Theriogenology 142 (2020) 291e295

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Theriogenology
journal homepage: www.theriojournal.com

Superovulation, embryo recovery, and pregnancy rates from

seasonally anovulatory donor mares treated with recombinant equine
FSH (reFSH)
Janet F. Roser a, Maria V. Etcharren b, Marcelo H. Miragaya c, Adrian Mutto d,
Mark Colgin e, 1, Luis Losinno b, Pablo J. Ross a, *
a
Department of Animal Science, University of California, Davis, CA, USA
b
Facultad de Agronomía y Veterinaria, Universidad Nacional de Rio Cuarto, Argentina
c
Facultad de Ciencias Veterinarias, INITRA, Universidad de Buenos Aires, Argentina
d gicas, Universidad Nacional de General San Martin, Argentina
Instituto de Investigaciones Biotecnolo
e
Aspen Bio Pharma Inc., Castle Rock, CO, USA

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

Article history: The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth,
Received 6 August 2019 multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-
Received in revised form winter season (JulyeAugust in Argentina, South America) forty light breed donor mares, presenting
18 October 2019
follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly
Accepted 28 October 2019
assigned (n ¼ 10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM)
Available online 29 October 2019
injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH,
Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline
Keywords:
reFSH
(control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10
Anestrus days. When the largest follicle reached 35 mm in diameter, treatment was discontinued and 2500 IU
Superovulation hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen
Embryo transfer every 48 h until ovulation(s) were detected or one dose of frozen semen (250  106 motile sperm) after
Pregnancy the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was
performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone
treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares
receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On
average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size
(>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in
none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of
embryos recovered, or pregnancy rates were observed among reFSH treatments. Of treated mares, 6, 7,
and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per
ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59%
pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus
results in follicular development, ovulation of fertile oocytes, and production of embryos that established
viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily
administrations, which allows for a simplified administration regimen.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction

Embryo transfer (ET) is a widely utilized assisted reproductive

* Corresponding author. Department of Animal Science, One Shields Ave, Uni- technique for mares to obtain the following: a. foals from show
versity of California, Davis, CA 95616, USA. mares in competition, b. multiple foals from the same mare in a
E-mail address: [email protected] (P.J. Ross).
1 single year, c. foals from mares with non-reproductive health or
Current address: Ceva Animal Health, Lenexa, KS, USA.

https://doi.org/10.1016/j.theriogenology.2019.10.030
0093-691X/© 2019 Elsevier Inc. All rights reserved.
292 J.F. Roser et al. / Theriogenology 142 (2020) 291e295

musculoskeletal problems and d. foals from mares with reproduc- lights with reFSH to induce superovulation followed by embryo
tive problems. The success rate of ET in a commercial operation is transfer and pregnancy in the Southern Hemisphere.
based on the percent of embryo recovery rate per ovulation
(50e80%) multiplied by the percent of pregnancy rate per trans- 2. Materials and methods
ferred embryo (50e80%). Thus, for a given cycle there is a 25e64%
chance of obtaining a pregnant recipient [1e5]. Factors that affect 2.1. Animal selection and management
embryo recovery include age of the donor mare, quality of the
semen, number of ovulations and day of recovery [2,6]. Factors that 2.1.1. Donor mares
affect pregnancy rate are management of the donor and recipient Clinically healthy deep-anestrous mares of light horse breeds
mares, age and health of the donor and recipient mares, non- between the ages of 5e13 y weighing between 900 and 1400 lbs
surgical vs surgical techniques, quality and age of the embryo, were selected for this study. Mares were housed in single large
synchrony of the recipient mare, transportation and storage of the paddocks at two facilities: La Irenita Equine Embryo Transfer Cen-
embryo, manual versus cervical forceps techniques and expertise of ter, Argentina and University of Buenos Aires, Argentina. All the
the clinician [2,4]. Additional factors that have limited the success animals had unlimited access to grass hay that was fed off the
of ET are the narrow window of time for breeding (breeding season ground in a separate area of the paddock and fed once daily with
only) and the ability to superovulate the mare. grain. At both facilities, fresh water was provided ad libitum. Animal
In the Northern and Southern Hemispheres, the natural care was approved by the ethics and animal welfare committee, Rio
breeding season for mares is April to September or October to May, Cuarto National University, and Buenos Aires University.
respectively. At the end of the breeding season, mares enter a state Mares were determined to be in deep anestrus by transrectal
of ovarian inactivity or seasonal anestrus. The goal of many per- ultrasound (Aloka SSD 500, Aloka Inc, Japan), examination of the
formance horse owners is to begin breeding mares in early reproductive tract and progesterone analysis. Specifically, mares
February in the Northern Hemisphere or early July in the Southern were selected based on their reproductive records to have no more
Hemisphere, a time when most mares maintained under ambient than 20% multiple ovulations per previous seasons, presenting
lights are not cycling. The practice of hastening the first ovulation of follicles <10 mm in diameter and no corpus luteum detected when
the year is carried out to produce early foals the following year that examined by ultrasound once per week for two consecutive weeks
have an economic advantage and competitive edge because of their prior to the start of the study. The initial examinations were per-
size and maturity over others born later in the year. Historically, an formed during the weeks of July 1e15th. Blood samples were
artificial lighting program has been used to advance the first collected once a week from each mare at the time of each pre-
ovulation of the year [7,8]. However, it takes 60e70 days to work liminary ultrasound examination. A third blood sample was taken
[9,10], is labor intensive and expensive, and not always effective or from each mare on July 15th, day of initial treatment.
utilized. Therefore, drugs have been utilized but have been less Forty mares were randomly assigned to one of the following
than consistently successful [11]. Pharmacological compounds have four treatment groups at each of the test facilities: Group 1: reFSH
included native gonadotropin releasing hormone (GnRH), GnRH at 0.65 mg IM twice daily (n ¼ 10), Group 2: reFSH at 1.3 mg IM once
agonists (buserelin, deslorelin, goserelin), dopamine antagonists daily (n ¼ 10), Group 3: reFSH at 0.32 mg IM twice daily (n ¼ 10), or
(domperidone, sulpiride), progesterone, progestins, prolactin, and Group 4: control, phosphate buffered saline (PBS) IM once daily
prostaglandins [11]. Disadvantages of these drugs that arose in (n ¼ 10).
previous studies were: the necessity of multiple injections, im-
plants or pumps for greater than 10 d, inconsistent success rates in 2.1.2. Recipient mares
different studies using the same drug, the necessity for follicles to The recipients at La Irenita Farm and the University of Buenos
be more than 20e25 mm in diameter, lower ovulation rates in Aires were clinically healthy anestrous mares between 3 and 15
deep-anestrous mares and return to anestrus after treatment [11]. years old and weighing between 900 and 1400 lbs. with no history
Equine pituitary extracts (EPE) and partially purified equine FSH of reproductive problems or were maiden mares. All mares were
(Bioniche Animal Health, Athens, GA) have been relatively effective maintained on mixed grass pasture and fresh water provided ad
in inducing superovulation in anestrous mares [12,13] and transi- libitum.
tional mares [14e17], respectively. However, embryo transfer uti- To synchronize the recipients with the donors, the mares were
lizing EPE preparations has been marginally successful when intramuscularly treated with estradiol cypionate (ECP Estradiol® -
carried out in deep-anestrous mares [18]. Treatment with EPE has Koning Laboratories, Argentina) during three consecutive days,
been suggested to have negative effects on embryo viability [19]. using decreasing doses of 10 mg, 6 mg and 4 mg beginning the day
The author is not aware of the use of eFSH treatment in a successful ovulation was detected in the donor mare. When mares showed
embryo transfer program in the deep-anestrous mare. Neither EPE evidence of endometrial edema by ultrasonography for at least 3
nor eFSH are commercially available. days, 1500 mg of long-acting progesterone (P4 LA300® - Labo-
Advancing the first ovulation of the year and inducing super- ratorios B.E.T., Rio de Janeiro, RJ, Brazil) were intramuscularly
ovulation at the same time in deep-anestrous mares under ambient injected on the fourth day.
lights has the potential for increasing embryo recovery and
decreasing the cost of embryo transfer. In the past few years, reFSH 2.1.3. Study design
has been shown to consistently induce superovulation followed by 2.1.3.1. Mare treatments. Mares were treated with reFSH in PBS
high pregnancy rates in both cycling and deep-anestrous mares given as an intramuscular (IM) injection according to their treat-
[20e22]. Recombinant eFSH was developed on a single chain ment group. Group 1 received 1.3 ml (0.65 mg) of reFSH twice daily
platform and is devoid of other hormones and contaminants (BID), 8 h apart; Group 2: received 2.6 mL (1.3 mg) of reFSH once
[23,24]. In one study, it was demonstrated that deep-anestrous daily (SID) in the morning; and group 3 received 0.65 mL (0.32 mg)
mares that did not get pregnant or lost their pregnancy kept of reFSH twice daily (BID), 8 h apart. Control mares (Group 4)
cycling or could be retreated successfully [22]. Recombinant eFSH received 1.3 mL of PBS as an IM injection once daily in the morning.
may be commercially available in the future. The objective of this Treatments were administered until either a) the mare developed a
present study was to determine the success rate of treating follicle 35 mm in diameter or b) for a maximum of 10 consecutive
seasonally deep-anestrous donor mares under natural ambient days. Ultrasound per rectum examinations were performed daily
 J.F. Roser et al. / Theriogenology 142 (2020) 291e295 293

during the morning treatment period. The diameter and position of 2.1.6. Statistical analysis
the largest follicle and all of those 30 mm on each ovary were Mean ± standard error of the mean (SEM) are presented in
recorded during each ultrasound examination. Mares detected with Table 1. Quantitative variables, including days of treatment, follicle
a follicle 35 mm in diameter during the treatment period were size, number of ovulations and number of embryos were analyzed
allowed to coast for 36 h and then subsequently administered 2,500 using unpaired Student’s t-test. Proportions, including ovulating
units of human chorionic gonadotropin (hCG;Ovusyn®- Syntex, mares, embryo recovery rate, and pregnancy rate, were compared
Buenos Aires, Argentina) intravenously (IV) to induce ovulation. using the chi-squared test. Analysis was performed in Excel 2016. A
Ultrasound examinations were performed and recorded once daily p-value of <0.05 was considered significant.
after detection of a follicle 35 mm in diameter to confirm the day
of ovulation. Mares that developed a follicle 35 mm in diameter 3. Results
and received hCG were bred every 48 h with fresh semen until
ovulation(s) were detected or with frozen semen (250  106 motile All mares were considered to be in deep anestrous based on the
sperm/dose) in the case of three mares, two in group 1 and one in presence of follicles <10 mm in diameter, no CL at ultrasound ex-
group 3. Eight days after the first ovulation, standard transervical amination and plasma concentrations of progesterone <1 ng/mL for
embryo recovery (see below) was performed and the embryos were three consecutive weekly examinations. All mares receiving reFSH,
graded according to McKinnon and Squires [25], harvested and but none receiving saline (Control), responded to the treatment
transferred (see below) to naturally synchronized or anovulatory with follicular growth, with all mares reaching ovulatory size
treated recipients. Pregnancy was diagnosed by ultrasonography 7, (>35 mm), except for two mares in group 3 that only achieved
14 and 21 days later. After embryo recovery, mares received 2 mL follicles of 20 and 25 mm size during their 10-day treatment period.
sodium cloprostenol (Estrumate®- Intervet, Germany) IM on two On average, 6.5 d of reFSH treatment were required for mares to
consecutive days. develop follicles of ovulatory size (>35 mm). There was no differ-
ence in the number of follicles greater than 35 mm in diameter
2.1.4. Embryo transfer among the groups. Ovulations were detected in 80% of mares in
2.1.4.1. Transcervical embryo recovery. Embryo recovery was per- Groups 1 and 2, 50% of mares in Group 3 and none in Group 4
formed by transcervical uterine flushes at day 8 after ovulation. The (Control). Of treated mares, 6, 7, and 5 produced embryos in Groups
flushing media was Ringer lactate sterile solution (average 2 L per 1, 2, and 3, respectively. Among ovulating mares, no differences in
mare). The embryos were evaluated using a stereomicroscope and number of ovulations, number of embryos recovered, or pregnancy
classified according size and morphology as described previously rates were observed among reFSH treatments. Combined, for mares
[25]. producing at least one embryo, the average number of embryos
recovered was 2.5 embryos/mare, with 6 mares producing 4 to 5
2.1.4.2. Nonsurgical transfer of embryos. Embryos were transferred embryos in a single flush. For the 0.65 mg dose BID, 1.3 mg dose SID
into a recipient mare between the third and sixth day after the first and 0.32 dose BID, embryo recovery rates per ovulated mare were
long-acting progesterone injection (P0). The mares were once more 75% (6/8), 88% (7/8), and 100% (5/5), respectively. Embryo recovery
treated with 1500 mg of long-acting progesterone the day of rates per ovulation were 37%, 47% and 45%, respectively. Consid-
transfer and every 10 days until 100 days of pregnancy. ering all the mares that produced at least one embryo, the embryo
recovery rate per ovulation based on number of ovulations per
2.1.5. Progesterone assay mare were, 59% for up to 4 ovulations (n ¼ 7 mares), 75% for 5 to 6
Concentrations of plasma progesterone were measured by a ovulations (n ¼ 6 mares), and 27% for more than 6 ovulations (n ¼ 5
validated RIA as previously described [19]. Tritiated progesterone (1, mares). Combined, mares with 1e6 ovulations had an embryo re-
2, 6, 7-3H-progesterone, NET381, specific activity 90e115 Ci/mmol; covery rate of 69% (n ¼ 13 mares). Overall, a 59% pregnancy rate was
Perkin Elmer Life Science, Boston, MA) was used as trace. Standards achieved with embryos collected and transferred during the
(Q2600; Steraloids, Wilton, NH) ranged from 0.1 to 20 ng/ml. Plasma anestrus season. Results are summarized in Table 1.
samples were diluted with PBS-G when necessary to fall within the
standard curve. The primary antibody was a sheep anit- 4. Discussion
progesterone-11alpha-hemisuccinate: BSA (1/13,000 dilution;
#8939 Stabenfeldt, UCD). Extraction efficiency for standards and This is the first study to present successful embryo production,
plasma were the same (75%), so no adjustments of the plasma values transfer and recipient pregnancy after treatment with reFSH from
using the extraction efficiency were necessary. The sensitivity of the deep-anestrous donor mares. All deep-anestrous mares adminis-
assay was 0.2 ng/ml and the intra- and inter-assay coefficients of tered reFSH under natural winter photoperiod exhibited marked
variation were 4.0% (n ¼ 6) and 8.3% (n ¼ 2), respectively. follicular development within 5e7 days after the start of treatment.

Table 1
Ovulation and embryo recovery rates of anovulatory mares treated with reFSH.

Dose of reFSH 0.65 mg BID 1.3 mg SID 0.32 mg BID Saline control

Days of treatment 5.9 ± 0.6a 7.1 ± 0.8a 7.3 ± 0.7a 10 ± 0b

Ovulating mares 8/10 8/10 5/10 0/10
Follicles >35 mm in diameter 5.2 ± 0.9a 6.4 ± 0.8a 4.1 ± 1.0a 0 ± 0b
Ovulations per ovulated mare 5.1 ± 1.0 5.5 ± 0.8 4.4 ± 0.9 -
Embryos recovered per ovulated mare 1.9 ± 0.5 2.6 ± 0.5 2.0 ± 0.4 -
Embryo recovery rate per ovulated mare 75% (6/8) 88% (7/8) 100% (5/5) -
Embryo recovery rate per ovulation 37% 47% 45% -
Embryos transferred 14 16 4 -
Pregnancy rate per transferred embryo 57% 56% 75% -

Values indicate mean ± SEM.

a, b: Different letters indicate p < 0.05.
294 J.F. Roser et al. / Theriogenology 142 (2020) 291e295

Ovulation rates were 80% for mares receiving 0.65 mg BID and ovulations. The oocyte recovery rate in EPE-treated mares with
1.3 mg SID and 50% for the mares receiving 0.32 mg BID. None of the three or fewer ovulations was the same as in untreated control
control mares ovulated. The follicular response and ovulation rates mares. The authors found that the ovulation fossa in the super-
for the higher doses (0.65 mg and 1.3 mg) were similar to that re- ovulated mares had large amounts of coagulated blood which was
ported in previous studies using EPE [12,18,26] in anestrous mares not observed in the control mares, and concluded that excessive
under natural photoperiod or partially purified eFSH [14,15,27] in hemorrhage in multiple-ovulating ovaries could be involved in the
transitional mares. Follicular response and ovulation rates were failure of the oocytes to enter the oviduct. Further refining of the
numerically lower in mares receiving a lower reFSH dose (0.32 mg protocol may increase embryo recovery rates. It is interesting to
BID), which is similar to what was reported for mares treated note that when LH was added to the treatment, there was an in-
during the breeding season with 0.35 mg reFSH BID [20]. Taken crease in embryo recovery per ovulation. Meyers-Brown reported
together these studies suggest that 0.35 mg BID is insufficient to that recovery after treatment with reFSH/reLH was 83% compared
induce a maximal follicular response. Also, the breeding season to 66% in cycling mares treated with reFSH alone [29]. The effect of
study indicated that 0.85 mg reFSH BID resulted in lower embryo LH on the ovulation fossa is unknown but it may enhance oocyte
recovery per flush compared to 0.65 mg BID, which in combination development and maturation [29]. Some of the variations in re-
with the current results indicates that a total daily dose of 1.3 mg of covery rates may be a function of whether the ovulations were from
reFSH, administered either as a single 1.3 mg injection or in two one ovary or both. Bilateral ovulations may prove to be a better
0.65 mg injections, is the optimal level of reFSH for inducing condition for the escape of oocytes through both ovulations fosses
follicular development in mares. It is important to note that giving resulting in higher embryo recovery per ovulation. The effect of
1.3 mg SID induced the greatest number of ovulations (5.5) sug- multiple follicular development and multiple ovulations on fertil-
gesting that a single administration of reFSH may be as good, if not ization rate, oocyte quality and early embryo development has not
better, than twice daily administration. been addressed, but given the altered hormonal environment
The number of follicles and ovulations per ovulated mare were resulting from multiple follicles/corpus lutea it is possible that
relatively high, especially considering that control mares showed these events may be affected.
no follicular activity during the treatment period. On average the Pregnancy rates in recipient mares are also quite variable
number of follicles that reached 35 mm in diameter was 5.2 and depending on the study. In a large study carried out by Jacob and
the number of ovulations was 5.0. Similarly, two previous studies coworkers (2012), in which donor mares did not receive any
carried out on deep anestrous mares given 0.65 mg of reFSH, the stimulatory agent, the average pregnancy rates for recipient mares
average number of follicles and ovulations were 4.0 and 3.6, (non-surgical transfer) between days þ1 to 3 was 69% [35]. The
respectively [21,22]. Moreover, the ovulations induced in deep- average pregnancy rate for recipient mares using the Wilsher
anestrous mares resulted in viable embryos, although the number technique (vaginal speculum and cervical grasping forceps) was
of embryos recovered was considerably lower than the number of 92.3% compared to 70.9% using the conventional technique [4].
ovulations. The average number of embryos recovered per ovulated Recipient pregnancy rate per non-surgically transferred embryo in
mare is comparable to the embryo recovery found in mares after mares that received eFSH was 33% compared to 67% in control
treatment with partially purified eFSH during the transition period mares [36]. Pregnancy rates in the present study were 56e75%
in two studies by Raz and coworkers (average: 2.2) [17,28]. Studies depending on the dose of reFSH received. Both recovery rates and
conducted during the breeding season also observed a similar pregnancy rates were quite acceptable considering the time of year
number of follicles, ovulations and embryos recovered after treat- this study was carried out. The ovaries of mares in deep anestrus
ment with reFSH [20,29]. were inactive with 10 mm in diameter follicles prior to the start of
Embryo recovery rates/ovulated mare in this study ranged from treatment. To be able to stimulate the ovary in 6e7 days is
75 to 100% depending on the dose of reFSH, which is higher than remarkable. Additionally, 14e16 embryos were transferred from
what is generally reported for non-stimulated cycles, ranging from superovulated mares compared to 0 in controls.
50% to 70% embryo recovery per cycle [30]. Similarly, treatment
with eFSH (Bioniche) resulted in a 90% embryo recovery rate/cycle 5. Conclusion
compared to 42% in mares receiving no treatment [31]. However,
embryo recovery rates per ovulation, which in this study ranged These results indicate that treatment with reFSH during deep
from 37 to 47%, are generally lower in superovulated mares anestrus results in follicular development, ovulation of fertile oo-
compared to non-stimulated mares. Logan and coworkers (2007) cytes, and production of embryos that established viable preg-
reported an embryo recovery rate per ovulation in cycling mares nancies after transfer. Also, a single daily administration of reFSH
that was lower after treatment with eFSH than in controls [32]. was as effective as two daily administrations, which allows for a
Speculation has it that the ovulation fossa prevents multiple oo- simplified administration regimen.
cytes from escaping the ovary into the oviduct [33]. In this study,
multiple mares produced 4-5 embryos in a single flush (n ¼ 6
Acknowledgements
mares), which indicates that mares are capable of ovulating at least
two fertile oocytes per ovary. The embryo recovery rates were high
Dr Jorge McDonoughy and Sebastian Bereterbide from La Irenita
(75%) for mares ovulating 5-6 follicles, which requires at least one
Embriones, Daireux, Argentina. AspenBio Pharma provided the
ovary undergoing a minimum of 3 ovulations. On the other hand,
reFSH used in this study.
mares with more than 6 ovulations presented greatly decreased
embryo recovery rates, suggesting that more than 3 ovulations per
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