Journal of Equine Veterinary Science 89 (2020) 102987

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Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Original Research

Historical Aspects of Equine Embryo Transfer

W.R. (Twink) Allen*, Sandra Wilsher
The Paul Mellon Laboratory of Equine Reproduction, Newmarket, Suffolk, United Kingdom
Sharjah Equine Hospital Reproduction Laboratory, Sharjah, United Arab Emirates

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

Article history: Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, En-
Received 1 November 2019 gland, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral
Received in revised form to the side of ovulation but the rates improved markedly when the whole uterus was flushed on real-
1 March 2020
ization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial
Accepted 2 March 2020
Available online 10 March 2020
transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the
cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%e90%
today. Experimental use of embryo transfer in horses and donkeys demonstrated the unique ability of
Keywords:
Embryo transfer
equids to carry to term a full range of interspecies hybrid conceptuses and extraspecies pregnancies
Interspecies created by embryo transfer. Furthermore, splitting of day 4e8 cell embryos and day 6 compact morulae
Extraspecies allowed the creation of genetically identical twin foals. But despite these and other significant advances
Placenta over the past 45 years, a persisting limitation is the relatively low embryo recovery rates from donor
Endometrial cups mares treated with exogenous gonadotropins in attempts to induce them to superovulate. This is due to
Equine chorionic gonadotropin (eCG) the toughness of the ovarian tunica albuginea which forces ovulation through the ventrally situated
ovulation fossa where multiple follicles compete with each other and luteinize before they can ovulate
properly.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction pregnancies from nonsurgical transfer in their first attempts [3],

Equine embryo transfer can be said to have begun in the early
1970s. The author (WRA) and his “boss”, Tim Rowson, at the
renowned Animal Research Station in Cambridge, adapted surgical
methods of embryo recovery and transfer developed previously
there in cattle and sheep to the horse and obtained a number of
pregnancies, including three from the transfer of interspecies
hybrid mule (\horse
_donkey) and hinny (\ donkey
_ horse)
embryos, into the opposite species of the dam of the embryo [1,2].
Simultaneously, a group of workers in Japan were using nonsurgical
methods to both recover and transfer horse embryos between
donor and recipient mares and although they failed to achieve any
Animal welfare/ethical statement: There are no ethical considerations as this is a
review and all data cited is from historic studies undertaken under UK Home Office
approval.
Conflict of interest statement: There are no conflicts of interest.
* Corresponding author at: W.R. (Twink) Allen, The Paul Mellon Laboratory of
Equine Reproduction, 18 Woodditton Road, Newmarket, Suffolk, CB8 9BJ, United
Kingdom.
E-mail address: twinkallen100@gmail.com (W.R. Allen).
they subsequently achieved a very encouraging 40% pregnancy rate
after transferring 15 day 6 morulae/early blastocysts to synchro-
nized recipient mares via a long needle passed through the anterior
fornix of the vagina and thence through the wall of the uterus,
thereby avoiding passage through the cervix which they assumed
had been the cause of their failure to achieve any pregnancies from
their first attempts.
Two major barriers existed in the 1970s which greatly limited
the early development of embryo transfer in horses compared with
the other domestic farm species such as cattle, sheep, goats, and
pigs. First, and perhaps most important at that time, the registra-
tion authorities for most pure “breeds” (rather than “types”) of
horses throughout the world, and very much led by the national
Jockey Clubs representing the Thoroughbred racehorse, would not
permit entry into their respective stud books of progeny conceived
by either artificial insemination or embryo transfer. Second, there
did not then, and nor does there still today, exist a practical and
efficient method for inducing superovulation in donor mares, as
existed for the female donors of the other farm species. But despite
these very real drawbacks, a number of significant advances in both
the methodologies and results of equine embryo transfer occurred

https://doi.org/10.1016/j.jevs.2020.102987
0737-0806/© 2020 Elsevier Inc. All rights reserved.
2 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987
during that early period and this paper attempts to review that
progress. But before doing so, it is pertinent to highlight some of the
unusual aspects of the estrous cycle and early pregnancy in the
mare which have impinged, both positively and negatively, on the
development and widescale use today of embryo transfer and
related embryo technologies in equids.
2. The Estrous Cycle of the Mare
The mare is a seasonally polyestrous animal with a distinct
breeding season during spring, summer, and early autumn. The
estrous cycle is normally 21e23 days in length, consisting of
16e17 days of luteal activity or diestrus and 4e6 days of estrus, the
duration of which differs markedly between individual mares and
is also influenced by season, being prolonged at the start of a
breeding season in early spring, shortest at the summer solstice,
and lengthening again in autumn [4,5].
Some 8e12 follicles >1 cm in diameter are usually present in the
ovaries on the first day of estrus, but only one of these, or occa-
sionally 2 or 3 in some breeds of horses, progress to ovulation. The
follicle reaches 3e5 cm in diameter before ovulating and is there-
fore readily palpable and ultrasonographically visible through the
wall of the rectum. Ovulation can only take place through the small
semicircular region on the ventromedial surface of the ovary
known as the ovulation fossa (Fig. 1) which is not covered by the
tough, fibrous tunica albuginea that envelops the rest of the ovary
[6] and it occurs usually 12e24 hours before cessation of the
behavioral signs of estrus [7]. Luteinization of the resulting corpus
hemorrhagicum occurs quickly so that peripheral plasma proges-
terone concentrations have generally risen to 1e2 ng/mL by
24 hours, and frequently reach as high as 3e5 ng/mL by 48 hours,
after ovulation. This rapid rise permits the use of daily plasma
progesterone measurements during estrus as an accurate method
for determining the day of ovulation for the purpose of embryo
transfer.
Pituitary gonadotropin release patterns in the cycling mare
differ markedly from those shown by the other large domestic
species. Serum FSH concentrations show a biphasic rise with a
pronounced mid-diestrous surge and a second, generally smaller,
peak which coincides roughly with ovulation [8]. Luteinizing hor-
mone concentrations on the other hand, remain baseline
throughout diestrus but rise steadily during estrus to reach a peak
1e2 days after, rather than before, ovulation. This prolonged release
Fig. 1. A sectioned horse ovary showing the preovulatory follicle adjacent to the
ventral ovulation fossa through which it will ovulate.
of LH during estrus in the mare contrasts sharply with the rapid,
more pulsatile release of LH that occurs some 24e30 hours before
ovulation in other farm species [9] and it is very likely responsible
for some of the twin and early diestrous second ovulations which
occur in some mares [10].
The marked variability which exists in both the duration of
estrus and in the occurrence of ovulation in relation to the onset of
estrous signs precludes any reference to estrus as a pivotal point on
which to calculate the age of embryos for recovery purposes. The
time of ovulation is the only meaningful parameter in this regard
and day 0 is usually taken as the day on which ovulation is first
detected transrectally as having occurred. However, because donor
mares were often only examined once daily by rectal palpation in
the 1970s, any estimate of the time of ovulation was subject to an
inherent error which could be as great as 24 hours. This was re-
flected in the considerable variation found in the stage of devel-
opment of horse morulae or blastocysts recovered on days 6e8 for
embryo transfer [11].
3. Synchronization of Ovulation
Tim Rowson, Bob Moor, and other colleagues at the Animal
Research Station in Cambridge demonstrated that the pregnancy
rate after surgical embryo transfer in sheep and cattle was highest
when ovulation was exactly synchronized in donor and recipient
animals, and began to drop off sharply when the degree of donor-
recipient synchrony exceeded 24 hours either way in the cow or
48 hours either way in the ewe [12]. Only the one published study,
that of Oguri and Tsutsumi [13], had seriously examined the
question of donor-recipient synchrony in relation to equine embryo
transfer. From the transfer of 79 horse blastocysts using two
different nonsurgical techniques, initial conception rates of 63%,
54%, 41%, 38%, and 0%, and live foal rates of 50%, 35%, 30%, 19%, and
0%, respectively, were obtained when the degree of synchrony
between recipient and donor mares was, respectively, 2, 1, 0, þ1,
and þ2 days. Although the situation was slightly confounded by the
use of two different transfer methods, and only half the number of
animals in the 2- and þ2-day groups compared with the other
three groups, the figures nonetheless gave a clear indication that a
degree of asynchrony as great as 48 hours or more was not disad-
vantageous in terms of pregnancy rate, provided the recipient had
ovulated after, not before, the donor.
In a more recent study [14] involving the nonsurgical transfer of
a total of 78 day 10 blastocysts to recipient mares which ranged
from 9 to þ 2 days with respect to the donor, pregnancy rates
remained high in recipients that had ovulated up to 4 days after, but
not before, the donor and 3 of 8 mares that were as much as 7 days
negatively asynchronous with the donor became pregnant
(Table 1). Interestingly, these 3 minus 7 day embryos slowed their
rate of development, as monitored ultrasonographically and from
measurement of the onset of equine chorionic gonadotropin (eCG)
secretion in the recipient mares, to the “ovarian stage” of the
recipient rather than that of the original donor (Fig. 2).
The critical period for recognition of pregnancy in the mare
occurs around day 12 after ovulation [15]. This important message
from the embryo to prevent luteolysis clearly involves suppression
of the normal, cyclical release of prostaglandin F2a by the endo-
metrium at the end of diestrus [16] and, surprisingly, it seems not to
be related to the large quantities of estrogens secreted by the
blastocyst from as early as day 10 [17e20]. It is conceivable that
handling, changes in temperature, and any other adverse condi-
tions which the blastocyst experiences during a nonsurgical re-
covery and transfer process may temporarily halt its development,
thereby enabling it to survive so well when transferred to a very
negatively asynchronous recipient uterus. Conversely, such delayed
 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987 3

Table 1
Influence of donor-recipient asynchrony on pregnancy rates and early embryonic loss rates after transfer of day 10 embryos.

Degree of Donor: Recipient Days After Ovulation of the No. of Recipient Mares Pregnant (%)
Asynchrony (Days) Recipient on Day of ET
2 Days Post ET 10 Days Post ET With Embryonic Heartbeat
a
þ4 14 3/6 (50%) 0/6 (0%) 0/6 (0%)a
þ2 12 6/6 (100%) 6/6 (100%) bc 6/6 (100%) bd
0 10 6/6 (100%) 6/6 (100%) bc 6/6 (100%) bd
1 9 6/8 (75%) 5/8 (63%) abc 5/8 (63%) abd
2 8 6/8 (75%) 6/8 (75%) bc 5/8 (63%) abd
3 7 6/8 (75%) 6/8 (75%) bc 6/8 (75%) bcd
4 6 8/8 (100%) 8/8 (100%) bc 7/8 (83%) bcd
5 5 6/8 (75%) 6/8 (75%) bc 6/8 (75%) bcd
6 4 5/6 (83%) 5/6 (83%) bc 5/6 (83%) bcd
7 3 8/8 (100%) 6/8 (75%) bc 3/8 (38%) abc
9 1 4/6 (67%) 2/6 (33%) ab 0/6 (0%)a
Control AI derived, non-ET pregnancies 9/9 (100%) 9/9 (100%)c 9/9 (100%)d

Abbreviations: AI, artificial insemination; ET, embryo transfer.

a,b,c,d
Values with different superscripts within a column are significantly different at P < .05.
Adapted from Wilsher et al [14].

development may prevent the blastocyst from liberating its
maternal recognition of pregnancy signal in time to prevent
luteolysis in an “advanced recipient”.
The development of efficient methods to control and synchro-
nize estrus and ovulation in the large domestic species provided a
keystone for the successful commercial exploitation of embryo
transfer in cattle [12]. The horse was no exception and the need to
be able to control ovulation in groups of donor and recipient mares
was perhaps even greater than in the cow because of the much
longer period of estrus and the marked individual variation be-
tween mares in the time from the onset of estrous behavior to the
occurrence of ovulation. The ability of a single injection of a rela-
tively low dose of prostaglandin F or various prostaglandin analogs
to induce rapid luteolysis in the diestrous mare [21,22] provided the
basis of the treatment regimens developed over the early years to
control ovulation in mares. Two injections of a prostaglandin
analog such as cloprostenol (Estrumate, ICI Pharmaceuticals Divi-
sion, Cheshire, UK), given 14 days apart to a group of randomly
cycling mares, resulted in >90% of them displaying estrus by 6 days
after the second injection (day 20) and approximately 80% of them
ovulating during a 4 day period between days 20 and 24 [23]. The
success of this double prostaglandin synchronization schedule,
with or without injection of human chorionic gonadotropin (hCG)
given on days 7 and 20 after the first prostaglandin treatment to
hasten ovulation in each prostaglandin-induced estrus, was
confirmed [24,25], and Eric Palmer showed that daily administra-
tion of the orally active synthetic progestagen, allyl trenbolone
(Regumate, Roussel Uclaf, Paris), to cycling mares for 10 days
accompanied by prostaglandin analog on the last day of progesta-
gen administration and hCG a further 10 days later, likewise
resulted in synchronized ovulation in over 75% of the treated ani-
mals [26].
4. Superovulation in the Mare
Equine chorionic gonadotropin, formerly called pregnant mare
serum gonadotropin, the high-molecular-weight glycoprotein
hormone which uniquely expresses both FSH-like and LH-like

Fig. 2. Peripheral serum progesterone (closed symbols) and eCG (open symbols) profiles in two recipient mares to which a day-10 embryo was transferred when they were only
3 days after ovulation to create a donor-recipient asynchrony of as much as 7 days. Note, how the eCG concentrations and the progesterone concentrations from the first secondary
ovulation do not begin to rise until conceptus age day 45 after ovulation, some 7 or 8 days later than normal.
4 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987
activities [27,28] remained the most widely used and effective
gonadotropin for stimulating ovulation in cattle, sheep, goats, and
pigs in the 1970s and 1980s. It was therefore somewhat ironic that
eCG, which is present in high concentration in the blood of mares
between 40 and 120 days of pregnancy [29,30], proved completely
ineffective with regard to stimulating follicle development when
given to cycling and pregnant mares, even in repeated doses as high
as 150,000 iu [7,31]. Studies by the late Francesca Stewart working
with the author [32,33] demonstrated that the gonadotropic re-
ceptors in equids, as distinct from those in other species, have
somehow evolved the ability to differentiate between the binding
properties of their own chorionic gonadotropin (eCG) and those of
their pituitary gonadotropins, FSH and LH. That such an unusual
mechanism allowing differential recognition of, and response to,
different types of gonadotropin molecules should have evolved in
the mare is perhaps not surprising when one considers that at the
time of peak eCG production around day 60 of gestation, a total
quantity in excess of 1,000,000 i.u eCG is likely to be circulating in
her blood. Hence, were such a protective system not to exist, her
ovaries would be hyperstimulated and “blown apart” each time she
became pregnantdas indeed can occur in the ovaries of a donkey
carrying either an interspecific hinny conceptus or an extraspecific
horse conceptus created by embryo transfer (Fig. 3; [31]). The
greatly increased ratio of FSH to LH found to exist in horse (1.4) and
hinny (0.8) CG compared with donkey CG (0.1) [28] was very likely
the cause of the marked differences in ovarian responses to these
variations in fetal genotype.
One early advance with regard to superovulation in the mare
came from experiments undertaken in Wisconsin [34] which
induced estrus and ovulation in 25 of 29 seasonally anestrous pony
mares by giving daily injections for 14 consecutive days of a crude
extract of equine pituitary glands. Based on rectal palpation of the
ovaries, the authors noted that 14 of the 25 treated mares which did
ovulate had two or more ovulations. Subsequently, the same group
[35] obtained a similar proportion of double and triple ovulations
when giving the same type of course of daily injections of equine
pituitary extract, with or without a subsequent injection of hCG, for
14 days to seasonally anestrous mares and for 6 consecutive days to
cycling mares, between either days 11e16 of diestrus or days 1e6 of
estrus. Once again, however, the ovulation figures were based on
rectal palpation alone and were not supported by any attempts at
embryo recovery. Furthermore, the repeated injections of a crude
pituitary extract caused the death of one mare and gave rise to
injection site abscesses of many of the others.
Fig. 3. Pregnant uterus and hyperstimulated ovaries in a Jenny donkey carrying a
transferred horse conceptus. One ovary has ruptured as a result of excessive growth of
secondary follicles and accessory corpora lutea stimulated by the high concentrations
of horse eCG secreted by the larger horse endometrial cups.
Subsequently, the same group [36] used a commercially avail-
able equine pituitary extract (Pitropin, Biological Specialties, Wis-
consin) to similarly treat cycling mares daily for 6 days during mid-
estrus or late diestrus/early estrus. As in the previous experiments,
ovulation rates in the treated and control mares were assessed by
rectal palpation only, and on this basis, 6 of the 8 treated mares
were considered to have produced between 2 and 4 ovulations each
during the estrus after treatment. However, when they were sub-
jected to nonsurgical uterine flushing between days 8 and 10 after
the induced ovulation, the embryo recovery rate was only 35% of
that expected based on ovulation rate in the 8 treated animals,
compared with 67% of that expected in 20 untreated control mares.
A subsequent study by the same group [37] found that the
conception rate was markedly reduced, compared with that in
untreated controls, in mares treated daily for 14 days with equine
pituitary extract to induce single and/or multiple ovulations early
in the breeding season (31% vs. 60%). In addition, only one
conceptus was still present at day 50 after ovulation in all 8 of the
27 treated mares that conceived during the estrus in which two or
more ovulations had been diagnosed per rectum.
The incidence of spontaneous twin ovulations in the mare is
strongly breed-dependent, ranging from as high as 20% in the
larger, more modern breeds such as the Thoroughbred, to zero in
smaller, more primitive breeds such as the Welsh Mountain Pony.
Although the Wisconsin studies demonstrated that administration
of equine pituitary gonadotropins in sufficient quantity could
induce some degree of multiple ovulations in a proportion of
treated mares, it became clear that this type of therapy would not
be advantageous for routine use in a commercial equine embryo
transfer program. The cost and impracticality of the treatment
regimen, the variability in response rates and the very significant
reduction in pregnancy rate in treated mares combined to
outweigh any possible advantages. However, the more recent and
encouraging results from attempted induction of multiple follicle
growth in donor mares by the administration of recombinant-
derived equine pituitary gonadotropins [38] may provide a useful
and practical regime for improving oocyte recovery rates when
undertaking ovum pick-up.
Back in the 1970s, the most practical solution to the failure of
superovulation was to increase the frequency of ovulation in
selected donor mares by repeatedly shortening their estrous cycle
with prostaglandins. In the author's laboratory, donor mares were
routinely injected with 250 mg of prostaglandin analog (Clopros-
tenol; ICI Pharmaceuticals Division) immediately after undergoing
nonsurgical embryo recovery on day 7 or 8 after ovulation. This
induced a return to estrus in 2e3 days when each mare was again
inseminated with the required semen and usually ovulated within
4e5 days. After a further 7 days, the mares were ready to be flushed
again and in this way the interval between successive embryo re-
covery attempts was shortened from the expected 21 or 22 days of
the normal estrous cycle to about 13e15 days. Repeated shortening
of the estrous cycle of donor mares in this manner may or may not
lower their fertility and from one pony mare flushed a total of 6
times during a period of 83 days in 1979, produced 5 normal
blastocysts. On the other hand, subsequent studies have indicated
that repeated embryo recovery attempts may induce deleterious
changes in the uteri of donor mares [39,40].
5. Surgical Embryo Recovery
Because the horse embryo does not enter the uterus until days
6e6.5 after ovulation [13,41], embryo recovery at any stage before
day 6 (late morula) necessitated entry into the abdomen via a
ventral midline laparotomy under general anesthesia followed by
flushing of the ipsilateral oviduct with 30e40 mL of medium. The
 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987 5

flushing technique was similar to that described for the cow [42]
although two factors complicated the process in the mare. First, the
ovarian ligament on the right side of the animal is considerably
shorter than that on the left so that, in nulliparous pony mares,
excessive traction was often necessary to exteriorize the right ovary
and oviduct sufficiently through a midventral incision to enable the
oviduct to be flushed. This interfered with the flushing technique
and carried the risk of rupture of the ovarian ligament with
resulting hemorrhage. Second, the uterotubal junction in the mare
takes the form of a firmly occluded papilla protruding into the
uterine lumen (Fig. 4) [43]. Although this permits fluids to flow
from the oviduct into the uterus, its very-effective valve-like action
completely prevented the reverse passage of fluid from the uterus
into the oviduct. At that time, it was not possible to achieve
retrograde oviduct flush by injecting medium into the uterine
lumen and then forcing it into the oviduct, as was carried out in the
cow.
Fig. 5. Illustration of the technique of surgical embryo recovery in equids. The rigid
The method used successfully for many years in the author's plastic cannula was held between thumb and forefinger in the ampulla of the oviduct
laboratory [1,2,31] involved the insertion of a 5e7 cm length of and the fluted end of the glass pipette was held also by digital pressure in the tip of the
firm, polystyrene cannula (5 mm O.D) into the dilated fimbria of the ipsilateral uterine horn adjacent to the uterotubal papilla.
oviduct. This was connected by a short length of flexible tubing to a
20 mL syringe containing the flushing medium. The cannula was
held in place in the oviduct and back-flow of fluid prevented by firm mares between 1 and 3 days after ovulation yielded 28 embryos
manual pressure. A 1-cm longitudinal excision was made in the between the 1- and 8-cell stages of development and 4 freshly
outer serosa and myometrium of the ipsilateral uterine horn about ovulated unfertilized oocytes. Many old, unfertilized oocytes from
3 cm posterior to the uterotubal junction. The endometrium was previous estrous cycles were also recovered in most flushes [46].
pierced with the blades of a pair of rounded scissors and the fluted
end of a short length of glass Pasteur pipette, previously shaped in a 6. Nonsurgical Embryo Recovery
Bunsen burner flame, was inserted into the incision and held firmly
against the uterotubal junction by manual pressure. The other end Certain features of the mare's reproductive tract make the
of the glass tube was tapered and connected to a length of soft whole process of nonsurgical uterine flushing much simpler than
rubber tubing which passed to the embryo collection dish (Fig. 5). its equivalent in the cow. The mare's cervix is relatively short and is
Some 30e40 mL of flushing medium [44] was forced through the more easily distended in diestrus than that in the cow, therefore
oviduct and collected into 2 or 3 embryo dishes. allowing the insertion of a much larger diameter catheter into the
Despite the impracticality of the method, a high recovery rate of uterus. Furthermore, the horns of the mare's uterus are not coiled
early-stage embryos and/or unfertilized oocytes could be achieved like those of a cow and they diverge from each other at a much
when flushing young, fertile mares [45]. For example, Allen and greater angle, thereby more easily permitting the passage of a
Rowson [2] obtained 23 embryos from 30 Welsh Pony mares (77% flushing catheter from the body of the uterus into each uterine
recovery rate) flushed between days 1 and 6 after ovulation. In a horn. Two nonsurgical flushing methods were used originally in the
subsequent experiment, surgical flushing of the left oviduct of 39 mare.

6.1. Single-Horn Flushing

A rigid instrument [3]; Fig. 6 [2]; or a flexible rubber or poly-

thene two-way catheter [11,47] was passed through the cervix into
the body of the uterus. With the operator's arm in the rectum the
catheter was then manipulated into the horn of the uterus ipsi-
lateral to the recently ovulated follicle so that the inflatable rubber
cuff was situated at the base of the horn. The cuff was inflated with
40e50 mL of air or water to isolate the lumen of the horn from the
remainder of the uterus (Fig. 7) and 50e60 mL of medium was
flushed rapidly into the horn using a catheter-tipped 60 mL syringe
attached directly to the free end of the catheter. While massaging
the fluid-filled uterine horn per rectum, the medium was then
either positively withdrawn into the same syringe or was allowed
to flow back out of the catheter into a suitable collecting vessel. The
whole process was repeated twice.

6.2. Whole Uterus Flushing

This method soon replaced the ipsilateral horn only flush

[13,36,48,49] and it simply involved passing the flushing instru-
ment, either the rigid instrument [46] or the flexible two-way
Fig. 4. The tightly closed and protruding uterotubal papilla or junction (UTJ) at the catheter with inflatable cuff through the cervix into the body of
anterior tip of a uterine horn in a normal diestrous mare. the uterus. The cuff was inflated and a slight backward tension
6 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987

Fig. 6. The older method of single uterine horn embryo nonsurgical embryo recovery
using a rigid, metal, 3-way flushing apparatus manipulated into the base of the ipsi-
lateral uterine horn with the operator's arm in the mare's rectum.

maintained on the catheter so that the cuff was kept adjacent to, and
hence occluded, the internal os of the cervix (Fig. 8). The flushing
medium (500e1000 mL of phosphate buffered saline containing 5%
v:v bovine serum albumin) was run into the uterus under gravity
until the lumen was distended (Fig. 8) and it was then allowed to run
back out into a suitable collecting vessel while the operator
massaged the uterus per rectum. The whole process was repeated
two or three times so that, eventually, a total in excess of 2 L of
Fig. 8. The whole uterus, nonsurgical, embryo flushing technique using the flexible
two-way 18-FG catheter shown in Fig. 7 inserted into the body of the uterus and
withdrawn so that the inflated cuff occluded the internal os of the cervix. Around
500 mL of flushing medium was run into the uterus and recovered by gravity while the
operator continued to massage the uterine horns per rectum to “flush” the embryo out
from between folds in the endometrium.

medium had been infused into and recovered from the uterus in
three stages. Because embryo filters did not exist in these early days,
horn flushes of pony mares and Jenny donkeys which had been
the medium was collected back into 500 mL measuring cylinders
covered naturally by either a fertile horse stallion or a fertile Jack
which were allowed to stand on the laboratory bench for
donkey, yielded 26 intraspecies and interspecies hybrid embryos to
15e20 minutes to enable the embryo to sink to the bottom of the
give an overall recovery rate of only 48%.
cylinder. The excess flushing medium was carefully aspirated or
In their later article, Oguri and Tsutsumi [13] reported greatly
poured off and the remaining 20e30 mL placed in a concave embryo
improved embryo recovery rates when using a similar, rigid three-
dish and searched for the embryo using a dissecting microscope.
way flushing instrument passed only through the cervix so that the
The whole uterus flushing technique, although it used consid-
whole uterus was flushed with a total of 1500 mL of medium in
erably more medium, immediately doubled the embryo recovery
three “lots”, each of 500 mL. In their first experiment they recov-
rate compared with the ipsilateral single horn method. In earlier
ered 18 blastocysts from 20 attempted flushes of pony and half-
studies [2,3] using the original rigid flushing instrument (Fig. 6) to
breed mares between 6 and 7 days after ovulation and in the
flush only the ipsilateral uterine horn in fertile pony mares, embryo
subsequent much larger study [48] involving the recovery of a total
recovery rates of only 49% and 40%, respectively, were achieved.
Likewise, Tischner [11] recovered only 11 embryos from 21 mares
(52%) by the ipsilateral horn method and, in the author's laboratory
during the 4-year period from 1976 to 1980, a total of 54 single-

Fig. 7. The infusion of 60e100 mL of the flushing medium into the ipsilateral uterine Fig. 9. Surgical transfer of a horse embryo via midline laparotomy using a Pasteur
horn via a catheter-tipped syringe connected to the flexible 18-FG catheter with its pipette passed through a hole punched in the uterine horn by a blunted 18 gauge
inflatable cuff occluding the exit from the ipsilateral uterine horn into the uterine body. needle.
 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987
Fig. 10. Genetic drift in the nursery. Donkey (2n ¼ 62), Przewalski horse (2n ¼ 66), and
a common zebra (2n ¼ 44) foals with their recipient horse (2n ¼ 64) mothers.
of 148 embryos from 200 attempted flushes between days 6 and 9
after ovulation, they made a direct comparison between the ipsi-
lateral uterine horn and whole uterus flushing methods. While the
recovery rate from the former was only 44%, it rose to 87% when the
whole uterus was flushed.
The whole uterus flushing method was also adopted in the
author's laboratory during the 1981 breeding season when
attempting to recover donkey blastocysts for surgical transfer to
mares. The same flexible plastic, 18 French gauge human degusta-
tion tube catheter used for single-horn flushes (Fig. 8) was used and
three “lots”, each of 300e500 mL, of medium were infused into the
uterus under gravity and allowed to flow out again while manip-
ulating the uterus per rectum to aid the fluid expulsion. In this
manner, a total of 27 donkey and 8 horse blastocysts were recov-
ered from the attempted flushing of 37 Jenny donkeys and 11 pony
mares between days 6 and 8 after ovulation, to give an overall re-
covery rate of 72% which compared favorably with the average
recovery rate of 40%e50% obtained during the previous 4 years
when using the ipsilateral horn flushing method. A similar recovery
rate was reported by Douglas [36] who obtained 14 embryos from
the attempted whole-uterus flushing of 20 pony mares between
days 8 and 16 after ovulation.
From the available figures, and especially those provided by the
Japanese workers, it was apparent that whole uterus flushing gave a
marked improvement in embryo recovery rate to that achieved by
flushing only the ipsilateral uterine horn [49]. And when Butterfield
and Matthews published their excellent article in 1978 describing
intrauterine migration of the equine conceptus, it became generally
realized that a high rate of transuterine migration of the conceptus
Fig. 11. Two-day old (A) donkey and (B) horse foals with their respective extraspecies recipient horse and donkey mothers. Note the large size of the donkey foal having benefitted
from gestation in the larger horse uterus.
7
(±50%) occurs spontaneously in all breeds of horses [7,50,51] with a
strong likelihood in lactating mares for the conceptus to implant in
the uterine horn contralateral to the implantation side of the pre-
vious conceptus [52]. Furthermore, if the blastocyst was destined to
migrate for whatever reason, it appeared to do so very soon after
entering the uterus on day 6 after ovulation, so it has already vacated
the ipsilateral horn by day 7 or 8. Transrectal ultrasonography and
intrauterine videoendoscopy have since shown convincingly that
the equine conceptus moves constantly throughout the entire
uterine lumen between days 6 and 16 after ovulation [53].
In due course, a more precise timing of 144e168 hours for the
entry of the embryo into the uterus was determined [54], and it was
also reported at the other extreme that larger embryos flushed at
day 9 or later were more likely to be damaged during collection and
transfer [55,56]. Hence, nowadays, in a clinical setting, most em-
bryos destined for transfer to recipient mares are recovered for
embryo transfer on day 7 or 8.
7. Embryo Transfer
7.1. Surgical Transfer
The method used for surgical transfer of embryos in the mare
was very similar to that described in cattle [42]. The ipsilateral
uterine horn was exteriorized through a mid-ventral laparotomy
incision and a small hole made in the wall of the uterus and
endometrium by forcing through a blunted 18 gauge needle, the tip
of which was filled with solder and rounded off on a grindstone.
The embryo was taken up in a minimal volume of medium
(0.05e0.01 mL) in the tip of a Pasteur pipette attached by a short
length of rubber tubing to a 1 mL tuberculin syringe. The tip of the
pipette was eased through the hole in the uterine wall (Fig. 9) and
passed posteriorly in the uterine lumen for 2e3 cm before gently
expelling the embryo.
Surgical transfer of equine embryos gave high pregnancy rates,
provided the transfers were carried out >3 days after ovulation. In
earlier experiments, the author and Tim Rowson [2] obtained
pregnancies in only 7 of 16 mares (44%) to which a single embryo
was transferred surgically between days 1 and 6 after ovulation;
embryos recovered on days 1e3 were transferred to the ipsilateral
oviduct, whereas those recovered on Days 4e6 were transferred to
the ipsilateral uterine horn. However, analysis of the results
showed that whereas only one of 8 embryos transferred on days
1e3 continued to develop, 6 of 8 (75%) transferred to the uterus on
days 4e6 gave rise to pregnancies that were still ongoing at 42 days
after ovulation.
Once the basic methods of nonsurgical recovery and surgical
transfer of embryos was established in the author's laboratory in
8 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987

Fig. 12. The elongated, toothed, Wilsher forceps used to grasp and straighten the recipient mare's cervix to facilitate nonsurgical embryo transfer.

the 1970s, relatively few intraspecific transfers between horses
were carried out, the great majority involving the transfer of
interspecific hybrid mule or hinny embryos to a synchronized
recipient of the opposite species (donkey and horse, respectively).
Subsequently this was extended to include reciprocal extraspecies
transfer of horse and donkey embryos to unmated synchronized
recipients of the other species and it also included transfer of
Przewalski's Wild Horse (2n ¼ 66) and Common Zebra (2n ¼ 44)
embryos of domestic horse recipient mares (2n ¼ 64; Fig. 10). Yet
despite the xenogeneic nature of these transfers, the conception
rate remained relatively high. For example, during 1979e1981, in-
clusive surgical transfer of 8 horse embryos to unmated Jenny
donkeys resulted in 5 pregnancies (62%) and transfer of a total of 34
donkey embryos to unmated pony mares during the same 3 year
period resulted in 24 pregnancies (71%; Fig. 11; [31]).
In these original studies ventral midline laparotomy under
general anesthesia was used and this remained the method of
choice for the author (WRA) until nonsurgical embryo transfer
methods gave equivalent pregnancy rates. However, midline lapa-
rotomy was not the only surgical method and other research fa-
cilities were transferring embryos through the flank using local
infiltration anesthesia in the standing, sedated recipient mare [57].
7.2. Nonsurgical Transfer
This was a very straightforward technique in the mare whereby
the embryo, in 0.2e0.5 mL of medium, was drawn into the lumen of
a rigid plastic cattle insemination pipette or similar device using a
syringe attached to the other end of the pipette. A 1e2 mL cushion
of air was drawn into the pipette before the medium to ensure
complete expulsion of the medium and embryo when the plunger
of the syringe was depressed. With a gloved hand protecting the
end of the pipette as it passed into the vagina, the operator's index
finger was used to open the external os of the cervix and direct the
catheter into the uterus. The embryo was then deposited either in
the body of the uterus or, by manipulation with the operator's free
hand in the rectum, in the ipsilateral horn.
Just as nonsurgical recovery of equine embryos gave rise to
marked differences in recovery rates between research groups, so
the conception rates resulting from nonsurgical transfers varied
considerably. In their original study, Oguri and Tsutsumi [3] failed
to obtain any pregnancies when transferring 10 blastocysts and one
morula via the cervix to the ipsilateral horn of precisely synchro-
nized recipient mares. In their later study, however, they achieved a
40% pregnancy rate after the nonsurgical transfer of 15 day 6
blastocysts to synchronized recipient mares via a long needle
passed through the anterior fornix of the vagina and thence
through the wall of the uterus, therefore avoiding passage through
the cervix, all as described previously. In pony mares, Allen and
Rowson [2] obtained five pregnancies from the nonsurgical transfer
of 7 blastocysts via the cervix. Also, as described previously, transfer
was carried out using a disposable plastic cattle insemination
pipette and the embryo was deposited in the body of the uterus
without any attempt to direct the pipette into either of the two
uterine horns.
In the later Japanese study [48], 82 blastocysts were transferred
nonsurgically to unmated recipient mares. Of these, 45 were
transferred transcervically as described previously and the
remaining 37 were transferred via the cervix and deposited in the
middle of the uterine horn ipsilateral to the previous ovulation after

Fig. 13. (A) Passing the Wilsher forceps into the vagina of the recipient mare facilitated by a duck-billed vaginoscope and visualization using a pencil torch. (B) The cervix has been
grasped by the Wilsher forceps and pulled backward to straighten the cervical canal and so allow easier passage of the insemination catheter carrying the embryo in 2e3 mL of
medium through into the body of the mare's uterus.
 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987
Fig. 14. A recipient mare and its Welsh pony transferred foal in Krakow, Poland, after
transport to Poland as a day 6 embryo in the oviduct of a rabbit.
positioning the pipette by manipulation per rectum. No significant
difference in conception rate resulted from the two different
transfer methods, the figures being 42% for transcervical transfer
and 44% by intracervical transfer. However, some pregnancy losses
occurred in both groups of recipient mares such that the initial
figure of 42% for overall conception rate was reduced to 33% in
terms of the birth of live foals.
As the desire to undertake nonsurgical embryo transfer
increased in the 1980s, much work was undertaken at other labo-
ratories, in particular, Colorado State University in Fort Collins,
University of California in Davis and Unite  Physiologie de la
Reproduction et des Comportements (INRA), Nouzilly, France, to
hone the nonsurgical technique. This included improving knowl-
edge regarding the synchrony of recipients, the use of and phar-
macological manipulation of noncyclic recipients, and elucidating
factors affecting pregnancy rates and embryonic death after
nonsurgical embryo transfer [58]. Much of this work was also
presented at the now established 4-yearly International Sympo-
sium on Equine Embryo Transfer, the first of which was held in New
York in 1984 under the sponsorship of the Dorothy Russell Have-
meyer Foundation.
Over the subsequent years as pregnancy rates improved, intra-
cervical embryo transfer became the norm with some European
Fig. 15. Two pairs of genetically identical twin foals produced by embryo bisection. (A) “Romulus” and “Remus” being held by the author and Mr Peter Burrell CBE, architect and the
Director of the British National Stud in Newmarket. (B) “Ophelia” and “Desdemona” with their recipient mothers at the Equine Fertility Unit in Newmarket.
9
countries even banning the use of surgical transfer. Nonsurgical
transfer methods typically involve transfer of the embryo held in a
minimum volume (0.1e0.3 mL) of medium in a 0.25 or 0.5 mL
semen straw inserted into a rigid embryo transfer gun passed
through the cervix via the operator's gloved hand has become the
worldwide norm and, depending on the experience and resulting
expertise of the operator, gives pregnancy rates of 75%e80% in
well-synchronized recipient mares. However, a more recent
modification of the method involving insertion of an autoclaved,
duck-billed speculum into the mare's vagina provides clear visu-
alization of the cervix, which in turn, enables insertion of a pair of
elongated, toothed Wilsher forceps (Fig. 12) to grasp the external
cervical os and pull the cervix backward to “straighten” its lumen.
This then allows the embryo handler to pass an insemination
pipette containing the embryo in a much larger volume of medium
(2e3 mL) easily through the cervix to deposit the embryo in the
body or horn of the uterus (Fig. 13). The great advantage of the
Wilsher forceps method is the insertion of only sterilized in-
struments into the vagina and through the cervix to maintain ste-
rility and ease of passage through the cervix. As a result, the
technique routinely achieves pregnancy rates of >90%, even when
performed by even relatively inexperienced operators [59,60].
7.3. Storage, Transport, and Manipulation of Equine Embryos
Little was published about the storage and transport of equine
embryos in the early days. In 1975, the author and colleagues [61]
recovered 6 morulae surgically from Welsh pony mares at the
Animal Research Station in Cambridge and transported them by car
to Krakow, Poland, in the ligated oviducts of two rabbits, the
journey lasting 33 hours. Five of the embryos had continued to
develop normally in the rabbits and four of these were transferred
(3 surgically and 1 non-surgically) to Polish recipient mares in
which ovulation had been synchronized to within þ-48 hours by
the prior use of prostaglandin and hCG; the fifth embryo was
accidently lost. Three pregnancies developed from which one foal
was aborted at day 292 of gestation and 2 colt foals were born live
at term (Fig. 14).
Early attempts in Cambridge to freeze surplus equine morulae
and early blastocysts proved unsuccessful. A freezing method used
routinely for sheep embryos [62] was used and although the em-
bryos appeared viable and relatively undamaged when thawed,
transfer of 9 of them to suitable recipients failed to result in any live
foals; 8 mares were suspected of having become pregnant but no
conceptus developed beyond 40 days of gestation. The resistance of
10 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987

pellucida. These were then embedded in a small chip of agar gel

Fig. 16. A 43-day donkey-in-horse conceptus showing the poorly developed donkey
chorionic girdle which had failed to detach and invade the horse maternal endome-
trium at days 36e38 to form eCG-secreting endometrial cups.
the mare to superovulation and the resulting difficulty in obtaining
sufficient numbers of embryos for investigation severely limited
the amount of useful experimental study that could be applied to
determining the optimum cooling and freezing requirements of
equine embryos.
During 1979 and 1980, eight 2- to 8-cell horse embryos, recov-
ered surgically from the oviducts of Welsh pony mares between
days 1 and 3 after ovulation had their zonae pellucida removed and
the individual blastomeres were reconstituted in varying pro-
portions of the original cell number in empty porcine zonae
and the whole placed in the ligated oviducts of anestrous ewes for
3e5 days as described by Willadsen [63]. Of the total of 20 divided
embryos made in this manner and placed in sheep, 18 (90%) had
continued to develop normally to the morula or blastocyst stage
when subsequently recovered from the ewe; these were trans-
ferred surgically to the uteri of synchronized recipient mares, some
of which had been the donor of the original embryo 4e5 days
previously. A total of 10 pregnancies resulted and in five instances
only one half of the original embryo continued to develop, the other
half failing to progress in its recipient mare; in all three cases where
the donor also served as the recipient for one half of its manipu-
lated embryo, pregnancy did not result. Two sets of monozygotic
twins were born alive, one pair of colts in 1980 and a pair of fillies in
1981 (Fig. 15). Both these sets of identical twins originated from
separation of the two 4-cell embryos into 4 one-cell embryos. With
the first of these, only 3 of the 4 reconstituted quarter embryos
continued to develop in the sheep and were, therefore, retrans-
ferred to mares. With the second manipulation, all 4 reconstituted
embryos progressed to the morula/blastocyst stage in the ewe and
were transferred to recipient mares. Although 3 pregnancies
commenced, one of these failed between 43 and 51 days of gesta-
tion [64].
These preliminary experiments demonstrated quite clearly that
the equine embryo like that of the ewe [63] and cow [65] remains
pluripotent until at least the 8-cell stage and may not be destroyed
by manipulation and a reduction in overall cell number. Because
the chances of live births of spontaneously occurring identical twin
horses is extremely remote, embryo micromanipulation could
provide a useful tool to produce multiple monozygotic equine

Fig. 17. Depicting the recovery of a day 6 mule embryo from its horse mother followed by its bisection using a micromanipulator and transfer of the resulting demi-embryos, one to
a horse and the other to a donkey recipient. (A and B) The small and typically mule-like endometrial cups in the endometrium of the horse recipient (A) compared with the much
larger and hinny-like endometrial cups that developed from the other demi-mule embryo transferred to the donkey recipient (B) and which produced much higher eCG con-
centrations in maternal blood (C) thereby demonstrating clearly the profound influence of maternal uterine environment on endometrial cup development.
 W.R. Allen, S. Wilsher / Journal of Equine Veterinary Science 89 (2020) 102987
offspring that would be genetically identical and therefore immu-
nologically compatible. These would have obvious potential value
as experimental animals.
7.4. Interspecies Embryo Transfer
Equidae is one of the few genera of mammals in which mating
between different member species with widely differing chromo-
some complements can result in the birth of live hybrid offspring
[66]. For example, a Mongolian Wild Horse with a karyotype of 66
could be inseminated with semen from a Cape Mountain zebra
with a karyotype of only 32 and produce a live hybrid foal [67].
Allen and Rowson [1] originally reported the successful transfer
of an interspecies mule embryo to a Jenny donkey and, vice versa, a
hinny embryo to a horse mare and subsequent attempts to transfer
intraspecific embryos between horses and donkeys were surpris-
ingly successful [31]. From a total of 8 horse embryos recovered
nonsurgically at the blastocyst stage and transferred surgically to
the uteri of recipient Jenny donkeys, 5 established pregnancies
resulted. Two of these pregnancies were subsequently terminated
surgically on days 63 and 71 of gestation for gross and histological
examinations of the endometrial cups and other fetal tissues, one
conceptus was aborted spontaneously on day 82 and the other two
were carried normally to term and born live at 346 and 364 days of
gestation, respectively ([31]; Fig. 11). And with extraspecies trans-
fers, for example, a donkey embryo transferred to a horse recipient,
an even higher success rate was achieved. For example, transfer of a
total of 34 donkey embryos into synchronized recipient horse
mares resulted in 24 established xenogeneic pregnancies, giving a
transfer success rate of 71%.
The horse conceptuses in Jenny donkeys gave rise to large and
active endometrial cups with resulting high concentrations of eCG
in the maternal circulation and they seemed readily able to implant
and continue normally to term. This was not the case with the
donkey conceptuses transferred to mares, however, and of the 24
such pregnancies established, only one resulted in the birth of a live
foal on day 346 (Fig. 11). In 12 other cases, abortion or resorption of
the already dead conceptus occurred between 47 and 105 days of
gestation and in the remaining 11 recipient mares, surgical removal
of the conceptus between days 60 and 87 showed definite evidence
of placental and fetal damage resulting from a generalized maternal
cell-mediated immune attack against the xenogeneic conceptus. In
marked contrast to the “horse-in-donkey” pregnancies, none of the
“donkey-in-horse” pregnancies showed any endometrial cup
development as a result of failure of the donkey chorionic girdle to
invade the horse endometrium between days 35 and 40 of gesta-
tion (Fig. 16).
The marked influence of uterine environment on development
of the equine conceptus was perhaps best shown by bisecting a day
6 mule morula and transferring one demi-mule embryo back to a
recipient horse mare and the other demi-mule embryo to a recip-
ient Jenny donkey (Fig. 17; [68]). Both conceptuses developed
normally until they were removed surgically at 60 days of gestation.
While the mule-in-horse pregnancy showed the typical small and
already necrotic mule endometrial cups (Fig. 17A) that produced
low concentrations of eCG in the maternal circulation (Fig. 17C), the
mule-in-donkey pregnancy showed much larger, still-active
endometrial cups (Fig. 17B) that had secreted very high concen-
trations of eCG into maternal blood (Fig. 17C). Thus, in the “wrong”
uterus, the mule conceptus developed like a hinny pregnancy.
8. Conclusions
Many features of the reproductive anatomy and physiology of
the mare make the technique of embryo transfer simpler and more
11
useful in equids than it is in other large domestic species. The
equine blastocyst is robust and reasonably undemanding in its
environmental requirements. It is easily recovered by nonsurgical
means and it survives well if transferred correctly, even in an
imperfectly synchronized recipient uterus. These features, coupled
with the desire to reproduce a whole variety of characters of
excellence in the various breeds of horses has driven the rapid
development and wide-scale application of equine embryo transfer
in recent years after the rather slow and uncertain start in the 1970s
and early 1980s. But a major stumbling block remains. Namely, the
resistance of the mare's ovaries to stimulation by exogenous go-
nadotrophins, combined with the unusually tough tunica albuginea
which surrounds her ovaries and limits ovulation and release of the
oocyte to the ovulation fossa, thereby preventing any synchronous
ovulation of multiple follicles such as can be achieved successfully
and repeatedly in other domestic animal species ranging from the
mouse to the cow.
Acknowledgments
The senior author (WRA) expresses his profound gratitude to
the late Thaddeus Mann FRS, Tim Rowson FRS and Cyril Adams, and
to Bob Moor FRS, all of the now defunct Animal Research Station in
Cambridge, for their kind practical support and wonderful aca-
demic stimuli nearly half a century ago.
The historic research detailed in this paper was supported by
funding from the Thoroughbred Breeders’ Association, the Horse-
race Betting Levy Board and the Dorothy Russell Havemeyer
Foundation of America.
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