Archivos Latinoamericanos de Producción Animal. 2023. 31 (4)
Correlation of two approaches to estimate the area of corpus luteum
using ultrasonography images in sheep
Recibido: 20230702. Revisado: 20230727. Aceptado: 20231023.
1Departamento de Biociencias Veterinarias, Facultad de Veterinaria, Universidad de la República
2Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile
Sol Lafourcade
Abstract. Ultrasonography (US) is a helpful tool for studying follicular and luteal dynamics, ovulation, and embryo
development. However, there is currently no gold standard reference technique for estimating the corpus luteum
(CL) size. The aim of this study was to compare the results from measuring ewes CL size based on its diameter or
its surface with the aid of image software. The study involved 19 ewes, recording the images of 50 CLs at different
stages of development, and therefore, calculating its size by both procedures. The results showed that measuring
the CL diameter to calculate its’ area is a rapid and reliable technique that can effectively replace the measurement
of the surface, which requires precision and time. The study concluded that measuring the two main axes of the CL
is a valid method for effectively estimating its size.
Keywords: Ovary, Follicular dynamics, Luteal dynamics
Departamento de Biociencias Veterinarias, Facultad de Veterinaria, Universidad de la República, Montevideo Uruguay
Juan Pedro Bottino1
Correlación de dos enfoques para estimar el área del cuerpo lúteo mediante
imágenes de ecografía en ovejas
Resumen. La ultrasonografía (US) es una herramienta útil para estudiar la dinámica folicular y luteal, la ovulación y
el desarrollo embrionario. Sin embargo, no existe una técnica de referencia estándar para estimar el tamaño del
cuerpo lúteo (CL). El objetivo de este estudio fue comparar los resultados de medir el tamaño del CL de ovejas
basándose en su diámetro o en su área con la ayuda de un software de imágenes. El estudio involucró a 19 ovejas,
registrando las imágenes de 50 CL en diferentes etapas de desarrollo y, por lo tanto, calculando su tamaño mediante
ambos procedimientos. Los resultados mostraron que medir el diámetro del CL para calcular su área es una técnica
rápida y confiable que puede sustituir eficazmente la medición de la superficie, que requiere precisión y tiempo. El
estudio concluyó que medir los dos ejes principales del CL es un método válido para estimar eficazmente su
Palabras clave: Ovario, Dinámica folicular, Dinámica luteal
Marcelo Ratto2 Rodolfo Ungerfeld
Correlação de duas abordagens para estimativa da área do corpo lúteo
utilizando imagens ultrassonográficas em ovinos
Resumo. A ultrassonografia (US) é uma ferramenta útil para estudar a dinâmica folicular e luteal, a ovulação e o
desenvolvimento embrionário. No entanto, atualmente não existe uma técnica de referência padrão para estimar o
tamanho do corpo lúteo (CL). O objetivo deste estudo foi comparar os resultados da medição do tamanho do CL de
ovelhas com base em seu diâmetro ou em sua superfície com o auxílio de software de imagem. O estudo envolveu
19 ovelhas, registrando as imagens de 50 CL em diferentes estágios de desenvolvimento e, portanto, calculando seu
tamanho por ambos os procedimentos. Os resultados mostraram que a medição do diâmetro do CL para calcular
sua área é uma técnica rápida e confiável que pode substituir efetivamente a medição da superfície, que requer
precisão e tempo. Concluiuse que medir os dois principais eixos do CL é um método válido para estimar
efetivamente o seu tamanho.
Palavras chave: ovário, ovelha, ultrassom, dinâmica folicular, dinâmica luteal.
Lafourcade et al.
Ultrasonography (US) has provided a noninvasive
technique to study in situ in real time the anatomy and
physiology of the reproductive system and characterize
reproductive events in different species (Griffin et al.,
1992). This tool is useful to study follicular and luteal
dynamics, ovulation, and embryo development
(Murillo, 2013, UlloaLeal et al., 2014, Silva et al., 2015).
For example, the size of the corpus luteum (CL)
(Pierson et al., 1984) and its surface variations over time
are used as possible estimators of functionality
(Murillo 2013) in cattle (Pierson et al., 1984, 1987),
buffalo (Brito et al., 2002), sheep (Viñoles et al., 2004),
goats (Simões et al., 2005), and pigs (Da Silva et al.,
2017). The CL secrete progesterone (P4), which is
essential for the establishment and maintenance of
pregnancy (Shah et al., 2007, Bachelot and Binart.,
2005). Measurement of P4 concentrations is relatively
expensive, and requires animal restraintment and
blood collection. However, as the size of the CL? is
related to serum P4 levels in cows (Rocha et al., 2018,
Kastelic et al., 1990, Assey et al., 1993), sheep (González
de Bulnes et al., 2000., Schrick et al., 1993), llamas
(Adams et al., 1991), and mares (Bergfelt et al., 1989),
measuring the CL size may substitute P4 measurement.
In this sense, using ultrasound allows realtime
datarelated to P4 levels (Rocha et al., 2018).
Therefore, measuring the CL allows to estimate P4
levels, the estrous cycle stage, and pregnancy status
rapidly and straightforwardly. This is crucial for
understanding the reproductive status of the flock and
becomes significant when applying new
biotechnologies such as artificial insemination and
embryo transfer. However, there are no standard
reference techniques for many of those determinations.
Even though it is a commonly used approach, to the
best of our knowledge, there is no standard reference
technique to estimate the size of the CL, and a wide
number of different methods are reported in the
literature. Although the CL is a threedimensional
structure, the diameter was used as the only
measurement in cows (Assey et al., 1993, Kayacik 2005,
Bicalho et al., 2008), sheep (Viñoles et al., 2004), llamas
(Adams et al., 1989, Ratto et al., 2006, Fernández et al.,
2014), sows (Da Silva et al., 2017), and goats (Simões et
al., 2005). In other studies, the area of the circumference
obtained by ultrasound was used in cows (Herzog et
al., 2010, Murillo 2013), or based on the formula of the
area of the circumference: 0.5 length x 0.5 width x π, in
cows (Kastelic et al., 1990), buffaloes (Brito et al., 2002)
and sheep (González de Bulnes et al., 2000). Finally,
other authors estimated the volume based on the
formula of the sphere volume (4/3).π.r3) in cows
(Echternkamp et al., 2009) and sheep (Bartlewski et al.,
1999, Bartlewski et al., 2000). Although this approach
considers the CL a threedimensional structure, it is
usually not a perfect sphere. Furthermore, it is
important to consider that many CLs have a cavity that
does not include secretory tissue, so Pierson et al. (1987)
presented the data considering the luteal cavity
As no gold reference technique exists so far to esti
mate the CL size, this study proposed to compare two
CL area estimators, measuring the major diameters or
the surface of the CL in ewes to determine which is the
simplest technique that provides a reliable estimation.
The present study was performed in December (late
spring in the SH) in Campo Experimental No.1 of the
Facultad de Veterinaria, Universidad de la República
(Uruguay 34° S). Nineteen multiparous Australian Merino
ewes were used during the nonbreeding season. Their
reproductive tract was examined by ultrasonography to
check their reproductive health before starting the study.
Ewes remained grazing natural pastures with good forage
The study was approved by the Comisn Ética en Uso
de Animales (CEUA) of the Facultad de Veterinaria
The estrous cycle of ewes was synchronized with an
intravaginal sponge containing 60 mg of medroxy
progesterone acetate (Progespon, Syntex, Buenos Aires,
Argentina) inserted for 7 days. When intravaginal sponges
were withdrawn, females were given an i.m dose of 0.0375
mg of a PGF2alpha analog (Veteglan, dCloprostenol
0.075 mg/mL, Calier, Argentina) and 300 IU of equine
chorionic gonadotropin (eCG) (Novormon, Zoetis,
Transrectal ultrasonography was performed every 24 h
to record the development of CL until day 10 of the cycle
(day 0 = day of ovulation) in only the females that
ovulated. Ovulation was defined as the sudden
disappearance of a large follicle (≥ 8mm) detected during
the previous examination and confirmed by subsequent
detection of a CL. For this purpose, ewes were placed
standing in a trap and a 7.5 MHz linear transducer coupled
to a monitor (United Imaging, IUStar 160 Vet model,
Beijing, China) was inserted into the previously lubricated
rectum. The images obtained in realtime were stored on a
Material and Methods
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Ultrasound Image Area Measurement: Method Comparison
There was a significant positive relationship between
both CL area estimators, based on the formula and
measurements obtained through Image (R² = 0.98, P <
0.0001) (Fig. 2).
Fig. 1. CL measurement with different methods: (A) Major CL diameters with ultrasound tool. (B) Major diameters of the CL
with ImageJ. (C) CL area with ImageJ.
The positive relationship between both techniques
allows us to argue that the estimation of the area based
on the radius values can be replaced with high
accuracy in the measurement of the area with specific
software with high accuracy. The measurement of the
diameters of the CL is easy and rapid, and it can even
be done during the ultrasound procedure, therefore,
having important practical advantages. The calculation
based on the radius allows a rapid, concise, and
representative evaluation of the CL size in sheep,
which can probably be useful in females from other
species. On the other hand, as the CL shapes are elliptic
but usually irregular, the manual measurement of the
surface requires precision and time. In farm
production, it is essential to know the reproductive
status of the animals in the shortest possible time after
natural mating or artificial insemination, to plan the
work and be able to make decisions such as removing,
reinseminating, or treating the nonpregnant animals
according to each case (España et al., 2004). In this
context, the evaluation of the CL in situ allows the
determination of its dynamics, as with repeated
measures would be possible to know if it is growing or
regressing without the need for subsequent image
USB memory for later analysis of the images. Overall, the
images of 50 CLs at different luteal stages (days 1 to 10
postovulation) were stored, and analyzed using the
imageJ™ program (US National Institutes of Health,
Bethesda, Maryland, USA) (Schneider et al., 2012).
Two different approaches were used, one calculating the
area based on the two major axes (Fig. 1A, 1B) of each CL
observed by the program, applying the formula for an
elliptic area (A = π. a.b), where a and b are each radius. The
second measurement was a direct measurement of the area
using the ImageJ software, surrounding the border of the
CL with the “Polygon selections” tool of this program (Fig.
1C) (Schneider et al., 2012). In the cases in which the CLs
presented a cavity, its area was measured with the same
technique, subtracting its area from that of the CL.
Linear regression was performed relating both calculated
Fig. 2. Relationship between corpus luteum area estimators based on formula and measurements obtained through ImageJ
ISSNL 10221301. Archivos Latinoamericanos de Producción Animal. 2023. 31 (4): 305309
It was concluded that the size of the CL can be effectively estimated based on the measurement of the two major
axes using transrectal ultrasound.
Adams, G. P., Griffin, P. G., & Ginther, O. J., 1989. In
situ morphologic dynamics of ovaries, uterus, and
cervix in llamas. Biology of Reproduction, 41: 551–558.
analysis, and to be able to work out the reproductive
management of eachfemale at the moment. For
example, Espa, et al. (2004) used US for early pregnancy
diagnosis in cows. This opens interesting possibilities for
practical management in highvalue flocks.
In addition to being able to apply it in field conditions,
the time required is much shorter (data not recorded).
Moreover, in cases where the borderline is diffuse with
only a slight difference in echogenicity between the luteal
and ovarian stroma, it is complex to measure the area with
software due to the difficulty of following the edge of the
CL. On the other hand, when the area is calculated based
on the radius, it is only necessary to determine the limit of
the luteal stroma at 4 well defined points. Additionally, not
all ultrasound machines have the tools to measure a
specific irregular surface (Murillo 2013). Therefore, the
image must be saved and analyzed later, losing the
advantage of obtaining the information in realtime.
However, with technological advancement, different types
of ultrasound equipment tend to incorporate this type of
tool. Standardizing the method to process the data would
allow to adjust the tools to assess the development of CL,
including its growth or regression over time, from
reference processes. The variability in the methods used to
estimate CL dimensions creates a misunderstanding in
interpreting the results among studies. In addition, Murillo
(2013) states that the measurement of a single diameter, as
has been done in various studies (Silva et al., 2014, Gajardo
et al., 2021), is not the most appropriate measurement, as it
presents less variation over time than the surface.
Conflict of interest: No competing interests declared.
Approval of the Animal Experimentation Committee: The study was approved by the CEUA of the Facultad de
Author contributions: SL discussed the general study design, organized the experimental procedures, analyzed the
images, collected data, revised, and worked on the preparation of the manuscript. JPB performed and analyzed
doppler ultrasonographic evaluation, revised and worked on the preparation of the manuscript, and approved the
final version. MR discussed the general design, revised and worked on the preparation of the manuscript, and
approved the final version. RU organized the study and revised and worked on the preparation of the manuscript,
and approved the final version.
We acknowledge Carla Faliveni, Director of the Experimental farm, and the workers of the farm for their help with
animal management.
SL received a postgraduate scholarsip from the Agencia Nacional de Investigación e Innovación (ANII), Uruguay.
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