
yard. During summer, a sprinkling and ventilation cool-
ing system was used (Flamenbaum et al., 1986). Maxi-
mum and minimum air temperature and relative hu-
midity were as follows: spring, 22.9 and 12.6°C, 67 and
52%; summer, 32.7 and 22.5°C, 69 and 53%; and fall,
27 and 15°C, 68 and 52%, respectively. Cows were fed
ad libitum a TMR containing 1.74 Mcal of NE
of DM and 17% protein. Cows were milked 3 times daily.
Monthly milk yields were recorded, and percentages of
fat and protein were determined by the central labora-
tories of the Israeli Cattle Breeding Association. Body
condition score on a 5-point scale was determined (Wild-
man et al., 1982) by the same person immediately after
calving and at 70 DIM. The experiment was conducted
Journal of Dairy Science Vol. 89 No. 12, 2006
in accordance with the guidelines of the local ethics
Estrus was synchronized in lactating cows at about
50 to 60 d postpartum by inserting an intravaginal
insert containing progesterone (CIDR, Eazi-Breed,
Hamilton, NZ) for 9 d and treating cows with PGF
analog (Cloprostenol, Estrumate 500 g i.m., Coopers,
Berkhamsted, UK) 2 d before removal of the insert.
To prevent any possible effects of the synchronization
procedure [e.g., the persistent follicle syndrome that
could be induced in cows that receive exogenous proges-
terone in the absence of endogenous corpus luteum (CL;
Smith and Stevenson, 1995)], we began the experimen-
tal examinations during the estrus subsequent to the
synchronized estrus (about 70 to 80 DIM), 3 wk after
terminating the initial estrus-synchronization protocol.
A total of 106 cows (one-third primiparous and two-
thirds multiparous) that had been subjected to the syn-
chronization protocol were observed for estrus 4 times
daily at the time they were expected to manifest estrus.
Cows that were not observed to be in estrus within 4 d
after CIDR removal (during the initial synchronization)
were treated with a single dose of PGF
20 d after CIDR
insert removal. Thus, 2 groups of cows were included in
the experiment: those that manifested estrus following
initial synchronization and those that did not; the latter
were treated with PGF
. A 5-d period of intensive ex-
perimental examinations began 20 d after the synchro-
nized estrus, or 36 h after PGF
. Of the 106 cows sub-
jected to the initial synchronization protocol, 74 cows
(70%) manifested estrus about 3 wk after the initial
protocol within the period of continuous observations
and were included in the study. Of these, 47 cows exhib-
ited spontaneous estrus and 27 exhibited PGF
During the experiment, visual indications of estrus
were monitored continuously (24 h daily during 5 d) by
a team of 2 people. Exact onset of estrus was determined
by recording the time of the ﬁrst standing event, provid-
ing that the individual cow went on to manifest several
standing events and exhibited other behavioral signs
typical of a cow in estrus. Transrectal ultrasonography
of the ovaries was conducted by using a 7.5-MHz probe
(Aloka 210, Tokyo, Japan). It was carried out once at the
beginning of the 5-d period to detect the preovulatory
follicle and to conﬁrm that the ovarian structures were
typical of that found in a normal follicular phase. Com-
mencing 20 h after standing estrus, ultrasonography
BLOCH ET AL.4696
of the ovaries was carried out every 4 h until ovulation
was detected, or until 50 h after the onset of estrus for
cows that failed to ovulate by that time. Based on the
4-h interval of ultrasonographic monitoring, the time
of ovulation was considered to be 2 h before the time
when ovulation could be discerned (Kaim et al., 2003).
From 10 d before expected estrus, blood samples were
collected every other day for progesterone determina-
tion. During the 5-d period of continuous observation,
blood samples for later estradiol determination were
collected every 8 h until the onset of estrus. From the
onset of estrus, samples for LH determination were
collected every 3 h for 24 h. Following ovulation, blood
samples for progesterone determination were collected
every other day fromd1to20ofthecycle.
Plasma samples for estradiol determination were ex-
tracted with diethyl ether as described previously (Bad-
inga et al., 1992), and extracted samples were analyzed
by RIA as validated in our laboratory (Shaham-Alba-
lancy et al., 2000). The antibody (Diagnostic Products
Corp., Los Angeles, CA) did not cross-react signiﬁcantly
with other major steroids. Assay sensitivity was 0.5 pg/
mL, and the intra- and interassay coefﬁcients of varia-
tion were 8.5 and 9.5%, respectively. Plasma progester-
one concentrations of unextracted samples were ana-
lyzed by using a solid-phase radioimmunoassay kit (Di-
agnostic Product Corp.) against a standard curve
prepared in our laboratory by dissolving progesterone
in plasma from an ovariectomized cow, as described
previously (Shaham-Albalancy et al., 2000). Assay sen-
sitivity was 0.2 ng/mL and the intra- and interassay
coefﬁcients of variations were 3.9 and 8.6%, respec-
tively. Plasma LH concentrations were measured by
enzyme immunoassay with a biotin-streptavidin ampli-
ﬁcation and validated for bovine plasma as described
previously (Mutayoba et al., 1990). The procedure was
validated recently in our laboratory (Kaim et al., 2003).
Intra- and interassay coefﬁcients of variation in the
assay were 8.5 and 10.6%, respectively, and its sensitiv-
ity was 7.8 pg/well. Values for LH are expressed in
nanograms of bovine LH (USDA bLH-B-6) per mil-
Data were analyzed by ANOVA (procedure GLM;
SAS Inst. Inc., Cary NC). Cows were sorted into 4
groups according to the E-O interval range (Figure 1A)
and on the basis of previous publications, as follows:
The ﬁrst group, with intervals between 22 and 25 h,
was designated as having a short (but normal) interval
Journal of Dairy Science Vol. 89 No. 12, 2006
(Figure 1B). The second group, with an E-O interval of
26 to 30 h (most common range reported in the litera-
ture), was designated as normal (Walker et al., 1996;
Rajamahendran et al., 1998, Roelofs et al., 2005). The
third group comprised cows having E-O intervals be-
tween 31 and 35 h; this range was considered long (but
normal), and the group was designated as having a long
interval. Finally, a fourth group of cows with an E-O
interval greater than 36 h was designated as having a
very long interval (Figure 1B). Sorting the experimental
cows into 4 groups according to the E-O interval range
enabled us to efﬁciently characterize the extended E-
O interval syndrome. This syndrome has been described
(Walker et al., 1996), but little is known about its endo-
crine characteristics. This syndrome was represented
in the present study by the group of cows with a very
long E-O interval. We tested the effects of the type of
estrus (spontaneous or PGF
-induced) on the distribu-
tion of cows among the 4 groups, and on hormone (estra-
diol, progesterone, and LH) concentrations. On the ba-
sis of these analyses, type of estrus was ignored in
subsequent analyses because it had no signiﬁcant effect
on any outcome of interest, and the results for cows
exhibiting the 2 types of estrus were combined, as in
studies by other workers (Walker et al., 1996). Data
related to intervals from estrus to the LH surge, the
LH surge to ovulation, and the E-O intervals, peak
concentration of the LH surge, milk yield and composi-
tion, and BCS were analyzed by one-way ANOVA. Ef-
fects of season and parity on distribution of cows among
the 4 E-O interval groups was tested by χ
. The statisti-
cal models for hormonal concentrations included the
effects of E-O interval groups, cows (within a group),
time (hours or days from estrus), milk yield, body condi-
tion, parity, and season.
Length and Distribution of Intervals Between
Estrus, LH Surge, and Ovulation
Distribution of the E-O interval ranged from 22 to at
least 50 h (Figure 1A). The group having normal E-O
intervals (26 to 30 h) comprised about half (47%) of the
examined cows (Figure 1B). The group with short E-O
interval (22 to 25 h) comprised 25% of the cows. These
2 groups taken together represented nearly three-quar-
ters of the cows that exhibited short or normal E-O
intervals of 22 to 30 h. The group with long E-O inter-
vals (31 to 35 h) comprised 17% of the cows. The fourth
group with very long E-O intervals (>36 h) comprised
7 cows (i.e., 10% of the experimental cow population
examined in this study). Three cows of this group failed
to ovulate by 50 h after the onset of estrus (scanning
ENDOCRINE CHANGES ASSOCIATED WITH DELAYED OVULATION 4697
Figure 1. A) Distribution of the experimental cow population (n = 74) sorted by estrus to ovulation intervals. Individual values are
subject to an estimated error of ±2 h. B) Distribution (%) of the experimental cow population among 4 groups of cows, sorted according to
their range of estrus-ovulation interval into short, normal, long, and very long intervals.
was not continued after 50 h), and these 3 cows were
designated as having an E-O interval of at least 50 h.
Further evaluation of these 3 cows clearly indicated
that each eventually ovulated and subsequently devel-
oped a CL. First, ultrasound examinations performed
twice, at about 8 to 10 d and 13 to 15 d after estrus,
indicated that in all 3 cows, the largest follicle disap-
peared and a new, normal-size CL was present in the
expected ovary. These CL were much larger than the
average size (about 17 mm in diameter) of correspond-
ing ovulatory follicles, and their average calculated vol-
ume (about 7,300 mm
) was within normal range for
CL at that stage of the cycle. Second, progesterone con-
centrations in 2 of these cows exhibited a normal post-
ovulatory pattern that did not differ from those shown
in Figure 3. In the third cow, the progesterone pattern of
increase was similar, but was delayed by 4 d. Individual
midluteal-phase concentrations of progesterone of the
3 cows were 4.8, 5.1, and 7.5 ng/mL. They were typical
and within the normal range for high-yielding cows and
were indicative of steroidogenically active CL. Collec-
tively, these ﬁndings conﬁrmed that the 3 cows that
failed to ovulate by 50 h after the onset of estrus did
The mean interval between the onset of estrus and
the peak LH surge was later (P < 0.01) at9hinthe
very long E-O interval group, compared with 3.5 h in
the normal interval group (Table 1). Interval from the
LH surge to ovulation was greater (P < 0.01) by about
4 h in the long and very long groups than in the normal
group (Table 1).
Journal of Dairy Science Vol. 89 No. 12, 2006
Milk, milk fat, and protein yields during the experi-
mental period did not differ among the 4 experimental
groups (Table 2). Similarly, BCS at calving or during
the experimental period (70 DIM) did not differ among
the 4 experimental groups (Table 2). Effects of season
on distribution of cows among the 4 E-O interval groups
were minor and not statistically signiﬁcant. Likewise,
we did not ﬁnd any signiﬁcant effect of parity on distri-
bution of cows among E-O interval groups (neither pri-
miparous compared with multiparous cows, nor com-
parisons among multiparous cows with differing num-
bers of lactations had any effect on the above). It is
noteworthy that the time of onset of estrus (day or
night) did not have any signiﬁcant effect on the E-O in-
Ultrasound examination of the ovaries at the begin-
ning of the 5-d period of intensive experimental exami-
nations indicated that, similar to experimental cows
with a normal E-O interval, the ovaries of the cows with
a very long E-O interval exhibited a normal appearance,
typical of the follicular phase, consisting of 1 or 2 large
follicle(s), a few medium-size follicles, and a regressing
CL. Likewise, later ultrasound examinations (during
the 5-d period, intended to determine the time of ovula-
tion) conﬁrmed the presence of a dominant follicle and a
CL in an advanced stage of regression. All experimental
cows ovulated, as indicated by the growth of new CL
and elevated concentrations of plasma progesterone.
Proportions of cows having spontaneous or induced es-
trus (63 and 36%, respectively) were similar in the
short, normal, long, and very long E-O interval groups.
BLOCH ET AL.4698
Table 1. Intervals between estrus and LH surge, LH surge and ovulation, and estrus and ovulation in the
4 groups of cows, sorted by range of intervals between estrus and ovulation (mean ± SE)
Interval from estrus to ovulation groups
Short Normal Long Very long Overall
Estrus to LH surge, h 0.0 ± 0.6
3.5 ± 0.5
3.9 ± 0.8
9.0 ± 3.5
2.8 ± 0.4
LH surge to ovulation, h 24.0 ± 0.5
24.7 ± 0.5
28.4 ± 0.8
28.2 ± 3.2
25.5 ± 0.5
Estrus to ovulation, h 23.9 ± 0.2
28.2 ± 0.2
32.3 ± 0.4
42.6 ± 2.6
28.6 ± 0.6
Means of the 4 interval groups within a row having different superscript letters differ (P < 0.01).
Among the last group, 4 and 3 of 7 cows (57 and 42%,
respectively) exhibited natural and induced estrus, re-
A weak correlation was detected between the E-O
interval and mean progesterone concentration during
the 10-d period before the examined estrus (r = −0.27;
P < 0.04). Correlations between the E-O interval and
peak estradiol concentration before estrus (r = −0.17;
P < 0.15) or peak LH surge concentration (r = −0.21;
P < 0.09) were slight and not statistically signiﬁcant.
Further analyses, described below, were applied after
the experimental cows had been sorted into 4 groups
according to their E-O interval range.
Progesterone concentrations before the examined es-
trus are presented in Figure 2. The very long interval
group exhibited reduced (P < 0.05) progesterone concen-
trations during the 10-d period before estrus than those
in the short, normal, and long groups (2.8 ± 0.6 vs. 4.2
± 0.2 ng/mL; pooled value for the latter 3 groups). Luteal
regression in the very long interval group started ear-
lier than in the other 3 groups, as manifested in the
decline in progesterone on d −5 rather than on d −3
(Figure 2). As in the cycle preceding ovulation, proges-
terone concentration curves during the midluteal phase
of the cycle following the examined ovulation (d 11 to
18; Figure 3) were less (P < 0.04) in the very long inter-
Table 2. Milk and milk component yields during the experimental period, BCS, and cycle durations of the
4 groups of experimental cows, sorted by range of intervals between estrus and ovulation (mean ± SE)
Interval from estrus to ovulation groups
Short Normal Long Very long
Milk yield, kg/d 47.1 ± 2.8 43.9 ± 1.4 44.7 ± 2.3 47.1 ± 3.5
Milk fat, kg/d
1.51 ± 0.11 1.55 ± 0.06 1.46 ± 0.11 1.67 ± 0.17
Milk protein, kg/d
1.35 ± 0.08 1.29 ± 0.03 1.30 ± 0.07 1.38 ± 0.08
BCS at calving 3.3 ± 0.1 3.1 ± 0.1 3.2 ± 0.1 3.3 ± 0.2
BCS at 70 DIM 2.6 ± 0.1 2.5 ± 0.1 2.5 ± 0.1 2.4 ± 0.1
Estrous cycle length, d 22.0 ± 0.6 21.6 ± 0.4 22.2 ± 0.5 22.3 ± 0.8
Means did not differ among the 4 groups of cows.
Mean percentages of milk fat and milk protein were 3.40 ± 0.06% and 2.92 ± 0.02%, respectively.
Journal of Dairy Science Vol. 89 No. 12, 2006
val group, and also in the long interval group, than in
the short or normal groups (4.4 vs. 5.6 ng/mL). As in
the preceding cycle, luteolysis started earlier in the
groups with longer intervals than in those with short
or normal intervals (Figure 3). Cycle durations were
similar in the 4 groups of cows (Table 2).
Concentrations of estradiol during the follicular
phase before the LH surge were less (P < 0.06) in the
very long interval group than in the other 3 groups
(Figure 4). Interestingly, mean diameters of the preovu-
latory follicles on the day before ovulation did not differ
between the short, normal, long, and very long interval
groups (16.8 ± 0.6, 17.0 ± 0.3, 16.3 ± 0.6, and 17.7 ± 0.5
Concentrations of LH at the time of the preovulatory
surge were normalized to the peak concentration of the
LH surge in each of the 4 groups of cows. As shown in
Figure 5, the peak of LH surge concentration in the
very long interval group was about 2.5 times less (P <
0.03) than those in the other 3 groups. Mean peak LH
surge concentration in the short, normal, and long
groups was 10 ng/mL, with no signiﬁcant difference
among them, and that in the very long interval group
was 4 ng/mL (Figure 5).
Short fertile half lives of the oocyte after ovulation
and of the spermatozoa in the female tract (Hunter,
ENDOCRINE CHANGES ASSOCIATED WITH DELAYED OVULATION 4699
Figure 2. Concentrations of plasma progesterone in the 4 groups
of cows sorted by estrus-ovulation (E-O) interval during 10 d before
estrus. Concentrations in the very long E-O interval group were less
(P < 0.03) than in the other groups before estrus. Pooled SEM = 0.73.
1994) narrow the optimal time window for maximal
fertilization. In the present study, we characterized an
abnormal group of cows that exhibited delayed ovula-
tion and an extended E-O interval. This group formed
one-tenth of the total number of cows. The present study
sheds light on changes in the endocrine milieu that
may be partly associated with reduced fertility in cows
having extended E-O intervals. These changes included
low concentrations of progesterone and estradiol and a
low LH surge before ovulation.
The proportion of cows found to exhibit an extended
E-O interval varies among studies. In the present
study, the very long interval group comprised 10% of
Figure 3. Concentrations of plasma progesterone in the 4 groups
of cows sorted by estrus-ovulation (E-O) interval during the cycle
following ovulation. Data were normalized according to time of ovula-
tion. Concentrations in long and very long E-O interval groups during
midluteal phase were less (P < 0.04) than in the other groups. Pooled
SEM = 0.69.
Journal of Dairy Science Vol. 89 No. 12, 2006
Figure 4. Concentrations of plasma estradiol before ovulation
in the 4 groups of cows sorted by estrus-ovulation (E-O) interval.
Concentrations in the very long E-O interval group were less (P <
0.06) than in the other groups. Pooled SEM = 0.49.
the experimental cows, whereas in earlier studies it
ranged from 8% in heifers (Hernandez-Ceron et al.,
1993) to 15 to 22% in cows (Walker et al., 1996; Roelofs
et al., 2005). The number of cows used in the present
study provided a sufﬁciently large study population to
support adequate statistical analyses. Continuous ob-
servation for estrus and frequent ultrasonographic
monitoring enabled us to accurately determine the time
of ovulation and the E-O interval for each cow, with an
estimated error of ±2 h. In this respect, repeated rectal
ultrasound examinations of dairy cows (as performed
herein for determination of ovulation) did not alter the
timing of estrus, durations of the E-O, and estrus to
Figure 5. Concentrations of plasma preovulatory LH surges in
the 4 groups of cows sorted by estrus to ovulation (E-O) interval.
Data were normalized according to peak LH in each group and are
presented relative to the onset of estrus. Concentration of peak LH
in the very-long E-O interval group was less (P < 0.01) than in the
other groups. Pooled SEM = 0.68.
BLOCH ET AL.4700
LH surge intervals, or the periovulatory concentrations
of plasma steroids and LH (Roelofs et al., 2004).
The present study provides clear evidence that an
extended E-O interval is associated with alterations in
the concentrations of progesterone, estradiol, and LH
surge in plasma. We found markedly different hor-
monal proﬁles in the extended E-O interval (very long
E-O) group of cows than in the other 3 “normal” groups
of cows. Saumande and Humblot (2005) found correla-
tions between the preovulatory estradiol concentra-
tions, the preovulatory follicle size, and the E-O inter-
val. The present study, however, indicated that alter-
ations in progesterone, estradiol, and LH surge
concentrations before ovulation were evident only in
cows with an extended E-O interval and not in the
entire population of experimental cows. Although pre-
ovulatory follicle size in the very long E-O interval
group did not differ from that in the normal interval
groups, the reduced estradiol concentrations found in
the former group were presumably due to development
of a less steroidogenically active, preovulatory follicle.
Reduced estradiol concentrations before estrus proba-
bly delayed the LH surge so that the estrus to LH surge
interval was about 6 h longer in the very long interval
group than in the normal interval group. Although it
is generally accepted that most of the variation in the
E-O interval can be attributed to variation in the estrus
to LH surge interval (Saumande and Humblot, 2005),
the present ﬁndings indicate that extension of the inter-
val from LH surge to ovulation also contributed to
The markedly small preovulatory LH surge ampli-
tude in the very long E-O group, which is reported for
the ﬁrst time in our study, could be related to low pre-
ovulatory estradiol concentrations. Indeed, we found a
close association between low preovulatory estradiol
concentrations and low-amplitude LH surges, which
occurred only in the group of cows with a very long E-
O interval, and not among the entire population of cows.
A less-than-normal LH surge peak could be associated
with a delayed timing of ovulation, which caused, in
turn, a signiﬁcant 4-h extension of the LH surge to
ovulation interval in the very long E-O interval group.
This interpretation is strengthened by ﬁndings that
induction of stress in sheep or cows by endotoxin (LPS)
administration during the follicular phase or at the
onset of estrus was associated with a decline in the LH
surge amplitude and with delayed ovulation (Battaglia
et al., 1999; Suzuki et al., 2001; Lavon et al., 2004). It
should be mentioned, however, that a similar extension
of the LH surge to ovulation interval also was noted in
the long E-O interval group. Collectively, the present
ﬁndings suggest that extension of the E-O interval is
probably associated with 1) delayed secretion of the
Journal of Dairy Science Vol. 89 No. 12, 2006
preovulatory LH surge relative to onset of estrus, possi-
bly because of inadequate concentrations of estradiol;
and 2) extension of the time between the LH surge and
the occurrence of ovulation, possibly because of limited
secretion during the preovulatory LH surge.
Luteal insufﬁciency in cows with a very long E-O
interval, which is reported here for the ﬁrst time, is
characterized not only by low progesterone concentra-
tion curves, but also by an earlier luteal regression
before estrus. The reason for this insufﬁciency is not
yet clear. Interestingly, the reduced luteal progesterone
concentration curve does not seem to be a random event
because this pattern was repeated in the next cycle
after ovulation. Low progesterone curves could result
from low estradiol concentrations and a consequently
smaller LH surge, which could be associated with sub-
optimal luteinization of the growing CL after ovulation.
This relationship has been found in primates (Zelinski-
Wooten et al., 1997). Studies in cows (Ambrose et al.,
1998; Less et al., 1998; Rajamahendran et al., 1998)
showed that an induced small LH surge was associated
with inadequate postovulation plasma progesterone
concentrations. Likewise, granulosa cells that were ex-
posed to an inadequate or short-lived LH surge at the
beginning of culture secreted less progesterone at the
end of luteinization (Biger et al., 2000). An alternative
reason for suboptimal progesterone secretion in these
cows does not necessarily involve a small LH surge. A
suboptimal CL could result from ovulation of a subopti-
mal preovulatory follicle, as indicated by the reduced
concentrations of estradiol during the follicular phase
in those cows. This hypothesis is supported by previous
ﬁndings that a less-well-developed preovulatory follicle
yielded a poor progesterone-secreting CL (Vasconcelos
et al., 1999), and also that heat-stress–impaired follicles
yielded poor progesterone-secreting CL (Wolfenson et
The likelihood that cows having the very long E-O
intervals will conceive seems improbable for several
reasons. First, as mentioned previously, the weaker
association between time of AI and time of ovulation
minimizes the probability of successful fertilization.
Second, a small preovulatory LH surge may impair the
normal process of resumption of meiosis and nuclear
maturation (Tsafriri et al., 2005), and therefore could
lead to development of an incompetent oocyte before
ovulation. Third, cows with a long E-O interval exhib-
ited low progesterone concentration curves, both before
and after ovulation, which occasionally have been found
to be associated with poor fertility (Kimura et al., 1987;
Folman et al., 1990). These points are supported by a
recent study (Kaim et al., 2003) in which lactating cows
treated with GnRH at onset of estrus exhibited im-
proved fertility. Greater conception was associated with
ENDOCRINE CHANGES ASSOCIATED WITH DELAYED OVULATION 4701
induction of a shorter, less variable E-O interval, as
well as with elevated postovulatory concentrations of
In conclusion, a group of cows with an extended E-
O interval was characterized by reduced concentrations
of estradiol and a small LH surge before ovulation, and
by low concentrations of progesterone before and after
ovulation. The extended interval between estrus and
ovulation, and the depressed hormonal secretions prob-
ably minimize the likelihood that these cows could con-
ceive. No simple means exists presently to identify this
group of cows that have an extended E-O interval.
Therefore, hormonal treatments of cows having an ex-
tended E-O interval, intended to improve speciﬁcally
their fertility, rather than general treatment of the
whole herd, are currently not applicable to commercial
herds, and further research is needed to achieve a suit-
able treatment. Such treatments include administra-
tion of GnRH at onset of estrus, or delaying the applica-
tion of AI relative to time of estrus.
The authors express their appreciation of the help
given by the staff of the dairy farm in Kibbutz Naan,
Israel, and to the USDA for the provision of bovine LH
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