Most bovine estrous synchronization protocols utilize progesterone plus estrogen to control ovulation timing. A drug that inhibits estrogen production (Letrozole) may be an alternative, steroid-free synchronization method (not yet commercially available). However, low estrogen can negatively affect the health of follicles/oocytes and impact fertility. To determine its effects, Letrozole was administered intramuscularly while tracking follicle growth and circulating hormones. Letrozole response was variable. Two of three cows experienced delayed luteolysis/ovulation and extended progesterone production. This preliminary data indicates that Letrozole treatment allows normal follicle progression but drug response may vary and little is known about effects on oocyte quality

Abedal-Majed, Mohamed A., Majed

Bergman, Jeffrey W.

Cupp, Andrea S.

Davis, John S.

Kurz, Scott G.

Romereim, Sarah M.

Tenley, Sarah C.

Wood, Jennifer R.

English

UNL | Libraries

University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Nebraska Beef Cattle Reports Animal Science Department
2018
Letrozole: A Steroid-Free Estrous Synchronization
Method
Sarah M. Romereim
University of Nebraska-Lincoln, sromereim2@unl.edu
Sarah C. Tenley
University of Nebraska - Lincoln, sctenley09@gmail.com
Mohamed A. Abedal-Majed Majed
University of Nebraska - Lincoln
Jeffrey W. Bergman
University of Nebraska-Lincoln, jbergman1@unl.edu
Scott G. Kurz
University of Nebraska-Lincoln, skurz2@unl.edu
See next page for additional authors
Follow this and additional works at: http://digitalcommons.unl.edu/animalscinbcr
Part of the Large or Food Animal and Equine Medicine Commons, Meat Science Commons, and
the Veterinary Preventive Medicine, Epidemiology, and Public Health Commons
This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has
been accepted for inclusion in Nebraska Beef Cattle Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.
Romereim, Sarah M.; Tenley, Sarah C.; Abedal-Majed, Mohamed A. Majed; Bergman, Jeffrey W.; Kurz, Scott G.; Davis, John S.; Wood,
Jennifer R.; and Cupp, Andrea S., "Letrozole: A Steroid-Free Estrous Synchronization Method" (2018). Nebraska Beef Cattle Reports.
960.
http://digitalcommons.unl.edu/animalscinbcr/960
Authors
Sarah M. Romereim, Sarah C. Tenley, Mohamed A. Abedal-Majed Majed, Jeffrey W. Bergman, Scott G. Kurz,
John S. Davis, Jennifer R. Wood, and Andrea S. Cupp
This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/animalscinbcr/960
2018 Nebraska Beef Cattle Report · 9 
©  The Board Regents of the University of  
Nebraska. All rights reserved.
luteum dimensions were measured along 
the longest axis and the perpendicular axis 
(the average of those two dimensions is 
presented). Ovulation was determined by 
the absence of the preovulatory follicle. 
Estrus was detected using Estrotect™ Heat 
Detector patches on the tail head. Blood 
samples were collected every other day for 
the first week then daily, and circulating 
progesterone was measured with radioim-
munoassays performed in duplicate. For 
this experiment, Day 1 of the reproductive 
cycle was defined as the day of ovulation 
detection after the second set of PGF2α in-
jections. Treatment (250µg/kg Letrozole in 
8- 10 mL of a 1:5 mixture of benzyl alcohol 
and sesame oil) or control injections (8 mL 
of 1:5 benzyl alcohol/sesame oil) began on 
Day 10 of the cycle.
Results
When using Letrozole to inhibit aro-
matase activity, an important consideration 
is the success and degree of circulating 
estrogen suppression. The purpose of 
the drug is not to completely prevent all 
estrogen production but instead to prevent 
or delay the peak in estrogen production 
that occurs prior to ovulation. In the three 
treated cows, there was still a moderate 
amount of circulating estrogen during and 
after the treatment period. However, the 
peak of estrogen from the dominant follicle 
that must occur before ovulation occurs 
was delayed in two of the three treated cows 
(Figure 1, Treated Cows 1 and 2). This sug-
gests the possibility that the processing of 
Letrozole by the liver may vary from animal 
to animal, thus making the effectiveness of 
the drug inconsistent.
The effects of Letrozole on the repro-
ductive cycle were also variable, with the 
successful suppression of the estrogen peak 
corresponding with a delay in ovulation. Of 
the three control cows that received vehicle 
injections only, two of the cows had a two- 
follicular- wave cycle and one of them had 
a three- wave cycle (Figure 2). The intervals 
methods using pharmaceuticals that are 
not hormone- based but instead control 
the synthesis of hormones in the treated 
animal. Having a lower concentration of 
estrogen within the bloodstream pre-
vents the sequence of events that result in 
ovulation (i.e. loss of the corpus luteum 
due to luteolysis, decreased circulating 
progesterone, and a surge in Luteinizing 
Hormone). Thus, some researchers have 
proposed Letrozole, an aromatase inhib-
itor that decreases estrogen production, 
as a steroid- free estrous synchronization 
method. These researchers have had suc-
cess in controlling the timing of ovulation. 
However, having lower concentrations 
of estrogens inside the dominant ovar-
ian follicle is associated with decreased 
oocyte quality and fertility. Plus, delaying 
ovulation with a method that changes 
gonadotropin pulsatility (as with altering 
circulating estrogen) can potentially cause 
the development of a persistent follicle 
and reduced quality of the oocyte within 
the follicle. To determine whether Letro-
zole treatment promotes persistent follicle 
formation, a pilot trial to test its effects 
on follicle development and circulating 
hormone concentrations was performed 
using a small group of beef cows.
Procedure
All procedures were approved by the 
Animal Care and Use Committee at the 
University of Nebraska- Lincoln. Six non- 
lactating, composite beef cows (25% MARC 
III [¼ Angus, ¼ Hereford, ¼ Pinzgauer, ¼ 
Red Poll] and 75% Red Angus) ages 2- 4 yr 
old from the beef physiology herd at the 
Eastern Nebraska Research and Extension 
Center (ENREC) were initially synchro-
nized with two injections (25 mg/cow; i.m.; 
12 h apart) of prostaglandin F2α (PGF2α) 
following by a second pair of PGF2α injec-
tions 14 d later. Transrectal ultrasound with 
an Aloka UST- 5541- 7.5 probe was conduct-
ed every other day for three weeks then dai-
ly for 50 d total, and the follicle and corpus 
Sarah M Romereim
Sarah C Tenley
Mohamed A. Abedal- Majed
Jeffrey W. Bergman
Scott G Kurz
John S Davis
Jennifer R Wood
Andrea S Cupp
Summary and Implications
Most bovine estrous synchronization 
protocols utilize progesterone plus estrogen 
to control ovulation timing. A drug that 
inhibits estrogen production (Letrozole) 
may be an alternative, steroid- free synchro-
nization method (not yet commercially 
available). However, low estrogen can neg-
atively affect the health of follicles/oocytes 
and impact fertility. To determine its effects, 
Letrozole was administered intramuscularly 
while tracking follicle growth and circu-
lating hormones. Letrozole response was 
variable. Two of three cows experienced 
delayed luteolysis/ovulation and extended 
progesterone production. This preliminary 
data indicates that Letrozole treatment 
allows normal follicle progression but drug 
response may vary and little is known about 
effects on oocyte quality.
Introduction
The benefits of reliable estrous syn-
chronization for timed artificial insem-
ination are well known. Since estrous is 
controlled primarily by the cyclical nature 
of steroid hormones, most synchroniza-
tion protocols administer steroids such 
as progesterone and estrogen to prevent 
ovulation until a desired time. However, 
there is a public desire to avoid hormone 
treatments in beef cattle including legal 
prohibition of estrogen use in some 
countries. For this reason, some research-
ers seek to devise new synchronization 
Letrozole: A Steroid- Free Estrous Synchronization Method
10 · 2018 Nebraska Beef Cattle Report
recommended to producers until further 
research assures the health and quality of 
the oocyte within the follicle and until larg-
er studies show that the animal- to- animal 
variability is acceptable.
Sarah Romereim, postdoctoral research 
associate
Sarah Tenley, former graduate student
Mohamed Abedal- Majed, graduate student
Jeffrey Bergman, agriculture research 
technician III
Scott Kurz, laboratory manager
John Davis, Professor, Department of 
OBGYN, University of Nebraska Medical 
Center, Omaha, NE
Jennifer Wood, associate professor Animal 
Science, Lincoln
Andrea Cupp, professor Animal Science, 
Lincoln
by inhibiting the animal’s own PGF2α 
production. Commercial PGF2α can then 
be administered to trigger luteolysis and 
the subsequent ovulation event. The results 
of this study indicate that there is little risk 
to the developmental progression of the 
follicle from the Letrozole treatment, since 
the dominant follicle during the treatment 
period will either ovulate or undergo atresia 
rather than becoming a persistent or cystic 
follicle. However, a follicle that ovulates af-
ter being exposed to Letrozole may not con-
tain a high quality oocyte. More research is 
needed to confirm that Letrozole does not 
adversely affect the health or capacity of the 
oocyte to be fertilized before it is incor-
porated into synchronization protocols. 
A controlled- release version of this drug 
(similar to a CIDR) has been patented, but 
this is not yet commercially available for 
timed AI purposes. This drug should not be 
between ovulation for the control cows 
were 20 d, 25 d, and 26 d. Comparatively, 
one Letrozole- treated cow had a two- wave 
cycle with no delay in ovulation (Cow 3, 21 
d), while the other two treated cows had 
three- wave cycles with ovulation intervals 
of 27 (Cow 2) and >32 (Cow 1, did not 
ovulate during the experimental period) 
(Figure 2). The ovulation delays were ac-
companied by an extended period of peak 
progesterone production and a delay in 
corpus luteum lysis. This suggests that the 
way in which Letrozole delays ovulation is 
by preventing/delaying luteolysis.
Conclusions
Letrozole estrous synchronization is 
feasible because the decreased circulat-
ing estrogen delays luteolysis, possibly 
Figure 1. Letrozole aromatase inhibition delays the circulating estrogen peak that occurs prior to estrus and ovulation. The top three graphs depict the daily es-
tradiol concentration (ng/mL) of the Control cows, and the bottom three graphs depict estradiol of the Letrozole- treated cows. The day of estrus is shown with a 
light- gray bar, and the six days of injections are depicted with dark- gray bars. Treated Cows 1 and 2 had delays in the pre- estrus estradiol peaks.
2018 Nebraska Beef Cattle Report · 11 
Figure 2. Letrozole- treated cows experience variable delayed ovulation and extended peak progesterone production. For the follicle diameter graphs, each 
point represents an individual follicle measurement and the tracked follicles are connected by lines. The top three graphs depict the ultrasound- tracked follicle 
measurements and ovulation (shown as a star- shaped icon) of the Control Cows, while the three graphs immediately below are the ultrasound- tracked corpus 
luteum diameter (primary vertical axis) and the circulating progesterone (ng/mL; secondary vertical axis) of the Control Cows. The six graphs at the bottom 
show the follicle measurements, corpus luteum measurements, and circulating progesterone for the Letrozole- treated cows. The day of estrus is shown with 
a light- gray bar, and the six days of injections are depicted with dark- gray bars. Treated Cows 1 and 2 had delayed luteolysis and extended peak progesterone 
concentrations.


Letrozole: A Steroid-Free Estrous Synchronization Method

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnNebraska Beef Cattle Reports Animal Science Department2018Letrozole: A Steroid-Free Estrous SynchronizationMethodSarah M. RomereimUniversity of Nebraska-Lincoln, sromereim2@unl.eduSarah C. TenleyUniversity of Nebraska - Lincoln, sctenley09@gmail.comMohamed A. Abedal-Majed MajedUniversity of Nebraska - LincolnJeffrey W. BergmanUniversity of Nebraska-Lincoln, jbergman1@unl.eduScott G. KurzUniversity of Nebraska-Lincoln, skurz2@unl.eduSee next page for additional authorsFollow this and additional works at: http://digitalcommons.unl.edu/animalscinbcrPart of the Large or Food Animal and Equine Medicine Commons, Meat Science Commons, andthe Veterinary Preventive Medicine, Epidemiology, and Public Health CommonsThis Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It hasbeen accepted for inclusion in Nebraska Beef Cattle Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.Romereim, Sarah M.; Tenley, Sarah C.; Abedal-Majed, Mohamed A. Majed; Bergman, Jeffrey W.; Kurz, Scott G.; Davis, John S.; Wood,Jennifer R.; and Cupp, Andrea S., "Letrozole: A Steroid-Free Estrous Synchronization Method" (2018). Nebraska Beef Cattle Reports.960.http://digitalcommons.unl.edu/animalscinbcr/960AuthorsSarah M. Romereim, Sarah C. Tenley, Mohamed A. Abedal-Majed Majed, Jeffrey W. Bergman, Scott G. Kurz,John S. Davis, Jennifer R. Wood, and Andrea S. CuppThis article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/animalscinbcr/9602018 Nebraska Beef Cattle Report · 9 ©  The Board Regents of the University of  Nebraska. All rights reserved.luteum dimensions were measured along the longest axis and the perpendicular axis (the average of those two dimensions is presented). Ovulation was determined by the absence of the preovulatory follicle. Estrus was detected using Estrotect™ Heat Detector patches on the tail head. Blood samples were collected every other day for the first week then daily, and circulating progesterone was measured with radioim-munoassays performed in duplicate. For this experiment, Day 1 of the reproductive cycle was defined as the day of ovulation detection after the second set of PGF2α in-jections. Treatment (250µg/kg Letrozole in 8- 10 mL of a 1:5 mixture of benzyl alcohol and sesame oil) or control injections (8 mL of 1:5 benzyl alcohol/sesame oil) began on Day 10 of the cycle.ResultsWhen using Letrozole to inhibit aro-matase activity, an important consideration is the success and degree of circulating estrogen suppression. The purpose of the drug is not to completely prevent all estrogen production but instead to prevent or delay the peak in estrogen production that occurs prior to ovulation. In the three treated cows, there was still a moderate amount of circulating estrogen during and after the treatment period. However, the peak of estrogen from the dominant follicle that must occur before ovulation occurs was delayed in two of the three treated cows (Figure 1, Treated Cows 1 and 2). This sug-gests the possibility that the processing of Letrozole by the liver may vary from animal to animal, thus making the effectiveness of the drug inconsistent.The effects of Letrozole on the repro-ductive cycle were also variable, with the successful suppression of the estrogen peak corresponding with a delay in ovulation. Of the three control cows that received vehicle injections only, two of the cows had a two- follicular- wave cycle and one of them had a three- wave cycle (Figure 2). The intervals methods using pharmaceuticals that are not hormone- based but instead control the synthesis of hormones in the treated animal. Having a lower concentration of estrogen within the bloodstream pre-vents the sequence of events that result in ovulation (i.e. loss of the corpus luteum due to luteolysis, decreased circulating progesterone, and a surge in Luteinizing Hormone). Thus, some researchers have proposed Letrozole, an aromatase inhib-itor that decreases estrogen production, as a steroid- free estrous synchronization method. These researchers have had suc-cess in controlling the timing of ovulation. However, having lower concentrations of estrogens inside the dominant ovar-ian follicle is associated with decreased oocyte quality and fertility. Plus, delaying ovulation with a method that changes gonadotropin pulsatility (as with altering circulating estrogen) can potentially cause the development of a persistent follicle and reduced quality of the oocyte within the follicle. To determine whether Letro-zole treatment promotes persistent follicle formation, a pilot trial to test its effects on follicle development and circulating hormone concentrations was performed using a small group of beef cows.ProcedureAll procedures were approved by the Animal Care and Use Committee at the University of Nebraska- Lincoln. Six non- lactating, composite beef cows (25% MARC III [¼ Angus, ¼ Hereford, ¼ Pinzgauer, ¼ Red Poll] and 75% Red Angus) ages 2- 4 yr old from the beef physiology herd at the Eastern Nebraska Research and Extension Center (ENREC) were initially synchro-nized with two injections (25 mg/cow; i.m.; 12 h apart) of prostaglandin F2α (PGF2α) following by a second pair of PGF2α injec-tions 14 d later. Transrectal ultrasound with an Aloka UST- 5541- 7.5 probe was conduct-ed every other day for three weeks then dai-ly for 50 d total, and the follicle and corpus Sarah M RomereimSarah C TenleyMohamed A. Abedal- MajedJeffrey W. BergmanScott G KurzJohn S DavisJennifer R WoodAndrea S CuppSummary and ImplicationsMost bovine estrous synchronization protocols utilize progesterone plus estrogen to control ovulation timing. A drug that inhibits estrogen production (Letrozole) may be an alternative, steroid- free synchro-nization method (not yet commercially available). However, low estrogen can neg-atively affect the health of follicles/oocytes and impact fertility. To determine its effects, Letrozole was administered intramuscularly while tracking follicle growth and circu-lating hormones. Letrozole response was variable. Two of three cows experienced delayed luteolysis/ovulation and extended progesterone production. This preliminary data indicates that Letrozole treatment allows normal follicle progression but drug response may vary and little is known about effects on oocyte quality.IntroductionThe benefits of reliable estrous syn-chronization for timed artificial insem-ination are well known. Since estrous is controlled primarily by the cyclical nature of steroid hormones, most synchroniza-tion protocols administer steroids such as progesterone and estrogen to prevent ovulation until a desired time. However, there is a public desire to avoid hormone treatments in beef cattle including legal prohibition of estrogen use in some countries. For this reason, some research-ers seek to devise new synchronization Letrozole: A Steroid- Free Estrous Synchronization Method10 · 2018 Nebraska Beef Cattle Reportrecommended to producers until further research assures the health and quality of the oocyte within the follicle and until larg-er studies show that the animal- to- animal variability is acceptable.Sarah Romereim, postdoctoral research associateSarah Tenley, former graduate studentMohamed Abedal- Majed, graduate studentJeffrey Bergman, agriculture research technician IIIScott Kurz, laboratory managerJohn Davis, Professor, Department of OBGYN, University of Nebraska Medical Center, Omaha, NEJennifer Wood, associate professor Animal Science, LincolnAndrea Cupp, professor Animal Science, Lincolnby inhibiting the animal’s own PGF2α production. Commercial PGF2α can then be administered to trigger luteolysis and the subsequent ovulation event. The results of this study indicate that there is little risk to the developmental progression of the follicle from the Letrozole treatment, since the dominant follicle during the treatment period will either ovulate or undergo atresia rather than becoming a persistent or cystic follicle. However, a follicle that ovulates af-ter being exposed to Letrozole may not con-tain a high quality oocyte. More research is needed to confirm that Letrozole does not adversely affect the health or capacity of the oocyte to be fertilized before it is incor-porated into synchronization protocols. A controlled- release version of this drug (similar to a CIDR) has been patented, but this is not yet commercially available for timed AI purposes. This drug should not be between ovulation for the control cows were 20 d, 25 d, and 26 d. Comparatively, one Letrozole- treated cow had a two- wave cycle with no delay in ovulation (Cow 3, 21 d), while the other two treated cows had three- wave cycles with ovulation intervals of 27 (Cow 2) and >32 (Cow 1, did not ovulate during the experimental period) (Figure 2). The ovulation delays were ac-companied by an extended period of peak progesterone production and a delay in corpus luteum lysis. This suggests that the way in which Letrozole delays ovulation is by preventing/delaying luteolysis.ConclusionsLetrozole estrous synchronization is feasible because the decreased circulat-ing estrogen delays luteolysis, possibly Figure 1. Letrozole aromatase inhibition delays the circulating estrogen peak that occurs prior to estrus and ovulation. The top three graphs depict the daily es-tradiol concentration (ng/mL) of the Control cows, and the bottom three graphs depict estradiol of the Letrozole- treated cows. The day of estrus is shown with a light- gray bar, and the six days of injections are depicted with dark- gray bars. Treated Cows 1 and 2 had delays in the pre- estrus estradiol peaks.2018 Nebraska Beef Cattle Report · 11 Figure 2. Letrozole- treated cows experience variable delayed ovulation and extended peak progesterone production. For the follicle diameter graphs, each point represents an individual follicle measurement and the tracked follicles are connected by lines. The top three graphs depict the ultrasound- tracked follicle measurements and ovulation (shown as a star- shaped icon) of the Control Cows, while the three graphs immediately below are the ultrasound- tracked corpus luteum diameter (primary vertical axis) and the circulating progesterone (ng/mL; secondary vertical axis) of the Control Cows. The six graphs at the bottom show the follicle measurements, corpus luteum measurements, and circulating progesterone for the Letrozole- treated cows. The day of estrus is shown with a light- gray bar, and the six days of injections are depicted with dark- gray bars. Treated Cows 1 and 2 had delayed luteolysis and extended peak progesterone concentrations.

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Letrozole: A Steroid-Free Estrous Synchronization Method

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