Journal of Equine Veterinary Science 76 (2019) 6e13

Contents lists available at ScienceDirect

Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Review Article

Enhancing Fertility in Mares: Recombinant Equine Gonadotropins

Janet F. Roser*, Geraldine Meyers-Brown
Department of Animal Science, University of California, Davis, CA

a r t i c l e i n f o a b s t r a c t

Article history: Advanced reproductive technologies have been developed to enhance fertility in mares and stallions.
Received 30 January 2019 Some of these technologies in mares include superovulation, embryo transfer (ET), intracytoplasmic
Received in revised form sperm injection (ICSI), oocyte transfer (OT), gamete intrafallopian transfer (GIFT), and cloning. Super-
13 March 2019
ovulation can provide multiple oocytes for these techniques. This review will focus on how recombinant
Accepted 13 March 2019
Available online 21 March 2019
equine follicle-stimulating hormone (reFSH) and recombinant equine luteinizing hormone (reLH) are
important for superovulation and ET and may be useful for ICSI, OT, GIFT, and cloning. Superovulation
would increase pregnancy rates in normal and subfertile mares and enhance reproductive efficiency
Keywords:
Mare
when using semen from subfertile stallions. Superovulation depends on a timely interaction of gonad-
Reproductive technologies otropins and gonadal feedback in the mare. Historically, several hormone protocols have been used to
Superovulation manipulate follicular waves to increase development and ovulations in cycling, anestrous, and transi-
reFSH tional mares. Attempts to superovulate cyclic mares or induce the first ovulation of the year in anestrous
reLH or transitional mares using preparations of equine chorionic gonadotropin, gonadotropin-releasing
hormone (GnRH), GnRH agonists, porcine FSH, domperidone, sulpiride, equine pituitary extracts,
native equine FSH, human chorionic gonadotropin, progesterone, and immunization against inhibin have
produced variable results. The use of recombinant technology has improved the ability to produce a
reliable product in substantial quantities that is free of other hormones and possible contaminants.
Several studies using reFSH and reLH that demonstrate their efficacy to superovulate the mare and
induce the first ovulation of the year will be discussed in this review.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction ovulations in seasonally anovulatory mares using equine pituitary

The success of advanced reproductive technologies (ART) in the
mare requires superovulation of mares to provide multiple oocytes
for such techniques as embryo transfer (ET), intracytoplasmic
sperm injection (ICSI), oocyte transfer (OT), gamete intrafallopian
transfer (GIFT), and cloning. Superovulation could increase preg-
nancy rates in normal and subfertile mares as well as in mares bred
to subfertile stallions. Historically, superovulation has been
attempted in the mare during the last 45 years beginning with
studies by Douglas et al [1] who reported the induction of multiple
Animal welfare/ethical statement: No live animals were used for the preparation of
this review article.
Conflict of interest statement: J.F.R. serves as a partner in the company Fertilplus
Partners, LLC, which is developing a product, reFSH, related to the research being
presented in this review article. G.M.-B. has no conflict of interest.
Authors’ contributions: Both authors participated in the writing and editing of this
manuscript.
* Corresponding author at: Janet F. Roser, Department of Animal Science, Uni-
versity of California, One Shields Ave, Davis, CA 95616.
E-mail address: Jfroser@ucdavis.edu (J.F. Roser).
extract (EPE). Further studies in the same laboratory (1977)
demonstrated that EPE could induce multiple ovulations in mares
during the ovulatory season [2]. Since then, many other in-
vestigators have used various hormones regimens to induce su-
perovulation in the cycling mare [3e8]. It is not the intent of this
review to go into a detailed description of each of the hormone
regimens previously studied and presented in those reviews [3e8].
The intent of this review is to first briefly describe ET, ICSI, OT, GIFT,
cloning, and superovulation and then present hormone treatments
using recombinant equine follicle-stimulating hormone (reFSH)
and recombinant equine luteinizing hormone (reLH) that could
potentially help to improve success of these ART.
2. Assisted Reproductive Technologies
2.1. Embryo Transfer
Embryo transfer refers to a process in which an embryo is
removed from the uterus of a donor mare either surgically or
nonsurgically and placed in the uterus of a recipient mare with the

https://doi.org/10.1016/j.jevs.2019.03.004
0737-0806/© 2019 Elsevier Inc. All rights reserved.
 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
intent to establish a pregnancy. Embryo transfer is now generally
accepted as a valuable tool for obtaining foals from performance
and show mares, multiple foals from the same mare in 1 year, foals
from mares with nonreproductive health or musculoskeletal
problems, and foals from mares with reproductive problems. One of
the problems with ET is obtaining one if not more embryos from the
donor mare. Whereas single ovulating mare results in a 50% re-
covery rate, and double or triple ovulations would result in a higher
embryo recovery rate [9,10]. Presently, there is no commercial
preparation available that would consistently induce multiple
ovulations. Recombinant eFSH may be an agent of choice and
studies demonstrating its efficacy are presented below in Section
3.2.2.
2.2. Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection involves the direct injection of
sperm into the cytoplasm of a mature oocyte derived from either a
mature follicle or immature follicles found in the ovary of a mare.
The reason to use ICSI is that the mare has cervical, uterine, or
oviductal abnormalities or disease but has good quality oocytes. In
addition, ICSI allows breeding of mares by top stallions that have
aged or died and have only limited supplies of frozen semen
available. There are two main approaches of recovering oocytes
from the donor mare: transvaginal ultrasound-guided follicular
aspiration (TVA) of the preovulatory follicle or dominant follicle
(DF) that has been stimulated with gonadotropins or TVA of
immature follicles (<25 mm in diameter). What is not known is
whether the process of superovulation would provide more SDF or
more immature follicles that are viable for ICSI. Superovulation
agents such as reFSH given in small amounts on certain days of the
cycle may rescue small follicles from going atretic and increase the
number of immature or mature oocytes. Manipulation of the ovary
after stimulation to carry out TVA may or may not be more difficult
due to the presence of too many large follicles. Future studies
treating mares with reFSH for ICSI procedures will help determine
its efficacy.
2.3. Oocyte Transfer
Oocyte transfer involves the transfer of a mature oocyte from a
donor mare that is surgically placed into the oviduct of a recipient
mare. The recipient is inseminated with semen by way of the uterus
and fertilization, embryo development, and fetal development
occur within the reproductive tract of the recipient mare. Oocyte
transfer is carried out in mares that have ovulatory problems,
persistent uterine infections, pyometra, and/or torn or scarred
cervices. As in the case of ET and ICSI, a good number of immature
oocytes for OT is essential for a successful procedure, albeit only
1e2 oocytes from stimulated DFs can be used. A superovulating
agent such as reFSH may be beneficial in increasing the number of
immature or mature follicles to obtain a sufficient number of viable
oocytes.
2.4. Gamete Intrafallopian Transfer
Gamete intrafallopian transfer is a technique in horses that in-
volves the transfer of both sperm and mature oocytes directly into a
recipient mare's oviduct. Since sperm are deposited near the site of
fertilization within the oviduct, the procedure has the potential to
be used when sperm numbers are low, for example, in cases of
subfertile stallions, frozen semen, and sex-sorted semen. With ICSI
becoming more popular, GIFT may not be the procedure of choice
for subfertile mares and stallions. In addition, superovulation of the
7
donor mare using agents such as reFSH may be important for the
success of obtaining oocytes for GIFT.
2.5. Cloning
Cloning is the production of a population of genetically identical
horses. A clone may be produced in the laboratory by putting the
nucleus of a diploid cell into an oocyte that has had its nucleus
removed (nuclear transfer). Since the closing of slaughterhouses in
the United States, it has been difficult to obtain oocytes for cloning.
Superovulation with reFSH and oocyte collection by TVA may be
the technique of choice.
2.6. Superovulation
Superovulation is the process whereby treatment with exoge-
nous factors or hormones stimulates the ovary to produce multiple
follicles. The success of ART in the mare may be benefited by su-
perovulation to provide multiple oocytes for such techniques as ET,
ICSI, OT, GIFT, and cloning. Further benefits of stimulating multiple
follicles include availability of extra embryos for oocyte and embryo
freezing, enhancement of fertility in subfertile mares and stallions,
and advancement of the first ovulation of the year. Management
techniques to optimize fertility in the horse have been limited by
mares ovulating only one follicle per cycle. As mentioned previ-
ously in Section 1, superovulation has been attempted in the cycling
mare over the last 45 years beginning with studies by Douglas et al
[1] reporting the induction of multiple ovulations in seasonally
anovulatory mares using EPE. Since then, many other investigators
have used various hormones regimens to induce superovulation in
the mare [3e8]. Attempts to superovulate cyclic mares or advance
the first ovulation of the year in anestrous or transitional mares
using preparations of equine chorionic gonadotropin (eCG),
gonadotropin-releasing hormone (GnRH), GnRH agonists, porcine
FSH, domperidone, sulpiride, EPE, native equine FSH, human cho-
rionic gonadotropin (hCG), progesterone, and immunization
against inhibin have produced variable and often disappointing
results [6,11e14]. With the advent of recombinant technology, two
relatively new stimulating agents, reFSH, with or without reLH,
have been developed and reported to superovulate the ovaries of
the cycling mare [7].
3. Recombinant Gonadotropins
3.1. Development of the Recombinants
The gonadotropins, LH and FSH, are heterodimer glycoprotein
hormones comprised of an alpha and beta subunit [15]. Glycopro-
tein hormones consist of a peptide backbone with carbohydrate
moieties added at specific sites [16]. Recombinant human FSH
(rehFSH) was reported to increase follicular activity in humans,
primates, rodents, and cattle, but when tested in mares, there was
no increase in ovulation rate or embryo recovery [17]. This may
have been due to the fact that the equine FSH receptors show dif-
ferences in their DNA sequence and structure compared with other
species [17]. In the horse, the common alpha subunit is 96 amino
acids long, with two N-linked glycosylation sites [18,19]. The equine
beta subunit is hormone specific for either LH or FSH and is 149 and
111 amino acids in length, respectively [19,20]. The beta subunit of
equine LH has one N-linked and multiple O-linked carbohydrate
chains on an extended C-terminus on the carboxyl end, whereas
the beta subunit of equine FSH consists of only O-linked glycosyl-
ation and no extension [18]. Recombinant, chimeric forms of each
equine gonadotropin have been engineered using cloned comple-
mentary DNA (cDNA) encoding for each subunit. These
8 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
recombinant gonadotropins are single chained molecules, distinct
from the native forms which are heterodimers. The cDNA of each
gonadotropin subunit has been fused by joining the carboxy-
terminal at the end of the b subunit with the amino end of the a
subunit through a C-terminal extension of the eLH beta subunit
[21]. This gene can be transfected into Chinese hamster ovary cells
for expression.
Recombinant equine gonadotropins, reLH and reFSH, exhibited
in vitro activity and binding to their corresponding receptors,
despite the structural differences from the native forms [21e23].
These recombinant proteins have sialic acid terminal groups added
at glycosylation sites as opposed to sulfate. The addition of sialic
acid serves to increase the half-life in systemic circulation. Others
have developed a form of recombinant equine LH but showed only
in vitro activity [24]. In vivo activity could not be demonstrated
perhaps due to inadequate sialic acid addition on the terminal ends
of carbohydrate chains resulting in a short half-life [24]. The use of
recombinant technology has improved the ability to produce a
reliable product that is free of other hormones and possible
contaminants.
3.2. Efficacy of the Recombinants
The development and efficacy of genetically cloned recombi-
nant equine gonadotropins (reLH and reFSH) have recently been
reported [21,25e30]. The single chain recombinants are not
contaminated with other hormones or prion disease and are bio-
logically active in stimulating follicular development and ovulation
in the mare.
3.2.1. Recombinant Equine LH
3.2.1.1. The Efficacy of a Single Chain reLH in Mares: Induction of
Ovulation, Hormone Profiles, and Interovulatory Intervals. An
ovulatory agent that consistently and reliably advances the time to
ovulation in mares is an important asset to the veterinarian and
researchers in situations where timed mating is critical for
enhancing breeding efficiency. Currently, hCG is widely used to
induce ovulation, but antibodies against hCG have been found in
mares treated with hCG [31]. The repeated use of hCG may lead to
an immune response and ovulatory refractoriness. Sullivan et al
[32] observed refractoriness to hCG on the third estrous cycle when
similarly administered for three consecutive cycles, suggesting that
other ovulatory agents should be used. A study was devised to
determine the efficacy of reLH in shortening the time to ovulation
in cycling mares and determine the effects of treatment on
endogenous hormones and interovulatory intervals [25]. In study 1,
mares of light horse breeds (aged 3e20 years) were treated with
either a vehicle, various doses or reLH (0.3, n ¼ 7; 0.6, n ¼ 20; 0.75,
n ¼ 10; or 0.9, n ¼ 20 mg) IV or hCG (2,500 IU) IV. Cycling mares
were examined by palpation and transrectal ultrasonography daily
or every 12 hours from the time of treatment to ovulation. In
studies 2 and 3, various doses of reLH were injected, and jugular
blood samples were collected daily or every 12 hours from time of
treatment to ovulation from either 10 or 12 cycling mares,
respectively, for analysis of LH, FSH, estradiol-17b, and progester-
one by radioimmunoassay (RIA). In terms of results, increasing
doses of reLH showed increasing effectiveness at inducing ovula-
tion within 48 hours of treatment. Treatments with the 0.75 and
0.9 mg doses resulted in 90% and 80% ovulations rates, respectively,
which were similar to hCG treatment (85.7%). Except for the early
rise in LH after treatment with 0.5, 0.65, and 1.0 mg of reLH, hor-
mone profiles appeared to be similar between control and treated
cycles. Interovulatory intervals were similar between control and
treatment cycles. In conclusion, reLH was found to be a reliable and
effective ovulatory agent that did not significantly alter endogenous
hormones profiles or affect interovulatory intervals.
3.2.2. Recombinant eFSH
Mares are a monovular species, with typically one follicle
becoming dominant and ovulation occurring from this follicle [33].
This selection mechanism also leads to the regression of the sub-
ordinate follicles (SFs) [34]. The gonadotropin, FSH, plays a critical
role in this selection, as FSH inhibition by the DF results in atresia of
SFs [35]. Increasing the number of ovulatory follicles by maintain-
ing increased circulating FSH concentrations during follicular
development may increase the number of oocytes, ovulation rates
and embryos for ART such as ET, ICSI, OT, GIFT, and cloning. At-
tempts to superovulate cyclic mares or induce the first ovulation of
the year in transitional mares using preparations with FSH-like
activity such as eCG, rehFSH, porcine FSH, EPE, and native equine
FSH have had limited success [3,6,11,12,14,17]. The following studies
on reFSH demonstrate the efficacy of the hormone to superovulate
the cycling mare and induce the first ovulation of the year in deep
anestrous mares under natural photoperiod.
3.2.2.1. The Efficacy of reFSH to Promote Follicular Growth in Mares
Using a Follicular Suppression Model. To generate reFSH, a single
chain platform was used similar to that used for reLH [21]. It had
already been demonstrated that reLH had biological activity in the
mare [25]. Thus, the following study was designed to test the hy-
pothesis that reFSH has specific in vivo biological activity in cycling
mares whose endogenous gonadotropins and follicular activity
were suppressed using a combination of progesterone and estrogen
(P&E) [26]. The specific objectives were to evaluate (1) efficacy of
reFSH to stimulate follicular growth and increase the number of
preovulatory follicles, (2) changes in hormone profiles before,
during, and after treatment, and (3) an effective reFSH dose for use
in further studies. Estrus was synchronized in 15 cycling mares
during the breeding season with prostaglandins F2a (PGF2a). The
day after ovulation, mares were treated once daily with P&E for
14 days. Mares received a second injection of PGF2a on Day 6 of the
synchronized cycle to induce luteolysis. On Day 8 postovulation,
mares were randomly assigned to three groups: low-dose reFSH
treatment group (0.5 mg reFSH IV, twice daily), high-dose reFSH
treatment group (0.85 mg reFSH IV twice daily), and control group
(saline IV, twice daily). Recombinant equine follicle-stimulating
hormone treatment occurred concurrently with the last week of
P&E treatment. Once a follicle or cohort of follicles reached 35 mm
in diameter, mares were injected with 0.75 mg of reLH to induce
ovulation. Posttreatment ovulation was assessed. Daily blood
samples were collected for analysis of FSH, LH, estradiol, proges-
terone, and inhibin by RIA. Daily teasing and transrectal ultraso-
nography were used throughout the trial to monitor follicular
activity, estrus, and ovulation.
A significant difference between the largest diameter follicles in
the reFSH treatment groups compared with controls occurred on
Day 14 postovulation, the daily treatments ended, and the differ-
ence continued to be significant up to Day 21 postovulation. ReFSH
treatment groups had significantly larger number of 20e29 mm
follicles (Days 13e18), 30e34 mm follicles (Days 15e20), and
35 mm follicles (Days 16e21) than controls. Mares treated with
reFSH, at either dose, took less time (average: 3.0 days) to develop
two to three times more preovulatory follicles than control mares
(7.8 days). The number of ovulations between treated mares and
controls was similar due to a higher incidence of ovulation failure in
reFSH-treated mares. The number of anovulatory follicles in treated
mares (0.85 mg reFSH: 2.4; 0.5 mg reFSH: 2.0) were significantly
higher than in the control group (0.0). During reFSH treatment,
concentrations of plasma FSH, inhibin, and estradiol were
 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
significantly higher compared with control levels. Plasma LH con-
centrations in reFSH-treated mares were suppressed and did not
exhibit the significant ovulatory surge seen in controls. Plasma
progesterone concentrations were not different across groups.
These findings demonstrate the specific effects of reFSH to in-
crease the number of total follicles including preovulatory follicles
in mares whose endogenous pituitary gonadotropins and follicular
growth were suppressed by a regime of P&E.
3.2.2.2. Superovulation in Mares Using reFSH: Ovulation Rates, Em-
bryo Retrieval, and Hormone Profiles. A follicular wave is a group of
follicles that emerges synchronously. Waves are categorized into
major (primary) and secondary waves in conjunction with minor
waves [36]. A minor wave may be a priming mechanism for
follicular growth in the next follicular wave. During a major
follicular wave, follicles diverge into dominant and SFs when they
reach approximately 23 mm in diameter, which is called the time of
deviation [37]. The DF continues to grow and develop, whereas the
growth of smaller SF become atretic and regress [38]. A significant
increase and decrease in FSH encompasses emergence of the major
wave and reaches a peak when the follicles are 12e15 mm in
diameter [36]. The declining portion of the FSH surge is accompa-
nied by divergence in diameter between the future DF and SF at the
time of deviation [39]. A study was designed to test the hypotheses
that treating cycling mares with reFSH when follicles were
22e25 mm in diameter postovulation would override the decrease
in endogenous FSH and rescue SF from regressing. The objective of
the following studies was to determine the number of preovulatory
follicles, ovulations, and subsequent embryos after treatment with
reFSH in cycling mares [27].
In study 1, 16 cycling mares of light horse breeds between the
ages of 4e15 years were randomly assigned to one of two groups:
control (sterile saline; n ¼ 8) IV and 0.85 mg of reFSH (n ¼ 8) IV.
Starting on the day follicles reached 22e25 mm in diameter post-
ovulation, mares were treated twice daily (12 hours apart) for 3 days
and once per day thereafter until a follicle or cohort of follicles
reached a size of ~32 mm in diameter. On the second day of treat-
ment, all mares received PGF2a to lyse the corpus luteum. Once
reaching 32 mm, treatment was discontinued, and the mares were
allowed to coast for 36 hours. When the follicles reached 35e38 mm,
the mares were treated with reLH followed by hCG 1 hour later to
ensure ovulation. The mares were inseminated with 1 billion pro-
gressively motile sperm (PMS) from a fertile stallion. A standard
transcervical embryo recovery flush was carried out on Days 7 and 8
postovulation. Jugular blood samples were taken daily for hormone
profiles. The number of preovulatory follicles (5.5), ovulations (3.75),
and the number of embryos recovered per flush (1.75) were greater
in the 0.85 mg reFSH group than in the controls (1.38, 1.25, and 0.5,
respectively). However, the embryo per ovulation ratios was similar.
Plasma inhibin and estradiol concentrations were greater in treated
mares around the time of ovulation compared with the control
group, whereas concentrations of LH remained low throughout. In
treated mares, concentrations of FSH increased during treatment
and before ovulation but decreased postovulation.
In study 2, 28 Quarter Horse cycling mares aged between 3 and
15 years were used. Mares in group 1 served as controls. Mares
were used in more than one treatment group and allowed to go
through an untreated cycle before being reassigned to a new
treatment group. Mares in group 2 received 12.5 mg of eFSH (Bio-
niche) IM twice daily, whereas mares in groups 3, 4, and 5 received
0.35, 0.5, and 0.65 mg reFSH IM twice daily, respectively. The
treatment protocol was the same as presented above in study 1.
Mares were inseminated with 1 billion PMS from a fertile stallion.
Varying doses of reFSH and eFSH increased the number of pre-
ovulatory follicles (4.8, 5.3, 4.0, and 3.6, respectively) compared
9
with controls (1.8). The greatest number of ovulations was induced
by treatment with 12.5 mg eFSH, 0.5 mg and 0.65 mg of reFSH (3.0,
4.6, and 3.3, respectively) compared with controls (1.3). The highest
number of embryos recovered per flush was found with treatments
of eFSH (2.4) and 0.65 mg reFSH (2.7) compared with controls (0.8).
However, the embryo per ovulation ratios was similar (<1.0) in all
treatment groups including the controls.
In study 3, 20 Quarter Horse cycling mares aged between 3 and
12 years were used. Mares were randomly assigned to one of four
treatment groups and administered 12.5 mg eFSH (Bioniche) IV
twice daily (n ¼ 5), 0.5 mg reFSH IV twice daily (n ¼ 5), 0.85 mg
reFSH IV twice daily (n ¼ 5), or 0.85 mg reFSH IV once daily (n ¼ 5).
Treatment regimens were similar to studies 1 and 2. Results indi-
cated a similar increase in the number of preovulatory follicles in all
four groups (3.2, 3.2, 4.2, and 3.0, respectively) and a similar
number of ovulations in all four groups (3.8, 3.2, 3.4, and 5.4). It was
noted that a number of anovulatory follicles remained on the ovary
in all four groups with the highest number in the 0.85 mg reFSH
group twice daily (2.6). In conclusion, our hypothesis proved to be
true: reFSH treatment could override atresia of SFs. In addition,
reFSH was as effective as eFSH in increasing the number of follicles
35 mm, ovulation rates, and embryo recovery rates per flush
compared with the control group.
3.2.2.3. Treatment With reFSH Followed by reLH Increases Embryo
Recovery in Superovulated Mares. The dynamics of follicular
development depend on a timely interaction of gonadotropins and
gonadal feedback. Previous studies above using reFSH demon-
strated higher number of preovulatory follicles, ovulation rates, and
embryo recovery rates per flush after treatment [26,27]. Yet, also
reported in these studies were a low LH surge, high inhibin and
estradiol concentrations, increase in anovulatory follicles, and no
difference in embryo per ovulation ratios compared with controls.
The low LH surge is most likely due to the higher estrogen pro-
duction from the increasing numbers of preovulatory follicles.
Ginther et al [40] demonstrated a negative effect of estradiol on LH
throughout the ovulatory LH surge in mares. In another study,
Ginther et al [41] reported a disruption of the periovulatory LH
surge by transient increase in circulating 17b-estradiol at the time
of ovulation in mares.
An optimum hormone milieu is important for normal oocyte and
follicle maturation. Oocytes require the presence of LH to stimulate
granulosa and cumulus cells to secret factors important for oocyte
maturation and competence for fertilization [42e49]. An increase in
LH receptors on granulosa and cumulus cells has been correlated
with an increase in follicular diameter and oocyte competence in the
mare [42]. Lindbloom et al [48] reported that reLH was effective in
increasing epidermal-like growth factors in granulosa cells and an
isoform of phosphodiesterase in equine oocytes within 9 hours after
incubation, which are essential for oocyte maturation. The studies
using reFSH, eFSH, and decreasing doses of EPE had higher systemic
concentrations of estrogen and inhibin [26,27,50]. High concentra-
tions of estradiol and inhibin acting as paracrine/autocrine factors
have been shown to be detrimental to oocyte maturation [49,51,52],
which may in turn eventually affect the embryo per ovulation ratio.
In addition, in vitro studies in cattle and swine indicate that oocyte
maturation and promotion of the cumulus cell-oocyte complex
expansion requires sequential exposure of FSH followed by LH
[53,54]. Therefore, one could hypothesize that a high FSH to LH ratio
after repeated injections of eFSH or reFSH may not be optimal for
oocyte maturation, fertilization, and an increase in the embryo per
ovulation ratio.
Several other factors can affect the embryo per ovulation ratio
such as oocyte quality [55e57] and the capability of the oocyte to
enter into the oviduct via the ovulation fossa [58]. The findings that
10 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
an increased number of anovulatory follicles were present after su-
perovulation regimens [8,26,27,59e61] support the concept that a
less than optimum embryo per ovulation ratio may be a function of
oocyte dysfunction. Carmo et al [58] reported that there was no
significant difference in the number of oocytes that appeared in the
oviduct from superovulated mares compared with control mares,
supporting the concept that oocyte transport may be a critical factor
affecting the embryo per ovulation ratio in superovulated mares.
When the genital tract was removed from mares, the presence of a
large amount of coagulated blood in the ovulation fossa of super-
ovulated mares was observed, suggesting a disturbance in the
ovulation process and a blockage at the ovulation fossa [58].
The next study was designed to test the hypothesis that a su-
perovulation regimen involving repeated injection of reFSH fol-
lowed by multiple injections of reLH would establish a hormone
environment conducive to maximum embryo recovery efficiency
[28]. The objectives of this study were to determine the efficacy of
treatment with reFSH followed by reLH (1) to increase the number
of multiple ovulating follicles and reduce the number of anovula-
tory follicles, (2) to increase the embryo recovery per flush and
embryo per ovulation ratio, and (3) to determine changes in the
hormone profiles of mares treated with reFSH/reLH compared with
those treated with reFSH/phosphate-buffered saline (PBS). Sixteen
normal cycling mares of light horse breeds between the ages of 4
and 12 years were used. Mares were randomly selected for the
following groups: Group 1; reFSH (0.65 mg)/PBS IV (n ¼ 8) and
Group 2; reFSH (0.65 mg)/reLH (0.75 mg) IV (n ¼ 8). On the day of a
22e25 mm follicle postovulation, mares were injected twice daily
with reFSH for 3 days (PGF2a given on the second day of treatment)
and once per day thereafter until a follicle or cohort of follicles
reached 29 mm after which either PBS or reLH was added, and both
groups injected twice daily IV until the presence of a 32 mm folli-
cles when reFSH was discontinued. Thereafter, mares were injected
three times daily (every 8 hours) with only PBS or reLH IV until a
majority of follicles reached 35e38 mm when treatment was dis-
continued. Mares were given hCG IV (2,500 IU) to induce ovulation.
Jugular blood samples were taken daily for hormone profiles.
Treatment with reFSH/reLH compared with reFSH/PBS had a
similar number of 35 mm follicles (4.50 and 5.0, respectively),
days of treatment to a 35 mm follicle(s) (8.12 and 7.75, respectively)
and days to ovulation after hCG (2.12 and 2.03, respectively). There
was an increase in the number of embryos recovered per flush for
mares treated with reFSH/reLH (3.87) compared with reFSH/PBS
(2.0). There was a decrease in the number of anovulatory follicles in
the reFSH/reLH (0.025) compared with reFSH/PBS (1.37) and a
tendency (P < .07) for a greater embryo per ovulation ratio in mares
treated with reFSH/reLH, suggesting a switch to LH dependence on
the growth of the DFs and maturation of the oocytes. Plasma con-
centrations of FSH were less in the reFSH/reLH treated mares on the
day of ovulation and certain days postovulation. Circulating con-
centrations of estradiol, inhibin, LH, and progesterone were not
statistically different. In terms of LH, there was a slight rise in both
groups, but daily sampling did not reflect the induced pulsatility of
repeated injection of reLH. However, the pulsatility of the reLH
injections may have been critical in stimulating oocyte develop-
ment and maturation [62e64].
3.2.2.4. Induction of Ovulation in Seasonally Anestrous Mares Under
Ambient Lights Using reFSH. The goal of many performance horse
owners is to begin breeding mares in early February in the North-
ern Hemisphere, a time when most mares maintained under
ambient lights are not cycling. This results in foals born early the
next year who are more mature and competitive in the show arena
than foals born later in the year. Traditionally, an artificial lighting
program has been used to advance the first ovulation of the year
[65e67]. The average duration from the onset of artificial light
exposure to ovulation is approximately 60e70 days [68]. However,
incorporation of a light program is labor intensive, not always
effective or used. Therefore, other options to induce a fertile
ovulation when mares are normally in winter anestrous and under
natural photoperiod would be highly beneficial. To date, other
drugs used to hasten the first ovulation of the year have been less
than consistently successful [6].
This study was to determine the efficacy of reFSH in stimulating
follicular development and advancing the first ovulation of the year
in deep anestrous mares maintained under ambient lights [29]. The
study was conducted simultaneously using exactly the same pro-
tocol at three university locations: UC Davis, Colorado State Uni-
versity, and University of Kentucky. A total of 60 mares of light
horse breeds in deep seasonal anestrus (20 mares per study site),
maintained under ambient lights, were used in this study. Mares
with <20 mm follicles and <1 ng/mL progesterone plasma con-
centrations at each site were randomly allocated to one of two
treatment groups of 10 mares each on January 31: Group A (0.65 mg
reFSH IM twice daily) or Group B (saline IM twice daily). Treatment
period continued until (1) a mare developed one or more follicles
35 mm in diameter or (2) for a maximum of 15 consecutive days.
Follicular activity was monitored by transrectal ultrasonography
three times a week. Once a  35 mm follicle or cohort of follicles
was present with appropriate uterine edema and cervical relaxa-
tion, treatment was discontinued for 36 hours after which hCG was
administered to induce ovulation. Ultrasound examinations were
subsequently performed once daily to confirm day and number of
ovulations. At the conclusion of the 15 days treatment period,
Group B mares were examined once per week until a 35 mm
follicle was detected. Mares were then examined daily until the first
spontaneous ovulation of the year was confirmed. Jugular blood
samples were collected daily throughout the 15 days treatment
period and three times a week thereafter for hormone profiles.
All 30 mares that received reFSH developed one or more follicles
35 mm in an average of 7.4 days after the onset of treatment.
During the 15-day treatment period, an average of 3.6 follicles,
35 mm in diameter, were present at the time of hCG adminis-
tration in Group A mares compared with zero 35 mm follicles in
saline-treated mares. The average number of ovulations per reFSH
mares was 3.8 compared with zero in saline-treated mares. The
saline-treated mares did not spontaneously ovulate until April.
Plasma concentrations of FSH, estradiol, and inhibin were signifi-
cantly higher than the saline group after the start of treatment.
Increased concentrations of progesterone were higher than the
saline mares after the start of treatment, which corresponded to
ovulations in the treatment mares. As observed in other studies,
plasma concentrations of LH in the treated mares were low and
similar to the saline mares. In conclusion, administration of 0.65 mg
of reFSH twice daily was effective in stimulating the development
of preovulatory follicles that ovulated after hCG in deep anestrous
mares housed under ambient lights.
3.2.2.5. Deep Anestrous Mares Under Natural Photoperiod Treated
With reFSH and reLH Have Fertile Ovulations and Become Pregnant.
In the above study (Section 3.2.2.4), it was demonstrated that
reFSH-induced ovulation in seasonally anestrous mares under
ambient lights but pregnancy was not evaluated. The study using
both reFSH and reLH (Section 3.2.2.3) in cycling mares demon-
strated a higher number of embryos per flush, a lower number of
anovulatory follicles, and a tendency for a higher embryo to
ovulation ratio. The objectives of this study were to determine (1)
the efficacy of reFSH and the combination of reFSH and reLH in
stimulating follicular development, ovulation, and pregnancy in
 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
deep-anestrous mares maintained under natural photoperiod and
(2) the return to cyclicity after termination of pregnancy.
In this study, a total of 30 university-owned mares of light horse
breeds (aged 4e19 years) in deep anestrus were maintained under
ambient lights or artificial lights [30]. Mares were randomly allo-
cated to one of three treatment groups of 10 mares each on
February 6. In Group A, mares received 0.65 mg reFSH IM twice
daily at 8 hours apart. When a follicle or cohort of follicles reached
32 mm in diameter, mares received PBS twice daily IM until a fol-
licle or a cohort of follicles reached 35 mm in diameter after which
mares were allowed to “coast” (i.e., not administered any exoge-
nous medications) for 36 hours and then treated with 2,500 IU hCG
IV to induce ovulation. Mares in Group B received 0.65 mg of reFSH
IM twice daily at 8 hours apart. When a follicle or cohort of follicles
reached 29 mm in diameter, mares continued to receive reFSH once
daily IM immediately followed by 1.5 mg reLH IV twice daily until a
follicle or cohort of follicles reached 32 mm in diameter. At 32 mm
in diameter, reFSH was discontinued while mares received reLH
three times daily IV at 8 hours apart. Once a follicle or cohort of
follicles reached 35 mm in diameter, mares were treated with hCG
IV to induce ovulation. Mares in Groups A and B were maintained
under ambient lighting conditions. Mares in Group C received
1.3 mL PBS IM as a placebo twice daily. All treatments continued
until a mare developed one or more follicles 35 mm in diameter
or for a maximum of 14 consecutive days. Mares in Group C were
maintained under artificial lights. Artificial lights were turned on in
the late afternoon on December 1 so that mares received 16-hour
light and 8-hour dark. Transrectal ultrasonography examinations
of the reproductive tract were performed daily on mares during the
treatment period and up to 14 days for controls. The diameter of the
largest follicle and diameter of all follicles 30 mm on each ovary
were recorded during each ultrasound examination. Mares were
then bred to one of two stallions of known fertility by artificial
insemination with at least 1 billion progressively motile fresh
sperm every other day until ovulation(s) occurred. Ultrasound ex-
aminations were performed once daily after administration of hCG
IV (2,500 IU) to confirm the day of ovulation and number of
ovulations.
At the conclusion of the treatment period or 14 days, all mares
were examined every other day until ovulation. Pregnancy and an
embryonic heartbeat were determined at 14 and 24 days post-
ovulation, respectively. Mares were treated on Day 25 with PGF2a
to terminate pregnancy and further monitored by transrectal ul-
trasonography to determine cyclicity and the next ovulation. Ju-
gular blood samples were collected once daily beginning on the day
before treatment, continuing through the end of treatment and
thereafter three times per week for hormone profiles.
Mares in the reFSH (Group A) grew a 35 mm follicle in
diameter in 5.9 days after the start of treatment and were not
significantly different from mares in the reFSH/reLH (Group B) at
5.7 days. The time to a 35 mm follicle in Group C control mares
was 15.8 days and was significantly longer than observed in the
treatment groups. There was no significant difference in the
percentage of reFSH mares (100%) and reFSH/reLH mares (100%)
that had 35 mm follicles, but a significant difference was
observed compared with controls (30%). There was no significant
difference in the number of follicles 35 mm between the reFSH
mares (4.4) and the reFSH/reLH mares (3.5). The control mares
had significantly fewer follicles 35 mm (0.3). There was no
significant difference in the percentage of ovulation between the
reFSH mares (100%) and the reFSH/reLH mares (100%). The
control mares were significantly lower (30%) during the 2-week
treatment period. There was a significantly higher number of
ovulations observed in the reFSH mares (3.4) than in the reFSH/
reLH mares (2.1) or the control mares (0.02). The number of
11
anovulatory follicles between the two treatment groups, reFSH
(0.8) and reFSH/reLH (1.4), was not significantly different,
whereas the number in the control group (0.01) was significantly
lower. There was no significant difference in the percentage of
reFSH mares (80%) that were pregnant compared with the reFSH/
reLH mares (80%) at 16 days postovulation. All three of the mares
that ovulated in the control group were pregnant (100%) during
the 2-week treatment period. There was no significant difference
in the percentage of reFSH mares (70%) and reFSH/reLH mares
(70%) that were pregnant at 24 days postovulation as observed
by an embryonic heartbeat, whereas all three of the control
mares that ovulated during the 2-week treatment period were
pregnant (100%). After treatment with PGF2a to terminate
pregnancy at 25 days, the seven reFSH mares grew a 35 mm
follicle within (5.3) days and ovulated within (9.8) days, whereas
the seven reFSH/reLH mares on average reached a 35 mm fol-
licle within (5.3) days and ovulated (11.4) days after PGF2a
administration.
Plasma concentrations of FSH were significantly higher in the
reFSH and reFSH/reLH mares after the start of treatment compared
with control mares. An increase in plasma LH concentrations was
not observed and did not coincide with ovulation(s) in the reFSH-
treated mares, but an increase was noted in the reFSH/reLH ma-
res. Some of the control mares continued to ovulate during the
blood collection period such that in those mares, a significant
second increase in LH was observed. Plasma inhibin concentrations
in both treatment groups were not different between each other
but were significantly higher compared with the control group.
Plasma concentrations of estradiol in both the reFSH and the reFSH/
reLH-treated mares were significantly higher compared with the
control mares but were not different between the two treated
groups. Plasma concentrations of progesterone in the reFSH/reLH-
treated mares were significantly higher compared with the
reFSH-treated mares and controls. There were no significant
changes in plasma hormone concentration between the two
treatment groups that continued to cycle after treatment with
prostaglandin to terminate pregnancy.
Two mares, one from each group (reFSH and reFSH/reLH-treated
mares) that did get pregnant at 14 days but did not maintain
pregnancy at 24 days continued to cycle. Two mares from the
reFSH/reLH treatment group failed to get pregnant. However, they
continued to cycle on their own. Two mares from the reFSH group
responded to treatment, ovulated, but failed to get pregnant at
14 days posttreatment ovulation. These mares stopped cycling and
fell back into winter anestrous and were monitored for 30 days
postovulation, at which time they were retreated using the reFSH/
reLH treatment protocol. Both mares ovulated and got pregnant at
14 days after ovulation and were pregnant on Day 24 when an
embryonic heartbeat was detected.
In conclusion treatment with reFSH or reFSH/reLH induced
formation of multiple follicles 35 mm in diameter within
5e6 days resulting in multiple ovulations within 8e10 days from
the start of treatment in deep anestrous mares under ambient
lights. Seven mares in each group went on to become pregnant as
observed by an embryonic heartbeat at 24 days. After treatment
with PGF2a to terminate pregnancy, all mares continued to cycle.
Repeated injections of reFSH or reFSH/reLH resulted in high plasma
estradiol concentrations, which may have affected the formation of
anovulatory follicles [69]. Although the number of mares that did
not become pregnant was low, it appears that treatment with
reFSH/reLH may be more beneficial in preventing these mares from
returning to anestrus. Treatment with recombinant gonadotropins
was significantly more effective in inducing cyclicity and preg-
nancies in anestrus mares in a shorter time frame than mares
exposed to an artificial photoperiod.
12 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
4. Summary
Advanced reproductive technologies such as ET, ICSI, OT, GIFT,
cloning, and superovulation serve to enhance fertility in mares and
stallions, particularly in those horses with reproductive problems.
Each technique comes with its own uncertainties for a successful
outcome. Each technique requires expert knowledge and a labo-
ratory specifically set up to carry out the procedures. Advanced
reproductive technologies will become more popular as the success
rate increases. These technologies would be benefited from su-
perovulation of mares by providing more oocytes. Various drugs
have been used to superovulate mares with variable success. With
the advent of recombinant technologies, two relatively new hor-
mones have been developed and used, reLH and reFSH. It has been
demonstrated that these recombinant gonadotropins can success-
fully induce multiple preovulatory follicles, ovulations, and em-
bryos in both cycling and deep anestrous mares. The question now
remains as to whether these recombinants will enhance repro-
ductive efficiency by increasing the number of viable oocytes for
ICSI, OT, GIFT, and cloning.
Acknowledgments
The authors would like to thank Dr Mark Colgin and AspenBio
Pharma Inc, Castle Rock, CO, for providing the reLH and reFSH to
carry out studies investigating the efficacy of the recombinant
gonadotropins.
Financial disclosure
No internal or external funding was associated with the writing
of this article.
References
[1] Douglas RH, Nuti L, Ginther OJ. Induction of ovulation and multiple ovulations
in seasonally-anovulatory mares with equine pituitary fractions. Ther-
iogenology 1974;2:133e42.
[2] Lapin DR, Ginther OJ. Induction of ovulation and multiple ovulations in
seasonally anovulatory and ovulatory mares with an equine pituitary extract.
J Anim Sci 1977;44:834e42.
[3] McCue PM. Superovulation. Vet Clin North Am Equine Pract 1996;12:1e11.
[4] Squires EL, McCue PM. Superovulation in mares. Anim Reprod Sci 2007;99:
1e8.
[5] Squires EL, McCue PM. Superovulation. In: McKinnon AO, Squires EL,
Vaala WE, Varner DD, editors. Equine reproduction. Second Edition. West
Sussex: Wiley-Blackwell; 2011. p. 1836e53.
[6] McCue PM, Logan NL, Magee C. Management of the transition period: hor-
mone therapy. Equine Vet Edu 2007;19:215e21.
[7] Roser JF, Meyers-Brown G. Superovulation in the mare: a work in progress.
J Equine Vet Sci 2012;32:376e86.
[8] Squires EL. Superovulation in mares. Vet Clin Equine 2006;22:819e30.
[9] Squires EL, McCue PM, Vanderwall D. The current status of equine embryo
transfer. Theriogenology 1999;51:91e104.
Slade NP, Takeda T, Squires EL, Elsden RP, Seidel Jr GE. A new procedure for
the cryopreservation of equine embryos. Theriogenology 1985;24:45e58.
[10] Squires EL, McClain MG, Ginther OJ, McKinnon AO. Spontaneous multiple
ovulation in the mare and its effect on the incidence of twin embryo collec-
tion. Theriogenology 1987;28:609e14.
[11] Niswender KD, Alvarenga MA, McCue PM, Hardy QP, Squires EL. Superovu-
lation in cycling mares using equine follicle stimulating hormone. J Equine Vet
Sci 2003;23:497e500.
[12] Williams GL, Thorsona JF, Prezottoa LD, Veleza IC, Cardosoa RC,
Armstaldenb M. Reproductive seasonality in the mare: neuroendocrine basis
and pharmacologic control. Domest Anim Endocrinol 2012;43:103e15.
[13] McCue PM, Hughes JP, Lasley BL. Effect of ovulation rate of passive immuni-
zation rate of mares against inhibin. Equine Vet J Suppl 1993;15:103e6.
[14] McCue PM, LeBlanc MM, Squires EL. eFSH in clinical practice. Theriogenology
2007;68:429e33.
[15] Combarnous Y, Richard F, Martinat N. Mammalian follicle-stimulating hor-
mone receptors and their ligands. Eur J Obstet Gynecol Reprod Biol 1998;77:
125e30.
[16] Alexander SL, Irvine CH. FSH and LH. In: McKinnon AO, Squires EL, Vaala WE,
Varner DD, editors. Equine reproduction. Second Edition. West Sussex: Wiley-
Blackwell; 2011. p. 1619e30.
[17] Tharasanit T, Colenbrander B, Bevers MM, Stout TA. Effects of recombinant
human follicle stimulating hormone on follicle development and ovulation in
the mare. Theriogenology 2006;65:1071e81.
[18] Bousfield GR, Butnev VY, Gotschall RR, Baker VL, Moore WT. Structural fea-
tures of mammalian gonadotropins. Mol Cell Endocrinol 1996;125:3e19.
[19] Bousfield GR, Butnev VY, Butnev VY. Identification of twelve O-glycosylation
sites in equine chorionic gonadotropin {beta} and equine luteinizing hor-
mone {beta} by solid-phase edman degradation. Biol Reprod 2001;64:
136e47.
[20] Saneyoshi T, Min K-S, Jing Ma X, Nambo Y, Hiyama T, Tanaka S, et al. Equine
follicle-stimulating hormone: molecular cloning of {beta} subunit and bio-
logical role of the asparagine-linked oligosaccharide at asparagine56 of
{alpha} subunit. Biol Reprod 2001;65:1686e90.
[21] Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M,
Boime I. Expression and bioactivity of a single chain recombinant equine
luteinizing hormone (reLH). Theriogenology 2007;67:311e20.
[22] Garcia-Campayo V, Jablonka-Shariff A, Boime I. A single-chain bifunctional
gonadotropin analog is secreted from Chinese hamster ovary cells as two
distinct bioactive species. J Biol Chem 2004;279:44286e93.
[23] Roser JF, Boime I, Colgin M, Niswender KD. Development and efficacy of re-
combinant eLH and eFSH. In: Proceedings West Coast Equine Reprod Symp II,
Buellton, CA; 2005. p. 39e52.
[24] Legardinier S, Duonor-Cerutti M, Devauchelle G, Combarnous Y, Cahoreau C.
Biological activities of recombinant equine luteinizing hormone/chorionic
gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines. J Mol
Endocrinol 2005;34:47e60.
[25] Yoon MJ, Boime I, Colgin M, Niswender KD, King SS, Alvarenga M, et al. The
efficacy of a single chain recombinant equine luteinizing hormone (reLH) in
mares: induction of ovulation, hormone profiles, and inter-ovulatory in-
tervals. Domest Anim Endocrinol 2007;33:470e9.
[26] Jennings MW, Boime I, Daphna-Iken D, Jablonka-Shariff A, Conley AJ,
Colgin M, et al. The efficacy of recombinant equine follicle stimulating hor-
mone (reFSH) to promote follicular growth in mares using a follicular sup-
pression model. Anim Reprod Sci 2009;116:291e307.
[27] Meyers-Brown GA, McCue PM, Niswender KD, Squires EL, DeLuca CA,
Bidstrup LA, et al. Superovulation in mares using recombinant equine follicle
stimulating hormone (reFSH): ovulation rates, embryo retrieval and hormone
profiles. J Equine Vet Sci 2010;30:560e8.
[28] Meyers-Brown G, Bidstrup LA, Famula TR, Colgin M, Roser JF. Treatment with
recombinant equine follicle stimulating hormone (reFSH) followed by re-
combinant equine luteinizing hormone (reLH) increases embryo recovery in
superovulated mares. Anim Reprod Sci 2011;128:52e9.
[29] Meyers-Brown GA, McCue PM, Troedsson MHT, Klein C, Zent W, Ferris RA,
et al. Induction of ovulation in seasonally anestrous mares under ambient
lights using recombinant equine FSH (reFSH). Theriogenology 2013;80:
456e62.
[30] Meyers-Brown GA, Loud MC, Hyland JC, Roser JF. Deep anestrous mares under
natural photoperiod treated with recombinant equine FSH (reFSH) and LH
(reLH) have fertile ovulations and become pregnant. Theriogenology 2017;98:
108e15.
[31] Roser JF, Kiefer BL, Evans JW, Neely DP, Pacheco CA. The development of
antibodies to human chorionic gonadotrophin following it repeated injection
in the cyclic mare. J Reprod Fertil Suppl 1979;27:175e9.
[32] Sullivan JJ, Parker WG, Larson LL. Duration of estrus and ovulation time in
non-lactating mares given human chorionic gonadotropin during three suc-
cessive estrous periods. J Am Vet Med Assoc 1973;162:895e8.
[33] Ginther OJ. Reproductive biology of the mare: basic and applied aspects. Cross
Plains, WI: Equisevices Publishing; 1992.
[34] Bergfelt DR, Ginther OJ. Relationships between FSH surges and follicular
waves during the estrous cycle in mares. Theriogenology 1993;39:781e96.
[35] Gastal EL, Gastal MO, Ginther OJ. Experimental assumption of dominance by a
smaller follicle and associated hormonal changes in mares. Biol Reprod
1999;61:724e30.
[36] Ginther OJ. Systemic and intrafollicular components of follicle selection in
mares. Domest Anim Endocrinol 2017;59:116e33.
[37] Gastal EL, Gastal MO, Beg MA, Ginther OJ. Interrelationships among follicles
during the common-growth phase of a follicular wave and capacity of indi-
vidual follicles for dominance in mares. Reproduction 2004;128:417e22.
[38] Jacob JC, Gastal EL, Gastal MO, Carvalho GR, Beg MA, Ginther OJ. Follicle de-
viation in ovulatory follicular waves with one or two dominant follicles in
mares. Reprod Domest Anim 2009;44:248e54.
[39] Bergfeldt DR, Ginther OJ. Relationships between circulating concentrations of
FSH and follicular waves during early pregnancy in mares. J Equine Vet Sci
1992;12:274e9.
[40] Ginther OJ, Utt MD, Beg MA, Gastal EL, Gastal MO. Negative effect of estradiol
on luteinizing hormone throughout the ovulatory luteinizing hormone surge
in mares. Biol Reprod 2007;77:643e50.
[41] Ginther OJ, Almamun M, Shahiduzzaman AK, Beg MA. Disruption of the
periovulatory LH surge by transient increase in circulating 17b-estradiol at the
time of ovulation in mares. Anim Reprod Sci 2010;117:178e82.
[42] Goudet G, Belin F, Bezard J, Gerard N. Intrafollicular content of luteinizing
hormone receptor, alpha-inhibin, and aromatase in relation to follicular
 J.F. Roser, G. Meyers-Brown / Journal of Equine Veterinary Science 76 (2019) 6e13
growth, estrous cycle stage, and oocyte competence for in vitro maturation in
the mare. Biol Reprod 1999;60:1120e7.
[43] Hinrichs K, Schmidt AL. Meiotic competence in horse oocytes: interactions
among chromatin configuration, follicle size, cumulus morphology, and sea-
son. Biol Reprod 2000;62:1402e8.
[44] Hillier SG. Gonadotropic control of ovarian follicular growth and develop-
ment. Mol Cell Endocrinol 2001;179:39e46.
[45] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of recom-
binant human FSH and LH on in vitro maturation of sheep oocytes; embryo
development and viability. Anim Reprod Sci 2004;81:77e86.
[46] Webb R, Garnsworthy PC, Gong JG, Armstrong DG. Control of follicular
growth: local interactions and nutritional influences. J Anim Sci Suppl
2004;82:63e74.
[47] Silvestre MA, Alfonso J, Garcia-Mengual E, Salvador I, Duque CC, Molina I.
Effect of recombinant human follicle-stimulating hormone and luteinizing
hormone on in vitro maturation of porcine oocytes evaluated by the subse-
quent in vitro development of embryos obtained by in vitro fertilization,
intracytoplasmic sperm injection, or parthenogenetic activation. J Anim Sci
2007;85:1156e60.
[48] Lindbloom SM, Farmerie TA, Clay CM, Seidel Jr GE, Carnevale EM. Potential
involvement of EGF-like growth factors and phosphodiesterases in initiation
of equine oocyte maturation. Anim Reprod Sci 2008;103:187e92.
[49] Rosen MP, Zamah AM, Shen S, Dobson AT, McCulloch CE, Rinaudo PF, et al. The
effect of follicular fluid hormones on oocyte recovery after ovarian stimula-
tion: FSH level predicts oocyte recovery. Reprod Biol Endocrinol 2009;7:35.
[50] Machado MS, Roser JF, Greco GM, Araujo GH, Alvarenga MA. Effect of
superovulatory treatment with equine pituitary extract and equine FSH on
endocrine profiles in mares. In: Allen WR, editor. 7th International Sympo-
sium on equine embryo transfer. Cambridge, England: Magdalene College;
2008. p. 59e60.
[51] Silva CC, Groome NP, Knight PG. Demonstration of a suppressive effect of
inhibin alpha-subunit on the developmental competence of in vitro matured
bovine oocytes. J Reprod Fertil 1999;115:381e8.
[52] Bing YZ, Naga T, Rodriguez-Martinez H. Effects of cysteamine, FSH and
estradiol-17beta on in vitro maturation of porcine oocytes. Theriogenology
2001;55:867e76.
[53] Mihm M, Baker PJ, Ireland JL, Smith GW, Coussens PM, Evans AC, et al. Mo-
lecular evidence that growth of dominant follicles involves a reduction in
follicle-stimulating hormone dependence and an increase in luteinizing hor-
mone dependence in cattle. Biol Reprod 2006;74:1051e9.
[54] Kawashima I, Okazaki T, Noma N, Nishibori M, Yamashita Y, Shimada M.
Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for
13
appropriate expression of steroidogenic and ovulation-related genes that
impact oocyte maturation in vivo and in vitro. Reproduction 2008;136:9e21.
[55] Carnevale EM, Ginther OJ. Defective oocytes as a cause of subfertility in old
mares. Biol Reprod Monogr 1995;1:209e14.
[56] Geary RT, Weber JA, Woods GL. Hastened transport of equine embryos
through the oviduct of the mare. Theriogenology 1989;31:973e8.
[57] Carnevale EM, Bozzola JJ, King SS, Schmitt SJ, Gates HD. Comparison of oocytes
from young and old mares with light and electron microscopy. Theriogenol-
ogy 1999;29:9.
[58] Carmo MT, Losinno L, Aquilar JJ, Araujo GHM, Alveranga MA. Oocyte transport
to the oviduct of superovulated mares. Anim Reprod Sci 2006;94:337e9.
[59] Alvarenga MA, McCue PM, Bruemmer J, Neves Neto JR, Squires EL. Ovarian
superstimulatory response and embryo production in mares treated with
equine pituitary extract twice daily. Theriogenology 2001;56:879e87.
[60] Welch SA, Denniston DJ, Hudson JJ, Bruemmer JE, McCue PM, Squires EL. 2006.
Exogenous eFSH, follicle coasting, and hCG as a novel superovulation regimen
in mares. J Equine Vet Sci 2006;26:262e70.
[61] Raz T, Gray A, Hunter B, Card C. Early effects of equine FSH (eFSH) treatment
on hormonal and reproductive parameters in mares intended to carry their
own pregnancy. Anim Reprod Sci 2009;115:76e87.
[62] Becker SE, Johnson AL. Effects of gonadotropin-releasing hormone infused in a
pulsatile or continuous fashion on serum gonadotropin concentrations and
ovulation in the mare. J Anim Sci 1992;70:1208e15.
[63] Bezard J, Mekarska A, Goudet G, Duchamp G, Palmer E. Timing of in vivo
maturation of equine preovulatory oocytes and competence for in vitro
maturation of immature oocytes collected simultaneously. Equine Vet J Suppl
1997;25:33e7.
[64] Takagi M, Kim IH, Izadyar F, Hyttel P, Bevers MM, Dieleman SJ, et al. Impaired
final follicular maturation in heifers after superovulation with recombinant
human FSH. Reproduction 2001;121:941e51.
[65] Burkhardt J. Transition from anoestrus in the mare and the effect of artificial
lighting. J Agri Sci Camb 1947;37:64e8.
[66] Palmer E, Driancourt MA, Ortavant R. Photoperiodic stimulation of the mare
during winter anoestrus. J Reprod Fertil Suppl 1982;32:275e82.
[67] Sharp D. Photoperiod. In: McKinnon AO, Squires EL, Vaala WE, Varner DD,
editors. Equine reproduction. Second Edition. West Sussex: Wiley-Blackwell;
2011. p. 1771e7.
[68] Guillaume D, Duchamp G, Nagy P, Palmer E. Determination of minimum light
treatment required for photostimulation of winter anoestrous mares. J Reprod
Fertil Suppl 2000;56:205e16.
[69] Gastal EL, Gastal MO, Beg MA. Conversion of a viable preovulatory follicle into
a hemorrhagic anovulatory follicle in mares. Anim Reprod 2006;3:29e40.