1.
Introduction
The use of embryo transfers
and development of embryo technology for the horse have increased steadily over
the past two decades [1].
Embryo Transfer (ET) is now generally accepted as a valuable tool for
increasing the number of progeny from genetically valuable mares, for producing
foals from competing mares without interrupting their sporting careers, and for
obtaining foals from mares incapable of carrying a pregnancy to term [2]. Furthermore, ET can be performed on yearling or 2 years old mares as
a method to get them into production 1
or 2 years earlier than traditional method [3]. Embryos are usually collected on day 6, 7 or 8 (day 0 is day of
ovulation) from naturally single
or occasionally multiple ovulations. Embryos are collected 1 day later if the
mare has been bred to frozen semen rather than fresh or cooled semen [4]. Recovery rates for days 7, 8 and 9 post ovulation are similar, but recovery of embryos
on day 6 slightly lower which attributed to failure to identify the embryo in recovery
medium, loss of embryo during recovery procedure due to its small size, failure
to obtain the embryo in the uterine flush because of its greater specific
gravity or failure of some embryos to enter the uterus by day 6 [5].
The major factor affecting embryo recovery is the mare's reproductive history.
Older mares with poor reproductive histories produce fewer embryos. Causes of
reduced embryo recovery from these older mares include uterine and oviductal
pathology and increases early embryonic death [6].
The majority of embryo transfer has been performed 6 to 8 days’ post ovulation.
Limited studies are available addressing the effect of embryo age on pregnancy
rate [5,7-9]. But in recent years there has been
a tendency by some veterinarians to flush mares on day 8 or 9 so that the embryo
may be visualized in the filter, enabling the flush to be stopped after just
one flush rather than three that are recommended. This practice can easily leave
a twin embryo in the uterus [10]. However, the team
in Goulburn Valley Equine Hospital (GVEH) produced a foal that transferred at
10 days old. Once they identify the mare pregnant using ultrasonography, they
then harvest and transfer the embryo. The catheter used to flush the mare is
necessary large [4,11] reported no pregnancies
after transfer of day 9 and 10 embryos, whereas pregnancy rates of 61%, 55%, and
25% were observed after of days 5, 7, and 8 embryos, respectively.
Conflicting results were found by Fleury et al (1989) [12] who achieved 69% pregnancy rate after transfer of
16 embryos collected 9 days’ post ovulation. It is noteworthy that these two
studies were performed while the procedure of equine embryo transfer was still
under development and had not been in practice for a long time, which could
have contributed to the divergent results. In recent study that carried out by
Jacob et al (2012) [13], they demonstrated that
embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g.,
63%), the degree of synchrony between donor and recipient mares does not need
to be as restricted as previously reported in horses. Acceptable pregnancy
rates (e.g., 70%) were obtained even when recipient mares ovulated 4 to 5 days
after the donors. Similar pregnancy rates were obtained when recipient mares
received embryos within a large range of days’ post ovulation (Days 3 to 8),
and Day 7 embryos produced higher pregnancy rates when compared with Days 8 and
9 embryos. There are few studies about transfer of 10 days’ embryo like that
carried out by Wilsher et al (2010) [14] who
mentioned that transfer of day 10 embryos to asynchronous recipient mares could
benefit the management of commercial equine transfer programs and may provide a
useful model for investigation of maternal influences on early embryogenesis.
Although they mentioned that the problematic aspect of the transfer of large
day 10 embryos is the use of transfer pipettes with an internal diameter ≥ 5 mm; the capillary forces required to hold the
embryo in its transfer medium in set columns within the pipette do not come
into play in these larger bore pipettes. Furthermore [14],
couldn't recover embryos ˃ 5 mm despite repeated times of flushing. Nearly all
embryos are transferred nonsurgically into synchronized recipients. For nonsurgical
transfer, embryos are loaded into 0.25- 0.5 ml straws and inserted into the
body of the uterus using an embryo transfer gun. Alternatively, the embryo is
loaded into a flexible insemination rod, which is covered with a protective
sheath and placed through cervix into the body of the uterus [1].
2.
Materials
and methods
2.1. Embryo Donor Mares
This study was carried out during (September, 2015- October 2016). The
donor mares were sub fertile mares with a history of low embryo recovery rates.
The donor mares (N= 36) were straight Egyptian mares weighing between 350-400
kg and between 7-24 years old. In addition to 6 mares were kept as a control.
The mares were fed green fodders mixed with hay, and were given barely, access
to mineral salt and fresh
water was provided ad libitum.
2.2. Ultra Sonographic Scan
Transrectal ultrasonography was applied for donor mares for determination
of proper time of insemination as well as for detection of ovulation time
(using 7.5 MHz linear endorectal probe, MYLAB 30, Italy
and Sonoscape; China)
2.3. Insemination and Breeding Management
Natural covering has been done for some mares and others have been freshly
inseminated from the stallions kept in the same ET facility. These stallions
were examined periodically for assessment of semen quality. All mares were
treated for infertility problems (using of ecbolics, uterine lavage,
intrauterine antibiotics and corticosteroids) for increasing embryo recovery
rates.
2.4. Embryo Recipient Mares
Recipient mares (N= 70) were kept in the ET facility as a recipient population
for our facility and for shipped embryos. They were between 5- 10 years old and
weighing between 350-450 kg. All mares were fed green fodders, hay and
concentrate. Estrous cycles were monitored using transrectal palpation and
ultrasonography to synchronize ovulation between the donor and recipient mares.
The degree of synchronization was-4 to-6 days (recipient ovulated 4-6 days
after donor). The degree of synchrony was achieved using Cloprostenol
(Estrumate; MSD, Animal Health) as a luteolytic agent, and 1500 IU of hCG
(Epifasi; EPICO, Egypt) as an ovulatory inducing agent.
2.5. Collection and Evaluation of Embryos
Beginning from day 9 post ovulation, the uterus of donor mare was scanned
daily using 7.5 MHz trans rectal ultrasound probe (MYLAB 30, Italy and
Sonoscape A5v, China). The uterine flushes were performed with detection of
embryonic sac by ultrasound scan (Figure 1-A, 1-B).
After washing the perineal area with mild soap and povidone iodine solution, a
36 Foley catheter (Bioniche Animal
Health, Belleville, ON, Canada),
was inserted into the vagina and through cervix. Passage of the catheter
through the cervix was performed manually by guiding it through using the operators
finger. Once into the uterine body, a cuff on the end of the catheter was
inflated with 30 ml of air then pulled caudally to ensure a tight seal against
the internal so of the cervix. Lactated Ringer’s solution (1 L) was infused
through the catheter and into the uterus then drained into a 65 μm EmSafe
filter for embryo recovery with integrated petri dish with grid (Minitüb, Germany). The process of flush continued until the
embryo recovered in the filter and was seen by naked eyes. Gross evaluation of
embryonic shape was carried out as well as microscopic evaluation of embryo
capsule and trophoblast cells.
2.6 Transfer of Embryos
After detection of the embryo in the EmSafe filter, it has been picked up
and washed in a holding media. The embryo was loaded in a catheter using a
special kind of pipette filler (A manual propipetter adjusted by turning the
wheel with the thumb) to control a suction and release to avoid rupture of the
embryo (Figure 2-A). A.I catheters, modified
A.I, ET catheters and external protecting sheath of double guarded uterine swab
have been used respectively for embryo transfer according to embryo size (Figure 2-B). Contamination was minimized by enclosing
the AI catheter in a sterile sheath. Passage of the pipette through the cervix
was mainly performed only transvaginally. The embryo was deposited into the
uterus and the catheter and sheath were withdrawn from the mare.
2.7. Pregnancy Diagnosis in Recipient Mares
Pregnancy diagnosis in recipient mares was carried out using transrectal
ultrasound scanning. Examinations were done 15, 30, 45, 60 and 90 days’ post
ovulation.
3.
Results
During this study, 36 donor mares were flushed after detection of embryos
using transrectal ultrasound scanning. No flushing was carried to donor mares
unless embryonic sac was recorded by ultrasound. Control mares (6 mares) were
scanned as well for detection of embryonic sacs and minoring pregnancy. All
recovered embryos were large expanded blastocysts with diameters ranged from 4
-6.5 mm (Figure 3-A, B).
The recovery rate was 94.4% (34/36) as there were 2 recovered intact
capsules enclosing collapsed trophoblasts of a day 10 and 11 embryos damaged
during flushing (Figure 4-A, B). There were 2
embryos recovered from uteri of mares that have been died suddenly at 11 days
of pregnancy, only one of them succeeded post-transfer (Table
1). The success rate post transfer was 73.5% for embryos transferred at
10 and 11 days’ post-ovulation. There were 12% (3/25) embryonic deaths have
been observed at 20, 35 and 45 days of pregnancy in recipient mares; so that
the conception rate was ultimately 64.7%.
The recovery rate was similar for 10 and 11 day old embryos. The effect
of day of ovulation in recipient mare on pregnancy rate post transfer was
studied.
4.
Discussion
In the vast majority of equine embryo transfer programs, the embryo recovery
usually carried out on days 6, 7 and 8 post ovulation and transferred into
recipient mares that ovulated 1 day before to 3 days after the donor mare. The
current study was greatly different, as it was depending on flushing of donor
mares when embryos were detected by ultrasound scanning (on day 10-11 post
ovulation) followed by transfer into a recipient mare depending on using a
large pore catheter to fit the embryo size. The current embryo transfer
programs do not depend on ultrasographic detection of embryo in the donor mare,
so that there are many flushes should be carried out to get the embryo from the
donor mare that means loss of large volumes of flushing media and stress on
mare's uterus through applying several washes of the uterus particularly, in
mares with a history reduced embryo recovery due to subfertility. Furthermore,
in old mares there was a delay in descend of the embryo into the uterus and
thus the determination of day of flush is not easy, but with using of the
modified method once the embryo detected by ultrasound, the flushing could be
performed. So the operator could safe flushing media and times of flushing
until he could be sure that is a formed embryonic sac.
David Hartman (2011) [10] mentioned that
veterinarians tend to flush on day 8 or 9 to visualize embryo in the filter to
stop flushing once they detect embryo, but in this practice twins may be lost.
Comparatively, in the current study twins can be detected with ultrasonography
and flushing continues until obtaining the twins and this result make this study
is more practical and precise in recovery of twins.
The studies that was carried out concerning transfer of old age embryos were
few as mentioned by [5,7-9] but there were
trials achieved by Vogelsang et al. (1985) [11] who
reported no pregnancy post transfer of day 9 and 10 embryos while Fleury et al.
(1989) [12] who achieved 69% pregnancy rate for
embryos collected 9 days’ post ovulation, these contrastive results might be
due to recipient factors or method of transfer. In the current study, the
success rate after transfer was 73.5% although the embryo size was larger
(10-11 days) than the previous studies. Based on the present findings, there
was a high degree of success in embryo recovery (94.4%) and pregnancy rate
73.5% compared to the results obtained by Jacob et al (2012) [13], who got recovery rate (63%) and pregnancy
rate (70%). Wilsher et al. (2010) [14] mentioned
a success in transfer in 10 days’ embryo and the problematic aspect of the
method of transfer as well as failure of recovery of embryo ˃ 5 mm after
detection with ultrasonography. The current study succeeded recovery of embryos
4- 6.5 mm in diameters, furthermore using different sizes catheters according
to the embryo size and using special kind of manual controllable pipettes to
manage embryo loading, amount of holding media as well as slow release of the
embryo during transfer to avoid
embryo burst.
Based on the current results, there is a new use of embryo transfer for large
equine embryos over 10 days that can work for mares that died suddenly or might
be subjected to euthanasia for urgent reasons and they were pregnant 9-11 days.
In conclusion, all previous studies concerning transfer of large equine embryo
ranged from 9 to 10 days and the results were contrastive. The current study
emphasized on transfer of large embryo that could be detected by ultrasound
scanning. This method can be used for mares with a history of low embryo
recovery to save times of flushing and avoiding loss of flushing media. As well
as it can be applied for mares died at 10-11 days’ pregnancy or have been
subjected to euthanasia.
Furthermore, the technique can be used for mares when the operator missed
the day of flushing for any reason or has to delay the time of flushing 1-3
days for availability of a recipient.
5.
Acknowledgements
Thanks are kept for all my team of assistants and labors in my lab. And owners
of the farms that helped me to achieve this work.
research article 
