Animal Reproduction Science 110 (2009) 128138

LH surge in Nelore cows (Bos indicus), after induced

estrus or after ovarian superestimulation
Fabio Morato Monteiro a,b, , Danilas Salinet Melo a ,
Marcelo Machado G Ferreira a , Luciano Mundim Carvalho a ,
Evandro Sartoreli e Sartoreli a , Bruno G Ederhardt a ,
Guilherme de Paula Nogueira c , Ciro Moraes Barros a
a Department of Pharmacology, Institute of Bioscience, University of Sao Paulo State, Botucatu, Sao Paulo, Brazil
b Institute of Zootecnia, Sert
aozinho, Sao Paulo, Brazil
c DAPSA - University of Sao Paulo State, Aracatuba, Sao Paulo, Brazil
Received 19 July 2007; accepted 9 January 2008
Available online 16 January 2008

Abstract
Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus),
the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and
after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly
important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore
cows (n = 12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B )
associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were
treated with PGF2 (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h).
Starting 38 h after the first PGF2 injection, blood sampling and ovarian ultrasonography took place every
4 h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of
LH secretion resulting in only 3 of 12 cows having ovulations at 46.7 4.9 and 72.3 3.8 h, respectively,
after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment
associated with FSH (Folltropin , total dose = 200 mg) administered twice a day, during 4 consecutive days,
starting on Day 5. Thirty-six hours after the first injection of PGF2, to minimize stress, only seven blood
samples were collected at 4 h interval each, and ultrasonography was performed every 12 h until ovulation.
In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6 1.6 and 59.5 1.9 h, respectively,
after removal of progesterone source. The maximum values for LH in those animals were 19.0 2.6 ng/ml
(mean S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol,

Corresponding author at: Rodovia Carlos Tonani, Km 94, Sert

aozinho 14160-970, SP, Brazil. Tel.: +55 16 3491 6156;
fax: +55 16 3491 6157.
E-mail address: monteiro@iz.sp.gov.br (F.M. Monteiro).

0378-4320/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.01.003
 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138 129

the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and
approximately 12 h before the LH surge observed after an induced estrus without ovarian superstimulation.
2008 Elsevier B.V. All rights reserved.

Keywords: LH; Superovulation; Bos indicus; Zebu cattle; Estrous cycle

1. Introduction

Nelore cattle (Bos indicus) are the predominant breed of cattle in Brazil and other tropical
countries due to the resistance to heat stress and parasites. The use of biotechnologies such as
fixed-time artificial insemination, and in vivo and in vitro embryo transfer have been increasing
in Brazil, particularly in Nelore breed, where total number in Brazil exceeds 90 million.
The detailed knowledge of ovarian follicular dynamics (Savio et al., 1988; Sirois and Fortune,
1988; Ginther et al., 1989) allowed the development of hormone treatments to regulate follicular
growth and the onset of ovulation, thus permitting fixed-time artificial insemination (i.e., artificial
insemination at predetermined times without the need for detection of estrus) in European (Bos
taurus; Thatcher et al., 1993; Pursley et al., 1995) and Zebu breeds (Bos indicus; Barros et al.,
2001; Fernandes et al., 2001; Baruselli et al., 2004). Similarly, ovarian follicular growth and
time of ovulation can be controlled pharmacologically to improve the superovulatory treatment
used in embryo transfer (Barros and Nogueira, 2001; Baruselli et al., 2006; Nogueira et al.,
2007).
For embryo transfer, protocols have been established to promote multiple ovulations that start
about 912 days after detection of estrus, with FSH being used to induce ovarian superstimulation
(Mapletoft and Pierson, 1993). However, the main limitation of these protocols continue are the
variation in the donor response to FSH. Despite a large number of studies conducted in different
countries, there is no protocol able that overcomes this problem.
An interesting approach to increase the superovulatory response is to change the onset of the
preovulatory LH surge. Differences in the maturation stage of follicles and oocytes are frequently
observed at the beginning of superovulatory treatment (Vos et al., 1994a,b). In addition, antici-
pation of the preovulatory LH surge, as well as the manifestation of estrus, is expected in cows
submitted to superovulatory treatment due to the increased secretion of estradiol by a larger num-
ber of growing follicles (Sanchez et al., 1995), which might result in the presence of follicles that
are not sufficiently developed to respond to the preovulatory LH surge, with a consequently lesser
ovulation rate (Vos et al., 1995; DOcchio et al., 1997). In this respect, some authors investigated
whether a delay in the LH surge would result in a larger number of follicles with ovulatory capac-
ity and, consequently, greater ovulation and embryo production rates. The results obtained were
conflicting and did not permit any consensus as to whether a delay in the endogenous LH surge
is beneficial (Rieger et al., 1990; DOcchio et al., 1997, 1998a,b), harmful (Vos et al., 1995; Van
De Leemput et al., 2001; Kohram et al., 1999; Baruselli et al., 2006) or irrelevant (Nogueira et
al., 2002) for embryo recovery rate.
In Brazil, studies conducted on Nelore females have shown no change (Nogueira et al., 2002)
or a reduction (Barros et al., 2001; Baruselli et al., 2006) in the number of viable embryos
after a delay in the endogenous LH surge, depending on the duration of the surge delay, hormone
treatments and animals used. However, these studies were based on data regarding the preovulatory
surge obtained in taurine cows, with no data regarding the preovulatory LH surge in Nelore
130 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138

cows being available in the literature. Therefore, the main objective of the present study was to
determine the onset of the preovulatory LH surge in Nelore cows after an ovarian superstimulation
protocol.

2. Materials and methods

2.1. Experimental conditions and animals

The experiments were conducted on a farm located in Botucatu, Sao Paulo, Brazil (latitude
22 51 S, longitude 48 26 W). Multiparous Nelore cows (n = 12) were kept in confinement and
fed with supplement containing 20% crude protein (about 3 kg of supplement/animal/day, Socil
Tech Pasto , Socil SA, Sao Paulo, Brazil), and supplemented with mineral salt (Socil 90 , Socil
SA, Sao Paulo, Brazil). All animals were first used in experiment 1 and then in experiment 2. The
cows body weight ranged from 312 to 475 kg (391 16 kg), age ranged from 3 to 4 years, and
body condition score ranged from 2.5 to 3.0 (2.9 0.8) on a scale from 0 to 5 (Lowman et al.,
1976).

2.2. Experiment 1 (LH surge after estrus induction)

Nelore cows (n = 12) at random stages of the estrous cycle received an intravaginal device
containing 1.9 g progesterone (CIDR-B , Pfizer Animal Health, Sao Paulo, SP, Brazil) and an
injection of estradiol benzoate (2.5 mg, i.m.; Estrogin , Farmavet, Sao Paulo, SP, Brazil) on day
0. Eight days later, all cows received a PGF2 analog (150 g d-cloprostenol, i.m.; Prolise ,
Tecnopec, Sao Paulo, SP, Brazil) in the morning (8:00 a.m.) and at night when the intravaginal
device was removed (8:00 p.m.).
Samples collection started 26 h after the removal of the intravaginal device and occurred at
4-h intervals over a period of 37 consecutive hours. Blood was collected from the jugular vein
into a 10-ml tube containing heparin (Vacutainer ) and immediately placed on ice. After about
1 h, the blood was centrifuged at 900 g for 20 min for plasma separation. The plasma samples
were divided into three aliquots and stored at 20 C until LH quantification by radioimmunoas-
say.
The presence of ovarian follicles and corpora lutea were evaluated by transrectal ultra-
sonography (Aloka 500 SSD with a 7.5-MHz linear transducer) at 48 h intervals starting
26 h after CIDR removal (Fig. 1). Behavioral signs of estrus were monitored over 30 min

Fig. 1. Hormonal treatment for estrous synchronization, blood sampling and ultrasonography in Nelore cows.
 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138 131

Fig. 2. Hormonal treatment for ovarian superstimulation, blood sampling and ultrasonography in Nelore cows.

immediately after each blood collection. The onset of ovulation was defined as the mid-
point between the last time the preovulatory follicle was detected via ultrasonography and the
first occasion the follicle was no longer observed on the ultrasonographic monitor. All ani-
mals were weighed at the beginning of blood collection and after the last ultrasonographic
exam.

2.3. Experiment 2 (LH surge after ovarian superstimulation)

Thirty days after experiment 1, the same cows (n = 12), at random stages of the estrous cycle,
simultaneously received an intravaginal device containing 1.9 g progesterone (CIDR) and an injec-
tion of estradiol benzoate (2.5 mg, i.m.) on day 0. From day 5 to day 8, the animals were injected
intramuscularly twice a day with pFSH (Folltropin-V , Bioniche Animal Health, Belleville, ON,
Canada) with decreasing doses of 40, 30, 20, and 10 mg. On day 7, the cows received a PGF2
analog (150 g d-cloprostenol, i.m.) at 8:00 a.m. in the morning and at 8:00 p.m. in the night
when the intravaginal device was removed.
Blood collections and ultrasonographic exams were performed as described in experi-
ment 1 but only 7 blood samples were collected at 4 h intervals, and ultrasonography was
performed every 12 h until detection of ovulation (Fig. 2). As in experiment 1, all ani-
mals were weighed at the beginning of blood collection and after the last ultrasonographic
exam.

2.4. Luteinizing hormone radioimmunoassay

LH concentrations were determined using the double-antibody radioimmunoassay adapted by

Bolt and Rollins (1983). All samples were analyzed in one assay. The intra-assay coefficient of
variation and sensitivity of the assay were 8.7% and 0.05 ng/ml, respectively.

2.5. Determination of the LH surge

Preovulatory LH surges were evaluated by graphic analysis of the results obtained for each
animal, adopting the criterion suggested by Cavalieri et al. (1997) and Callesen et al. (1990,
1993), who used the maximum concentration of LH during a preovulatory wave of follicular
development as the time of the preovulatory LH surge.
132 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138

Table 1
Interval between removal of CIDR and the detection of estrus, removal of CIDR and LH peak, removal of CIDR and
ovulation (Ov), LH peak and ovulation (LHOv), and maximum concentration of LH (ng/ml), in Nelore cows after
treatment to induce ovulation
Animal Interval (h)

CIDRestrus CIDRLH CIDROv LHOv LH (ng/ml)

36 43 38 72 34 27.68
18 48 47 79 32 36.87
09 55 66 11 17.15
Mean S.E.M. 45.5 2.5 46.7 4.9 72.3 3.7 25.6 7.4 27.2 5.7

3. Results

3.1. Experiment 1

Only 4 of the 12 animals had ovulations. However, cow 6 had ovulations at 95 h after removal
of the CIDR and the preovulatory LH surge could not be determined in blood samples collected
between 26 and 63 h after removal of the progesterone device. Thus, the data obtained for this
animal were not included in Table 1. The characteristic sign of estrus (mounting acceptance) was
observed in only two cows. The animals lost on average 23.5 2.4 kg of body weight over the 3
days of blood collection and ultrasonography.

3.2. Experiment 2

All animals had ovulations after hormone treatment. However, no preovulatory LH surge could
be determined in cow 32 because the animal had an increase in LH concentration only in the last
blood sample (48 h after removal of the CIDR). Ovulation was detected in this animal at 78 h after
the removal of the progesterone device (Fig. 3).
The mean interval between removal of the intravaginal device and preovulatory LH surge
was 34.6 1.6 h and ovulation occurred 24.9 1.1 h after the LH surge. The mean LH concen-
tration found with this blood collection regimen at 4-h intervals was 19 2.6 ng/ml (Table 2).

Fig. 3. LH concentration (ng/ml) in Nelore cows treated with an ovarian superestimulatory protocol. Blood samples were
collected every 4 h from 24 to 48 h after CIDR removal.
 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138 133

Table 2
Interval between removal of CIDR and LH peak, removal of CIDR and ovulation (Ov), LH peak and ovulation (LHOv)
and maximum concentration of LH (ng/ml), in Nelore cows after treatment to induce superovulation
Animal Interval (h)
CIDRLH CIDROv LHOv LH (ng/ml)

02 36 66 30 15.92
03 44 66 22 23.76
06 36 66 30 25.17
09 32 54 22 13.37
18 40 66 26 30.4
31 40 66 26 19.11
33 32 54 22 9.01
36 28 54 26 31.93
43 36 54 18 23.64
46 28 54 26 10.94
48 28 54 26 6.06
Mean S.E.M. 34.6 1.6 59.5 1.9 24.9 1.1 19.0 2.6

Concentrations of LH in plasma remained elevated for 812 h in most animals (Fig. 3).
The hormone treatment regimen used was effective in promoting ovarian superstimulation as
demonstrated by the large number of follicles (1560 follicles per animal) measuring 8 mm in
the ovaries, in addition to a greater induced ovulation rate (Table 3). Animals lost 15.0 1.9 kg
on average over the 2 days of blood collection and ultrasonography.

4. Discussion

In Nelore cows submitted to estrous synchronization (experiment 1), the preovulatory LH

surge was observed 46.7 4.9 h after removal of the progesterone-releasing device. However,

Table 3
Number of ovarian follicles (8 mm) 48, 60 and 72 h after CIDR removal and ovulation rate (determined by ultrasonog-
raphy), in Nelore cows after treatment to induce superovulation
Animal Hours after CIDR removal Ovulation rate (%)

48 h 60 h 72 h

02 35 29 3 89.6
03 23 24 2 91.6
06 29 30 100
09 15 1 100
18 56 54 100
31 30 28 100
32a 48 46 49
33 57 6 100
36 27 5 100
43 44 100
46 60 100
48 40 5 100
a It was determined through palpation per rectum that this animal had ovulations between 72 and 84 h after CIDR

removal. However it was not possible to accurately estimate the ovulation rate using palpation per rectum.
134 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138

only 4 of the 12 treated animals had ovulations and a LH surge was detected in only 3 of these
animals.
This lesser ovulation rate was probably due to the stress caused by the intensive handling of
the animals (blood collection and ultrasonography examination every 4 h). These results are in
agreement with those reported by Echternkamp (1984), who observed a preovulatory LH surge
in only 30% of animals (taurine heifers) submitted to intensive blood collection. The stress in the
most animals of experiment 1 may have negatively influenced hypothalamic GnRH secretion, not
allowing the preovulatory LH surge and ovulation. Dobson and Smith (2000), submitting sheep
to stress situations, observed a reduction of hypothalamic GnRH secretion and, consequently,
of LH pulse amplitude and frequency of LH release from the anterior pituitary. This reduction
compromised ovarian follicular growth, and consequently less 17-estradiol was present in blood
which was insufficient to stimulate the preovulatory LH surge.
Despite the small number of animals that had a preovulatory LH surge, the time of LH surge
(46.7 4.9 h) similar to those reported by Cavalieri et al. (1997; 47.4 h) and Hanlon et al. (1997;
39.9 h) where estrus was induced with progestagens and estrogens with or without PGF2 treat-
ment. These results differ, however, from those obtained after estrous induction with one or two
applications of PGF2. There is a wide variation reported in the literature in interval between
administration of a luteolytic agent and the preovulatory LH surge, ranging from 59 to 90 h (Refsal
and Seguin, 1980; Walters and Schallenberger, 1984; Evans et al., 2003). This variation may be
explained by the different estrous synchronization protocols used in these experiments, which
may have influenced the time of LH surge. Different estrous synchronization protocols can affect
the timing of the preovulatory LH surge by causing variation in the synchrony of follicular devel-
opment and differences in the timing of preovulatory increases in peripheral concentrations of
estradiol that are associated with follicular maturation.
The use of progesterone or progestagen in combination with estrogens reduces the secretion of
gonadotropins by a negative feedback mechanism in the hypothalamus/pituitary, thereby promot-
ing ovarian follicular atresia and the onset of a new wave of ovarian follicular development about
45 days later (Bo et al., 1991, 1996, 2003), i.e., synchronizing the timing of the follicular wave
in females in such a way that when PGF2 is administered and the progesterone or progestagen
source is removed (79 days after the beginning of treatment), most animals possess a dominant
follicle that is releasing enough estrogen into the blood to induce the preovulatory LH surge.
When estrus is induced with one or two applications of PGF2, however, a wide variation
in time of the LH surge is observed due to the fact that PGF2 does not synchronize the stage
of ovarian follicular development. Thus, if the luteolytic agent is applied when the dominant
follicle is beginning to undergo atresia, the preovulatory LH surge will be induced later after the
emergence of a dominant follicle from a new wave of ovarian follicular development. However,
if a dominant follicle in the full phase of growth is present, the preovulatory LH surge will be
induced earlier due to the greater amounts of estradiol produced by this follicle (Kastelic et al.,
1990; Sirois and Fortune, 1990; Savio et al., 1990).
The interval between removal of the progesterone-releasing device and ovulation observed in
the present study (72.3 3.8 h) was similar to those reported by Hanlon et al. (1997) for taurine
cows (65.8 3.9 h) and by Cavalieri et al. (1997) for Zebu cows (68.8 h). These investigators used
progesterone (CIDR-B ) and progestagen (Norgestomet, Crestar ) for estrous induction, respec-
tively. Likewise, the interval between the preovulatory LH surge and ovulation (25.7 7.4 h)
was similar to those described for females treated with PGF2 with or without progestagen or
estrogens treatments by Cavalieri et al. (1997) (26.2 1.3 h), Randel (1984; 21.3 h), Bernard et
al. (1983; 27.3 1.6 h) and Hanlon et al. (1997; 29.9 3.9 h).
 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138 135

The preovulatory LH surge was observed 34.6 1.64 h after removal of the progesterone
source in 11 of the 12 treated cows. In the second experiment, the period of blood collection was
reduced from 37 to 24 h to reduce the stress caused by the intensive handling of the animals.
In addition, the number of ovarian ultrasonographic exams was reduced from 15 to 5. With the
introduction of these changes all animals (n = 12) had ovulations 59.5 1.9 h after removal of the
device and the interval between the preovulatory LH surge and ovulation was 24.9 1.1 h.
Results obtained in the present study (34.6 1.6 h) were similar to those reported by Lafri
et al. (2002) and DOcchio et al. (1997) who used ovarian superstimulation protocols similar to
those of experiment 2. Lafri et al. (2002) observed an interval of 31.0 1.5 h between removal
of the intravaginal device and preovulatory LH surge in taurine cows, whereas DOcchio et al.
(1997) reported an interval of 35.0 4.4 h in three Brahman cows (Bos indicus).
Several investigators, inducing ovarian superstimulation with FSH between the tenth and
twelfth day of the estrous cycle associated with the application of PGF2 about 2 days later,
observed a variation from 41 to 52 h in the interval between the administration of PGF2 and the
preovulatory LH surge (Yadav et al., 1986; Lafri et al., 2002; Takagi et al., 2001). With similar
treatments using eCG instead of FSH, intervals between the administration of PGF2 and the
preovulatory LH surge ranging from 34 to 44 h occurred in taurine females (Jensen et al., 1982;
Bevers and Dieleman, 1987; Yadav et al., 1986). Similar to experiment 1, the variations in the onset
of the LH surge after ovarian superstimulation reported in the literature were probably related
to the extent of synchronization of follicular growth promoted by the different superovulation
protocols.
The interval between the preovulatory LH surge and ovulation was similar in experiment 1
(25.7 7.4 h) and experiment 2 (24.9 1.1 h) and agrees with those reported in the literature after
the use of hormone treatment for estrous synchronization with or without ovarian superstimulation
(range of 2427 h; Callesen et al., 1988; Wubishet et al., 1991; Mizuta, 2003). These data suggest
that ovulation will occur approximately 24 h after the LH surge, irrespective of the hormonal
conditions (natural or induced) that give origin to the preovulatory LH surge.
In experiment 2, the preovulatory LH surge (34.6 1.6 h) occurred about 12 h earlier than in
experiment 1 (46.7 4.9 h). This might have occurred due to a greater plasma estradiol concen-
tration in animals submitted to superovulatory treatment when compared to non-superovulated
animals. The elevated estradiol concentrations induced by superovulatory treatment serve as a
trigger for an earlier preovulatory LH surge (Hunter, 1988; Callesen et al., 1990; Wubishet et
al., 1991) although differences in the number of blood samples collected and ultrasonographic
examinations cannot be disregarded as factors that may have had some influence on the pattern
of LH secretion and the subsequent timing of the LH surge.

5. Conclusion

In Nelore cows submitted to treatment for ovarian superstimulation, the preovulatory LH surge
occurred 34.6 1.6 h after removal of the exogenous progesterone source, i.e., about 12 h earlier
than that observed in animals not submitted to superstimulation with FSH (46.7 4.9 h).

Acknowledgement

The authors wish to thank CAPES (Brasilia, Brazil) for providing fellowships for F.M. Monteiro
and D.S. Melo.
136 F.M. Monteiro et al. / Animal Reproduction Science 110 (2009) 128138

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