Theriogenology 153 (2020) 48e53

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Theriogenology
journal homepage: www.theriojournal.com

A practical guide to estimate the age of the early CL by

ultrasonography in mares examined for the first time to be used as
recipients in a commercial embryo transfer program
Juan Cuervo-Arango a, b, *, M. Soledad Martín-Pela
ez a, Anthony N. Claes a
a
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 112, Utrecht, the Netherlands
b
Cantero de Letur S.L., Letur, Spain

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

Article history: The echogenicity of the early CL undergoes obvious changes during the first 5 days of luteal development.
Received 30 November 2019 This phenomenon could potentially be used to estimate the age of the CL in mares which have not been
Received in revised form checked frequently for the diagnosis of ovulation. The objective of this retrospective study was to
1 May 2020
determine the accuracy of estimating the age of the early CL (Day 0 to Day 4; Day 0 ¼ Day of ovulation) in
Accepted 2 May 2020
Available online 6 May 2020
recipient mares examined for the first time in the breeding season to be used in a commercial embryo
transfer (ET) program. The post-transfer pregnancy rate and embryo loss of 28 recipient mares whose CL
age was estimated at the first scan of the breeding season (at least 2 months gap from the previous
Keywords:
Embryo transfer
examination) were compared to that of 41 recipients in which the Day of ovulation had been determined
Estimation of CL age by daily examinations and that received an embryo on the respective day. There was no difference
Echogenicity (P > 0.1) in pregnancy (85.7 and 85.3%) or early embryonic loss (12.5% and 11.4%) between groups,
Ultrasound respectively.
Recipient mare © 2020 Elsevier Inc. All rights reserved.

1. Introduction window of asynchrony between the frozen/thawed ICSI embryo

Equine embryo transfer allows an unusually large window of
(negative) asynchrony between the embryo and the recipient’s
uterine stage (i.e. þ1 to 4 days) [1,2]. In large embryo transfer (ET)
centers, where the number of recipients exceed that of donor mares
by a factor of 3, recipient mares are often examined for diagnosis
of ovulation every 2e3 days [3]. This protocol reduces the labor
costs, because recipient mares are examined less frequently, but
also reduces the number of days for which a recipient can be used
to receive an embryo within the optimal window of synchrony.
On the other hand, when the number of recipients available for a
donor mare is reduced (1:1 or 2:1), more frequent examinations for
the diagnosis of ovulation are required (i.e. every 24 he48 h) to
optimize the number of recipients available to receive an embryo.
Furthermore, when the ET program involves the use of in vitro-
produced (ICSI) cryopreserved embryos, it is even more important
to diagnose accurately the Day of ovulation, since the optimal
* Corresponding author. Department of Equine Sciences, Faculty of Veterinary
Medicine, Utrecht University, Yalelaan 112, Utrecht, the Netherlands.
E-mail address: copicuervo@hotmail.com (J. Cuervo-Arango).
and the uterine stage of the recipient mare is very narrow (i.e. 24 h)
[4] compared to that of in vivo derived embryos [5]. Therefore, a
method to estimate accurately the age of the CL of mares without
the need of frequent examinations to detect ovulation would be a
great advantage.
Over three decades ago (80s), Pierson and Ginther [6] described
the changes in echogenicity observed by transrectal ultrasonogra-
phy of the CL. Few years later, Ginther’s group showed in more
detail the association between CL age and luteal echogenicity
during the first 5 days of its development [7]. Luteal echogenicity
was determined in a subjective (visual scoring system) and objec-
tive (pixel computerized analysis) ways. Both approaches
concluded that the luteal echogenicity of the CL increases gradually
from 8 to 12 h (minimal or hypoechoic) to 48 h after ovulation
(maximal or hyperechoic). Then, it reaches a plateau until 72e96 h
after ovulation. Finally, the luteal echogenicity decreases gradually
until 120 h after ovulation [7]. The changes in luteal echogenicity
occurred similarly in both types of luteal morphologies (solid
corpora lutea and those with a central cavity). In the following
decade, Newcombe [8] described, in a ‘practical way’, the ultraso-
nographic changes (morphology and echogenicity) of the

https://doi.org/10.1016/j.theriogenology.2020.05.003
0093-691X/© 2020 Elsevier Inc. All rights reserved.
 J. Cuervo-Arango et al. / Theriogenology 153 (2020) 48e53 49

Fig. 1. Representative ultrasonograms of 4 different mares in early diestrus showing the ovary (upper panel) and uterine horn (lower panel). The number of days from ovulation was
estimated based on luteal echogenicity and morphology of the CL and echotexture and morphology of the uterus. The Day of ovulation (Day 0) was allocated to mares in which the
post-ovulatory site was an ill-defined area with similar echogenicity to the ovarian stroma, the uterus showed slight edema and the uterine horn was flat. The echogenicity of the CL
and the prominence of the luteal borders increased gradually until Day 2. Day 1 mares showed an increased luteal echogenicity compared to Day 0 mares, but the prominence of the
luteal border was not well defined yet compared to Day 2 or Day 3 mares. Day 3 mares had a rounder uterus and a more prominent separation between the myometrium and the
endometrium compared to that of Day 2 mares. All images are taken at the same magnification.

borders are clearly visible all around the CL [8].

Despite these early reports and continuous improvements in the
image quality of ultrasound machines, no study attempted to
evaluate the feasibility of estimating the age of the CL by studying
its ultrasonographic morphology and echogenicity. The objective of
this retrospective study was to determine the accuracy of esti-
mating the age of the early CL (Day 0 to Day 4; Day 0 ¼ Day of
ovulation) in recipient mares scanned for the first time in the
breeding season to be used in a commercial ET program. The post-
ET pregnancy rate of recipient mares whose CL age was estimated
before transfer was compared to that of recipients scanned daily for
the diagnosis of ovulation. It was hypothesised that the pregnancy
rate would not differ between groups.

2. Materials and methods

2.1. Recipient mare management

All recipient mares belonged to the Equine Science Department,

Fig. 2. Ultrasonograms of the ovary containing the CL of the same mare on Day 1 (left
lower panel) and Day 2 (left upper panel). The right images represent the prominence
of the luteal border as shown by a red line depicting the separation between the luteal
tissue and ovarian stroma. The Day 1 CL has an ill-defined border (right lower panel)
represented by a discontinuous red line: the border is not clear throughout the luteal
outer line. In contrast, the Day 2 CL has a well-defined border represented by a
continuous red line which can be easily drawn all over the luteal border.
developing CL to aid in the estimation of its age, in absence of daily
examinations to detect ovulation. In the latter study, in addition to
changes in luteal echogenicity, a new end point was introduced: the
prominence of the luteal border [8]. The post-ovulatory area re-
mains hypoechoic with no clear luteal borders until 15e20 h post-
ovulation. Then, the developing CL becomes a moderately dense
area with ill-defined borders by 24 h after ovulation. By 36e48 h
post-ovulation the luteal echogenicity is maximal and the luteal
Utrecht University, The Netherlands. All mares were warmbloods
(mean age of 7.1 ± 3.3 years old, range of 3e14). Recipient mares
were sent to a winter facility at the end of each breeding season,
after weaning from the previous foaling or after purchase (as new
recipients). At the beginning of the breeding season (starting in
February) and continuing until June, mares were brought back to
the clinic in groups of 6 mares until approximately a group of 40
recipients (non-pregnant) were available for transfer. As the re-
cipients were used (became pregnant after ET), more mares were
brought from the winter facilities until the end of the breeding
season.
On the Day that the recipients were brought back to the clinic,
they were examined by transrectal examination with an ultrasound
scanner (Esaote MyLab Delta, The Netherlands) equipped with a
7e10 MHz linear probe. During 4 breeding seasons (2016e2019), a
group of 28 recipients estimated to be in early diestrus at the first
scan of the breeding season were used for ET in the same cycle.
These 28 mares made of the experimental group. They were mares
with a fresh ovulation (i.e. < 24 h) or an early CL of 24e120 h old
50 J. Cuervo-Arango et al. / Theriogenology 153 (2020) 48e53

Fig. 3. Representative ultrasonograms of 3 different mares showing the uterine horn and the corresponding ovary containing an estimated Day 4 CL, the uterine horn of the same
Day 4 mare, a mature CL (Day 9), and a corpus albicans (old CL). The Day 4 CL shows increased luteal echogenicity of the luteal tissue compared to the borders and ovarian stroma.
The mature CL shows homogenous echogenicity of the luteal tissue and an overall size significantly smaller than the Day 4 CL. The old CL is assumed to be inactive since it has no
color-flow signal and the uterus shows an estrous-like echotexture; the inner part of the old CL shows increased echogenicity and a smaller diameter than the Day 4 and mature CL.
All images were taken at the same magnification.

2.2. Criteria for diagnosis of early diestrus (Day 0 to Day 4)

- Day 0: The mare showed a relaxed cervix and uterus on rectal

Fig. 4. Representative images of four Day 8 embryos of different grades (I to III). The
Grade III embryos (lower left and right panels) were transferred into recipient ID 23
and 24, respectively, and resulted in no pregnancy.
(Day 1 to Day 4, Day of ovulation ¼ Day 0). Since each mare was
examined for the first time after the winter (at least 2 months gap
from the previous examination), the exact age of the CL was un-
known. Instead, the age of the CL was estimated based on the
echogenicity of the post-ovulatory area [7], prominence of the
luteal borders [8], rectal palpation of the post-ovulatory area, and
uterine echotexture, tone and shape of the uterus. These mares
were examined by four veterinary surgeons, but the majority of
mares (64%) were scanned by the same veterinary surgeon with
previous training on estimating the CL age. The rest of vets were
trained by the first one and participated in the study from the third
breeding season. Due to the low number of observations for the
other vets, no attempt to analyse the effect of operator on preg-
nancy rate was made.
palpation. The ultrasound examination of the uterus showed a
flat uterine horn and slight endometrial edema. The ovaries
presented no obvious CL or large dominant follicles but a
hypoechoic area with ill-defined borders (similar to the ovarian
stroma) equivalent to a fresh ovulation (Fig. 1). Often, the rectal
palpation of the assumed fresh ovulation revealed a soft and
slightly painful area.
- Day 1: As in the previous section (Day 0), but the luteal tissue of
the post-ovulatory area showed increased echogenicity,
compared with the surrounding ovarian stroma. Yet, the borders
of the developing CL were still ill-defined, remaining as an
echoic (dense) area for some parts of the CL (Figs. 1 and 2).
- Day 2: The CL was highly echoic with well-defined borders all
over the CL. They were easily distinguished from the rest of the
ovarian stroma (Fig. 2). The cervix and uterus felt more tonic
(score 2 from a scale of 1e3; 1 ¼ closed cervix and toned uterus;
3 ¼ wide open cervix and relaxed uterus). The ultrasound ex-
amination of the uterine horns showed no clear separation be-
tween the endometrium and myometrium layers (Fig. 1).
- Day 3: The CL appeared highly echoic and with prominent luteal
borders as in Day 2 mares. In contrast to Day 2, the uterus and
cervix had acquired a diestrous-like shape and tone (score 1).
The ultrasound examination of the uterine horn showed a round
uterus with a clear separation of the endometrium and myo-
metrium layers (Fig. 1).
- Day 4: The echogenicity of the well-defined CL was reduced
compared to Day 2 and Day 3 mares, but it was still slightly more
echoic than a mature CL and ovarian stroma (Fig. 3). In addition,
the luteal region of increased echogenicity was more evident
towards the center, while the CL border had lower echogenicity,
similar to that observed in a mature CL. The overall size of the
Day 4 CL was significantly larger than an old CL, which may have
a similar luteal echogenicity (Fig. 3).
 J. Cuervo-Arango et al. / Theriogenology 153 (2020) 48e53 51

Table 1
Reproductive performance of recipient mares examined daily for diagnosis of ovulation (control group) or whose CL age was estimated (experimental group) at the first
examination of the season which received an embryo.

Ovulation diagnosis n Recipient age (years) Donor age (years) Embryo size (mm) Grade 3 embryos (%) Day of OV at ET (days) Pregnancy (%) EEL (%)

Daily scans 41 7.6 ± 3.6 (3e14) 10.9 ± 5.3 (3e19) 575 ± 330 (160e1500) 0/41 (0.0a) 6.7 ± 1.6 (4e9) 35/41 (85.3) 4/35 (11.4)
Estimated CL age 28 7.9 ± 3.7 (3e15) 11.9 ± 5.8 (3e21) 537 ± 307 (160e1200) 3/28 (10.7b) 6.8 ± 1.7 (4e10) 24/28 (85.7) 3/24 (12.5)

Pregnancy: percentage of pregnant recipients 4 days after embryo transfer; EEL: early embryonic loss by 45 days of pregnancy. Within column, different letters (a,b) indicate a
significant tendency (P ¼ 0.06).

Table 2
Characteristics of embryo transfers performed in recipients from the experimental group, in which the age of the CL was estimated at the first examination of the breeding
season.

Recipient ID CL age estimated at 1st scan (days) Day of OV at ET Embryo age (d) Embryo grade Embryo size (mm) Pregnant EEL

1 0 10 9 1 1280 Yes Yes

2 0 6 8 1 288 Yes No
3 0 4 8 1 544 Yes No
4 0 7 8 2 512 Yes No
5 0 7 8 1 544 Yes No
6 0 6 8 1 192 Yes No
7 0 7 8 1 736 Yes No
8 1 6 8 1 704 Yes No
9 1 8 8 1 832 Yes No
10 1 6 8 2 608 Yes No
11 1 9 8 1 800 Yes Yes
12 2 7 8 1 320 Yes No
13 2 9 8 1 448 Yes No
14 2 7 8 1 960 Yes No
15 2 7 9 1 1088 Yes No
16 2 9 8 1 224 Yes No
17 2 9 8 1 704 Yes No
18 2 7 8 1 800 Yes No
19 2 5 8 1 384 Yes No
20 3 7 8 1 576 Yes Yes
21 3 7 8 1 480 Yes No
22 3 6 8 1 480 Yes No
23 3 8 8 3 160 No N/A
24 4 5 8 3 192 No N/A
25 4 5 8 1 288 Yes No
26 4 4 8 2 192 No N/A
27 4 5 8 3 160 No N/A
28 4 9 8 1 608 Yes No

Embryo grade: embryos were scored according to its morphology (1 ¼ excellent; 2 ¼ fair; 3 ¼ poor). EEL: early embryonic loss by 45 days of pregnancy. N/A: not applicable. ET:
embryo transfer.

2.3. Embryo transfer and pregnancy diagnosis 2.4. Experimental design and end points

Day 8e9 in vivo-derived (flushed) embryos were used for the
recipient mares. All transfers were performed during the immedi-
ate estrous cycle following the first examination of the recipient
mare in each breeding season. Embryo donor mares were managed
and flushed as previously reported [9]. All embryos were trans-
ferred transcervically by one of four board certified theriogenolo-
gists by a modified Wilsher’s technique with the aid of a cervical
grasping forceps and speculum [10]. Embryos were transferred into
Day 4 to Day 10 (4 to þ1 asynchrony) recipient mares, respec-
tively (Day 0 ¼ Day of ovulation).
Flushed embryos were measured and graded according to their
morphology and quality assessment [11]: grade I (excellent quality
embryos, no morphological abnormalities), grade II (minor imper-
fections, such as few extruded cells or slightly irregular surface),
grade III (poor quality embryos, with definite, distinct abnormal-
ities). An example of embryos with different grades are shown
(Fig. 4).
Pregnancy diagnoses were performed 4 days after transfer, and
then again at 45 days of pregnancy. Embryo losses between the first
and second pregnancy diagnosis were used to calculate the early
embryonic loss (EEL).
The experimental unit of this study was the estrous cycle of the
recipient mare following the first examination of the breeding
season, which was scheduled for potential embryo transfer.
The end points calculated were the first pregnancy diagnosis
(%): number of pregnant recipients at the first pregnancy diagnosis
divided by the total number of transfers, and the EEL: number of
pregnancy losses within 45 days of pregnancy divided by the
number of pregnant recipients at the first pregnancy diagnosis.
In order to determine the accuracy of the technique to estimate
the CL age in recipients in early diestrus, the pregnancy rate and EEL
of these recipients (n ¼ 28) were compared to that of a control
group. The control group was composed of 41 recipients whose
ovulations had been diagnosed by daily examinations and received
an embryo on the same day that mares from the experimental
group. These mares were managed in a similar way that recipients
from the experimental group (feeding and housing), except that
they were checked every day, once in estrus, for diagnosis of
ovulation. These mares entered estrus either spontaneously or
following administration of a luteolytic treatment of 75 mg d-clo-
prostenol (0.5 mL Genestranvet, Dechra, The Netherlands). The
embryos used in the recipients from the control and experimental
groups were transferred by the same veterinary surgeon on each
52 J. Cuervo-Arango et al. / Theriogenology 153 (2020) 48e53
respective day. The size and grade of embryos, the age of the donor
mares, and the degree of synchrony between the recipient and
embryo were determined and compared between groups.
2.5. Statistical analyses
Sequential data (mare age, embryo-recipient synchrony and
embryo size) were checked for normality of distribution and ana-
lysed by unpaired student t-test. Categorical data (pregnancy, EEL
and proportion of Grade III embryos) were analysed by chi-square
test. Sequential data are reported as mean ± S.E.M, unless stated
otherwise. Significance was set at a  0.05. A P value between 0.1
and 0.05 meant that a difference approached significance.
3. Results
The direct comparison of the reproductive performance be-
tween recipient mares from the experimental (CL age was esti-
mated) and control (ovulation diagnosis by daily examinations)
groups is shown in Table 1. There was no difference (P > 0.1) in
pregnancy (85.7 and 85.3%) or early embryonic loss (12.5% and
11.4%) between the experimental and control groups, respectively
(Table 1). The reproductive characteristics of the 28 recipient mares
that received an embryo after estimation of the CL age are shown in
Table 2. Overall, 7 (25%), 4 (14.3%), 8 (28.6%), 4 (14.3%) and 5 (17.8%)
mares were estimated to be in Day 0, Day 1, Day 2, Day 3 and Day 4,
respectively (Table 2). On average, recipients from the experimental
group received an embryo 5.1 ± 0.4 days (range 0e10 days) after the
time their CL age was estimated. The mean estimated Day of
ovulation on the Day of ET in mares from the experimental group
was 6.8 ± 1.7, which was not different from that (6.7 ± 1.6) of mares
from the control group (Table 1).
4. Discussion
The hypothesis that the post-transfer fertility of recipient mares
whose ovulation Day was estimated would not differ from re-
cipients in which the ovulation was diagnosed by daily examina-
tions is substantiated by the results of this study. The experimental
design of this retrospective study could not determine the degree of
accuracy of the technique to estimate the CL age within the first 4
days of luteal development. However, the level of accuracy of this
technique appeared to be good enough to provide an acceptable
post-transfer fertility of recipients whose CL age was estimated,
which was not different from that of mares from the control group.
The authors acknowledge that the positive pregnancy diagnosis of a
recipient mare from the early diestrus group does not imply that
the estimation of the age of the CL was totally accurate, as the
flushed embryos tolerate a wide window of negative asynchrony.
However, since the fertility was similar between groups, a sufficient
degree of accuracy can be assumed to justify the use of this tech-
nique to estimate the age of the CL. Furthermore, the use of these
recipient mares to receive an embryo straight away after the esti-
mation of the CL age, results in a reduction in labor costs (fewer
examinations) and time to pregnancy.
The criteria used in this study to estimate the age of the early CL
were based on the results reported in a previous study [7], but they
were modified and adapted to field conditions to make the age
estimation technique more practical. In the reported study [7], the
authors used a computerized digital analysis and a subjective sys-
tem based on comparing the luteal echogenicity with an eight-
section gray-scale display located adjacent to the image. In the
current study, we just used a direct comparison of the luteal
echogenicity with that of the surrounding ovarian stroma, to assess
whether the echogenicity of the post-ovulatory area was increased.
Furthermore, relevant information was gathered by rectal palpa-
tion of the uterus, cervix and ovaries as well as by studying the
uterine echotexture and horn shape, so that a wider picture of each
case could be obtained to perform a more accurate estimation of
the CL age.
It appeared that recipients estimated to be on Day 4 of the cycle
had the poorest pregnancy rates (only 2 out of 5 mares became
pregnant). However, 2 of those failing embryos were scored as
Grade 3, and the third one was small for age (192 mm in diameter),
which are known to have a reduced viability [2]. An interesting
observation was that around 10% of embryos (3/28) transferred to
recipients from the experimental group were Grade 3, compared to
0% (0/41) of embryos transferred to recipients from the control
group. It is possible that there was certain bias to use lower quality
embryos in recipients which were not completely ‘reliable’ as the
CL age had been estimated, as a ‘better alternative’ to throw them in
the bin.
A limitation of the study was that the experimental design did
not allow studying the effect of the operator and the recipient’s Day
of ovulation on the accuracy of estimating the CL age, due to a
relatively low number of observations and the lack of a ‘gold
standard’. In this case, the exact Day of ovulation of the recipient
mare was unknown as they had not been checked for at least two
months previously. Further research should be carried out to
elucidate the potential of this technique involving a larger number
of mares with the help of a blinded operator, before this technique
can be recommended for practitioners.
In conclusion, the estimation of the Day of ovulation of recipient
mares during the first 4 days of luteal development based on the
echogenicity of the luteal tissue and assessment of the uterus and
cervix was a useful technique to reduce the frequency of exami-
nations of recipient mares without affecting post-ET pregnancy
rates.
Declaration of competing interest
The authors declare no conflict of interest.
CRediT authorship contribution statement
Juan Cuervo-Arango: Data curation, Writing - review & editing.
M. Soledad Martín-Pelaez: Data curation, Writing - review &
editing. Anthony N. Claes: Data curation, Writing - review &
editing.
Acknowledgments
This study received no funding. The author Juan Cuervo-Arango
thanks Prof. John R. Newcombe for his guidance and practical
teaching on how to assess the early CL for age estimation.
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