Theriogenology 141 (2020) 180e185

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

Effects of recombinant bovine somatotropin on pregnancy per

artificial insemination, corpus luteum cellular composition and
endometrial gland morphometry in beef cattle

lia Paulozzi Costa a, Angela Gonella-Diaza b, Guilherme Pugliesi c,
Nata
Maria^ngela Bueno Cordeiro Maldonado d, Saara Carollina Scollari c,
^ Castilho g,
Barbara Piffero Mello c, Isabella Feltrin e, Renato Girotto f, Calie
d , e, *
Claudia Maria Bertan Membrive
a ~o Paulo State University - UNESP, Araçatuba, SP, 16015-050, Brazil
Sa
b
University of Florida, North Florida Research & Education Center, Institute of Food and Agricultural Sciences, Marianna, FL, USA
c ~o Paulo, Pirassununga, SP, 13635-900, Brazil
University of Sa
d ~
Sao Paulo State University - UNESP, Dracena, SP, 17900-000, Brazil
e ~
Sao Paulo State University - UNESP, Botucatu, SP, 18618-689, Brazil
f

RG Advanced Genetics, Agua Boa, MT, 78635-000, Brazil
g ~o Paulo West, Presidente Prudente, SP, 19050-920, Brazil
University of Sa

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

Article history: The aim of this research was to evaluate the effect of recombinant bovine somatotropin (bST) on
Received 6 February 2019 pregnancy per artificial insemination (P/AI), cellular composition of the corpus luteum (CL) and endo-
Received in revised form metrial gland morphometry. In Experiment 1, Nelore cows (n ¼ 587) received a fixed-time artificial
11 September 2019
insemination (FTAI) protocol and, at insemination, received 0, 250 or 500 mg of bST subcutaneously (SC).
Accepted 16 September 2019
Available online 17 September 2019
In Experiment 2, Nelore cows (n ¼ 243) received 0 or 500 mg of bST, SC, on D7 (D0 ¼ day of FTAI). Blood
samples were collected on D7 and D16 to measure progesterone (P4) concentrations. In Experiments 1
and 2, pregnancy diagnosis was performed 30 days after FTAI. In Experiment 3, Nelore heifers (n ¼ 20)
Keywords:
bST
received a FTAI protocol, but were not inseminated, and on D0 (ovulation day), they received 0 (bST 0;
Fertility n ¼ 9) or 500 mg of bST (bST 500; n ¼ 11), SC. The heifers were slaughtered on D15 (D0 ¼ ovulation day),
Corpus luteum at which time the CL was evaluated for diameter, weight, a percentage of large (LLC) and small (SLC)
Endometrium luteal cells, and the concentration of progesterone in plasma measured. The number, perimeter and area
Bos indicus of superficial and deep endometrial glands were evaluated. There was no difference in P/AI when bST
was applied on D0 and D7. In Experiment 1, P/AI did not differ among treatments, with 59.28% (115/194),
58.38% (115/197) and 65.82% (129/196) for the bST 0, 250 and 500 treatments, respectively. In Experi-
ment 2, P/AI did not differ between treatments, with 57.3% (71/124) and 60.5% (62/119) for the bST 0 and
500 treatments, respectively. Plasma progesterone concentrations on D16 was greater in the bST 500
(11.63 ± 0.84 ng/mL) than bST 0 (9.83 ± 0.88 ng/mL). In Experiment 3, there was no difference in ovarian
diameter and weight, CL diameter, percentage of SLC, P4 concentrations and endometrial gland
morphology. Heifers in the bST 500 treatment had heavier CL (3.11 ± 0.32 vs. 2.25 ± 0.20 g); however, the
bST 0 treatment heifers had a greater percentage of LLC than did the bST 500 treatment (13.72 ± 1.16% vs.
8.60 ± 1.52). It was concluded that the doses of bST used in this study do not increase P/AI; however, they
do cause changes in P4 concentration and the cellular composition of the CL.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction

~o Ribeiro de Barros (SP 294),

* Corresponding author. Rodovia Comandante Joa
~o Paulo State University (UNESP), Dracena, SP, 17900-000, Brazil.
Km 651, Sa
E-mail address: claudia.bertan@unesp.br (C.M. Bertan Membrive).
The use of hormonal protocols for fixed-time artificial insemi-
nation (FTAI) provides a service rate of 100% without the need for
estrous detection. In addition, FTAI induces cyclicity in females

https://doi.org/10.1016/j.theriogenology.2019.09.023
0093-691X/© 2019 Elsevier Inc. All rights reserved.
 N.P. Costa et al. / Theriogenology 141 (2020) 180e185
during the postpartum anestrus [1,2]. Despite the fact that FTAI is
currently used on a large scale, the pregnancy per artificial
insemination with this biotechnology in commercial beef herds in
Brazil is 49.1% [2,3]. In dairy and beef bovine herds, early embryonic
mortality is a major cause of reproductive failure [4,5]. In beef cows,
fertilization rates normally range from 94.0 to 100% while the major
portion of early embryo loss occurs after day 8 and before days
16e18 after FTAI. During this period, the embryos must elongate
and produce interferon Tau to prevent luteolysis [6]. During this
period, 20e40% embryonic losses are reported in the female zebu,
most of which are attributed to the inability of the conceptus
(embryo and the extra-embryonic membranes) to signal pregnancy
recognition [7].
In bovine, the uterus and the embryo have receptors for growth
hormone (GH, also known as somatotropin) and insulin-like
growth factor (IGF-I), which, through endocrine, paracrine and
autocrine actions, have important impacts during pregnancy
establishment and maintenance [8e10]. Indeed, experiments per-
formed in vivo and in vitro have highlighted the positive effects of
GH and IGF-I on uterine functions, increasing mRNA expression for
proteins that form the histotroph and on conceptus development
during the pre- and peri-implantation stages in many species
[11e15]. Recombinant bovine somatotropin (bST) is commonly
utilized in dairy operations to improve the milk production of
lactating dairy cows [16]. The administration of bST increases
plasma concentrations of IGF-1 in both beef and dairy cattle
[17e19]. However, the literature presents contradictory results
regarding the effect of bST on improving fertility, specifically P/AI
[17,20]. Ribeiro et al. [21] reported that low doses of bST during the
pre- and peri-implantation periods enhanced conceptus develop-
ment, reduced embryonic losses and improved fertility in dairy
cows. They also reported an increase of IGF-I for two weeks after
applying 325 mg of bST on D0, and for four weeks when applying it
on D0 and D14; however, such doses were not sufficient to alter the
plasma progesterone concentrations. Also, Moreira et al. [22]
administered 500 mg of bST to embryo donors and/or recipients
and found that the administration of bST increased the percentage
of transferable embryos, the number of blastocysts obtained by
uterine flush, and pregnancy per embryo transfer. In contrast,
Rivera et al. [20], using primiparous and multiparous Holstein cows,
studied the effect of 6 consecutive subcutaneous injections of
500 mg of bST given at 10-d intervals, starting around 60 days
postpartum. They concluded that this bST treatment improved
lactation performance but had no effect on P/AI.
In beef cattle, some researchers have evaluated the effect of bST
on P/AI using different doses and times of application. Starbuck
et al. [23] administered 500 mg bST at the time of AI on lactating
dairy cows, dairy heifers and lactating beef cows; they found an
increase in conception rates only in dairy cows. Mercadante et al.
[19] administered a dose of 325 mg of bST at TAI, 14 days after TAI or
on both days to multiparous, suckled beef cows. They found an
increase in IGF1 plasmatic concentration; however, they did not
report any improvement in P/AI, fetal size or plasma progesterone.
Oosthuizen et al. [24] used 650 mg of bST at the moment of CIDR
insertion (9 days before TAI) in crossbred beef heifers. They re-
ported greater P/AI in the control group (no bST) compared to bST
heifers. Cooke et al. [25] evaluated the effect of 250 mg of bST in
pubertal beef heifers on plasmatic glucose, insulin, IGF1 and pro-
gesterone concentration. They found that bST failed to affect the
concentrations of insulin and P4, but increased those of glucose and
IGF1.
In this context, it was hypothesized that treatment with bST on
the day of FTAI or 7 days later would increase P/AI in Bos indicus
cows compared with untreated controls. It has also been hypoth-
esized that bST could promote an increase in the size of the
181
endometrial glands, but not in the size of the corpus luteum (CL) or
plasma progesterone concentrations. In order to test our hypothe-
ses, three experiments were designed. The aim of Experiment 1 was
designed to evaluate pregnancy per artificial insemination (P/AI) in
cows treated with 0, 250 or 500 mg of bST on the day of the FTAI
(Day 0). In Experiment 2, the aim was to evaluate the effect of
administering 500 mg of bST 7 days after the FTAI (Day 7). Finally, in
Experiment 3, the aim was to evaluate the effect of 500 mg bST,
administered on the ovulation day (Day 0), on the cellular
composition of the CL and the morphometry of the endometrial
glands 15 days after administration (Day 15) in heifers.
2. Materials and methods
2.1. Experiment 1
2.1.1. Animals and management
All experimental procedures were approved by the Ethics
Committee on Animal Experimentation of Sa ~o Paulo State Univer-
~o Paulo, Brazil (Protocol number
sity (UNESP), Araçatuba, state of Sa
2013/00602). The experiment was conducted in Araguaiana, state
of Mato Grosso, Brazil. Multiparous Nelore cows (Bos taurus indicus;
n ¼ 587) between 40 and 70 days postpartum were used in this
experiment. The means and standard deviation (SD) for body
condition score (scale 1e5, where 1 ¼ emaciated and 5 ¼ over-
conditioned) were 3.01 þ 0.21 for all treatments. The cows were
kept on Brachiaria brizantha pastures with water and mineral
supplementation ad libitum. The experiment was conducted using a
randomized block design: first cows were randomly divided into
eight lots for management purposes and all were submitted to FTAI
on the same day. Then, within each lot, cows were assigned
randomly to the bST 0, bST 250 or bST 500 treatments.
2.1.2. Synchronization of ovulation
Intravaginal devices containing 1 g of progesterone (DIB®; MSD
Animal Health, Sa ~o Paulo, SP, Brazil) were used, associated with the
intramuscular (IM) injection of 2 mg of estradiol benzoate (Gona-
diol®, 1 mg/mL; MSD Animal Health) on the day of the device
insertion. The devices were removed after 8 days, at which time the
cows were treated with 0.530 mg of sodium D-cloprostenol (Cio-
sin®, 0.265 mg/mL; MSD Animal Health) and 300 IU of equine
chorionic gonadotropin (eCG; Novormon®, 200 IU/mL; MSD Ani-
mal Health). Cows were treated with 1 mg of estradiol benzoate
(Gonadiol®; MSD Animal Health) IM 24 h after removal of the de-
vices; 30 h after the last injection, the cows were submitted to FTAI.
Ovaries were examined using ultrasonography (Aloka Ultrasound
Diagnostic Equipment, Model SSD-500, Tokyo, Japan) on the day of
the progesterone device placement and 30% of the cows presented
a visible CL.
2.1.3. Fixed timed artificial insemination procedure
AI was performed using frozen semen straws containing sperm
from 3 Aberdeen Angus bulls. Inseminations were performed by
three inseminators, equally divided between the different
treatments.
2.1.4. Treatment with bST
During the FTAI procedure, cows were randomly assigned into
one of the experimental treatments. The cows received 0 mg (bST
0; n ¼ 194), 250 mg (bST 250; n ¼ 197) or 500 mg (bST 500;
n ¼ 196) of bST (Boostin®, 250 mg/mL; MSD Animal Health),
administered subcutaneously (SC) in the ischiorectal fossa.
2.1.5. Pregnancy diagnosis
Pregnancy diagnosis was performed 30 days after FTAI (D30)
182 N.P. Costa et al. / Theriogenology 141 (2020) 180e185
using ultrasonography (Aloka Ultrasound Diagnostic Equipment,
Model SSD-500, Tokyo, Japan) with a 5 mHz linear transducer. Fe-
males were classified as pregnant (presence of an amniotic vesicle
containing a live embryo) or not pregnant.
2.1.6. Statistical analysis
Data were analyzed by logistic regression (GLIMMIX procedure
of SAS Institute, Cary, NC, USA). The initial model for the analysis of
P/AI included the effects of treatment, lot, body condition score and
interactions. In order to obtain the final statistical model, the
explanatory variables were sequentially removed according to the
Wald statistic criterion, removing variables with P > 0.20; in the
end, only the effect of treatment remained in the model. In the
analysis, P < 0.05 was considered significant.
2.2. Experiment 2
2.2.1. Animals and management
All experimental procedures were approved by the Ethics
Committee on Animal Experimentation of the UNESP, Dracena, S~ ao
Paulo state, Brazil (Protocol number 02/2008). The experiment was
conducted in Sa ~o Fe
lix do Araguaia, Mato Grosso state, Brazil.
Multiparous Nelore cows (Bos taurus indicus; n ¼ 243) between 35
and 70 days postpartum were used. The means and SD for body
condition score (scale 1e5, where 1 ¼ emaciated and 5 ¼ over-
conditioned) were 2.82 ± 0.23 and 2.9 ± 0.02 for the bST 0 and bST
500 treatments, respectively. The females were kept on Brachiaria
brizantha and Panicum maximum pastures with water and mineral
supplementation ad libitum.
2.2.2. Synchronization of ovulation
Intravaginal devices containing 1 g of progesterone (DIB®; MSD
Animal Health) were used, associated with an IM injection of 2 mg
of estradiol benzoate (Gonadiol®; MSD Animal Health) on the day
of the placement of the devices. The devices were removed after 8
days, when the cows were treated with 112.5 mg of D-cloprostenol
(Preloban®; 0.075 mg/Ml; Intervet Schering-Plough, S~ ao Paulo,
Brazil) and 300 IU of eCG (Folligon®; 200 IU/Ml; Intervet Schering-
Plough), IM. Cows were treated with 1 mg of estradiol benzoate IM
(Gonadiol®; MSD Animal Health) 24 h after removal of the devices
and were submitted to FTAI 30 h after the last injection.
2.2.3. FTAI procedure
AI was performed using the semen of 14 bulls. Each bull was
distributed equally among all the treatments and randomly be-
tween the cows. Inseminations were performed by two farm
technicians, who were also distributed equally among all the
treatments.
2.2.4. Treatment with bST
Seven days after FTAI (D7), cows were randomly distributed to
receive 0 mg (bST 0; n ¼ 124) or bST (Boostin®; MSD Animal
Health) at a dose of 500 mg (bST 500; n ¼ 119), SC in the ischiorectal
fossa.
2.2.5. Collection of blood samples
A random selection of 39.5% (96/243) of the cows was used to
perform progesterone concentration analyses. Blood collections
were performed on D7 and D16 after FTAI. Approximately 4 mL of
blood was collected with a vacutainer (Becton, Dickinson and
Company; BD) containing 175 mL of 30% sodium citrate by veni-
puncture of the median caudal vein or artery; these were stored at
4  C. The samples were centrifuged at 2900g for 30 min at room
temperature (Centribio Centrifuge, Model 80-2B). After centrifu-
gation, the plasma was removed, put into microtubes and stored
at 20  C.
2.2.6. Radioimmunoassay for the measurement of progesterone
The progesterone concentrations were measured by radioim-
munoassay using commercial kits (Count-a-Count®; DPC, Diag-
nostic Products Corporation, Los Angeles, USA). Samples were
measured in three runs; samples containing standard high con-
centrations (HCS) and low concentrations (LCS) of progesterone
were used. For HCS, the intra-assay coefficients of variation were
1.60, 1.56 and 1.39% for runs 1, 2 and 3, respectively. For LCS, the
intra-assay coefficients of variation were 0.15, 0.19 and 0.11% for
runs 1, 2 and 3, respectively. The inter-assay coefficients of variation
were 5.30% for HCS and 4.54% for LCS. The sensitivity limits for runs
1, 2 and 3 were 0.058, 0.054 and 0.158 ng/mL, respectively.
2.2.7. Pregnancy diagnosis
Pregnancy diagnosis was performed 30 days after FTAI (D30)
using ultrasonography (Aloka Ultrasound Diagnostic Equipment,
Model SSD-500, Tokyo, Japan) with a 5 mHz linear transducer. Fe-
males were classified as pregnant (presence of an amniotic vesicle
containing a live embryo) or not pregnant.
2.2.8. Statistical analysis
Statistical analysis was performed using SAS statistical software.
The initial model for the analysis of P/AI included the effects of
treatment, lot, body condition score and interactions. In order to
obtain the final statistical model, the explanatory variables were
sequentially removed according to the Wald statistic criterion,
removing variables with P > 0.20; in the end, only the effect of
treatment remained in the model. In the analysis, P < 0.05 was
considered significant. Using the PROC MIXED procedure of SAS,
ANOVA analyses were used to compare the progesterone concen-
trations on D7 and D16 between the 0 and 500 mg of bST groups.
Therefore, progesterone concentrations on D7 were used as the
covariate in the statistical analysis of progesterone on Day 16. The
residuals were checked for heteroscedasticity, which proved
adequate. The significance level used for all data was P  0.05. All
results were presented as means ± standard errors of the mean
(SEM).
2.3. Experiment 3
2.3.1. Animals and management
All experimental procedures were approved by the Ethics
Committee on Animal Experimentation of the UNESP, Araçatuba/SP,
Brazil (Protocol number 2013/00602). The experiment was con-
~o Paulo, Brazil. Non-pregnant, cyclic
ducted in Birigui, state of Sa
nulliparous Nelore heifers (Bos taurus indicus; n ¼ 24) were used. Of
the 24 heifers, 20 ovulated and remained in the study. Females
were previously evaluated for age, weight and body condition
score. The means and SD for body condition score (scale 1e5, where
1 ¼ emaciated and 5 ¼ over-conditioned) were 2.87 ± 0.31 for both
treatments. Ovarian condition was evaluated by ultrasonography
(Aloka Ultrasound Diagnostic Equipment, Model SSD-500, Tokyo,
Japan) on the day of placement of the devices, when 40% of females
presented a CL. The females were kept in confinement, receiving a
diet containing 40.7% sugar cane, 28.3% corn germ, 16.7% orange
pulp, 11.3% cottonseed meal, 1% urea and 2% mineral core.
2.3.2. Synchronization of ovulation
Intravaginal devices containing 1 g of progesterone (DIB®; MSD
Animal Health) were used, associated with an IM injection of 2 mg
of estradiol benzoate (Gonadiol®; MSD Animal Health) on the day
of placement of the devices. The devices were removed after 8 days,
when cows were treated with 0.530 mg of D-cloprostenol (Ciosin®;
 N.P. Costa et al. / Theriogenology 141 (2020) 180e185
MSD Animal Health) and 300 IU of eCG (Novormon®; MSD Animal
Health). The heifers were treated with 1 mg of estradiol benzoate
(Gonadiol®; MSD Animal Health) intramuscularly 24 h after the
removal of the devices. Considering that ovulation occurs 36e40 h
after the administration of estradiol benzoate, D0 was determined
to be the day of ovulation. Ovulation was confirmed by ultraso-
nography 48 h after the application of estradiol benzoate. The fe-
males were not inseminated.
2.3.3. Treatment with bST
Thirty hours after the last injection of estradiol benzoate
(D0 ¼ ovulation day), heifers received 0 mg (bST 0; n ¼ 9) or bST
(Boostin®; MSD Animal Health) at a dose of 500 mg (bST 500;
n ¼ 11), applied SC in the ischiorectal fossa. The heifers were
slaughtered on D15 (D0 ¼ ovulation day).
2.3.4. Collection of blood samples
Blood collections were performed immediately after slaughter.
Approximately 4 mL of blood was collected with a vacutainer
(Becton, Dickinson and Company; BD) containing 175 mL of 30%
sodium citrate by venipuncture of the median caudal vein or artery;
these were stored at 4  C. The samples were subjected to centri-
fugation at 2900g for 30 min at room temperature (Centribio
Centrifuge, Model 80-2B). After centrifugation, the plasma was
removed, put into microtubes and stored at 20  C.
2.3.5. Radioimmunoassay for the measurement of progesterone
The progesterone concentrations were measured by radioim-
munoassay using commercial kits (Count-a-Count®; DPC, Diag-
nostic Products Corporation, Los Angeles, USA). All samples were
analyzed in a single run. Samples containing standard high con-
centrations (HCS) and low concentrations (LCS) of progesterone
were used. For HCS and LCS, the intra-assay coefficient of variation
was 1.49% and 1.62%, respectively. The sensitivity limit for the assay
was 0.15 ng/mL.
2.3.6. Collection of luteal and uterine tissue
Immediately after slaughter, the ovaries were removed, weighed
and their diameters were measured separately. The CL was isolated,
weighed and its diameter was measured. A segment of the uterine
horn was removed in the mid-third of the horn, ipsilateral to the CL.
Both tissues were immediately packaged in buffered 4% formalin
(pH 7.0) for 24 h. The fragments were washed six consecutive times
every 12 h with PBS solution pH 7.5.
2.3.7. Histological preparation of tissues
The tissues were dehydrated by immersion in 70% alcohol for
60 min, then 90% alcohol for 60 min, followed by two 60-min
washes in absolute alcohol, and, finally, washed twice for 60 min
in xylene. The tissue was ‘embedded’ in paraffin. The CL was cut
into 6- to 8-mm-thick cross sections and the uterine segment was
cut into 4-mm-thick endometrial sections. Slides were placed in the
incubator for 2 h at temperatures of 56e58  C, then stained with
hematoxylin-eosin (HE).
2.3.8. Assessment of steroidogenic luteal cells
The stained slides were subjected to a computerized image
analysis system (Leco 2001, MI, USA), coupled with a camera (ZEISS
Axioplan 2, Model: MC 80 DX - Microscope Camera, Zeiss, Ober-
kochen, Germany). Using a light microscope, size, shape and
morphology were used to distinguish luteal cells from non-luteal
cells, as well as small luteal cells (SLC) from large luteal cells
(LLC). LLC appear polyhedral in shape and contain lipid droplets,
whereas SLC were stained darker than LLC cells. The cell diameter
was determined by individual distance measurements. The large
183
and small diameters of each cell were individually measured, and
the mean between both measurements was taken in order to rank
each cell. An arbitrary size cutoff of 20 mm was used for separating
SLC from LLC [26,27]. To estimate the percentages of SLC and LLC,
200 cells were counted in 16 slides/animal and a single mean per
cow was calculated; this evaluation was performed automatically
using Windows Select Measurements by measuring randomly
selected fields.
2.3.9. Morphometric analysis of endometrial glands
After obtaining the histology slides, they were subjected to
computerized image analysis programmed in a light microscope
with a camera (ZEISS Axioplan 2, Model: MC 80 DX - Microscope
Camera). Photos of 10 randomly selected optical fields were taken,
five of which were from the endometrial superficial region and five
from the deep region, with a 10x magnifying lens. Afterward, the
photos were analyzed using the software Image-Pro® PLUS-The
Proven Solution™ to calculate the number, perimeter (mm) and
area (mm2) of ducts of the endometrial glands in the superficial
endometrium and the deep endometrium.
2.3.10. Statistical analysis
Statistical analysis was performed using SAS statistical software.
The residuals were checked for heteroscedasticity, which proved
adequate; only the progesterone concentration data were trans-
formed using a natural logarithm. Using the GLM procedure, the
effect of the treatment on the dependent variables was evaluated
with an ANOVA. The treatment effect (0 or bST 500 treatment) was
evaluated, considering cow as a random effect and cow within each
treatment. The cows were considered as an experimental unit. In all
analyses, a level of P  0.05 was considered significant. All results
were presented as means ± standard errors of the mean (SEM).
3. Results
3.1. Experiment 1
As shown in Table 1, pregnancy per artificial insemination did
not differ between treatments (P ¼ 0.26). These values were 59.28%
(115/194), 58.38% (115/197) and 65.82% (129/196) for the bST 0, bST
250 and bST 500 treatments, respectively.
3.2. Experiment 2
As shown in Table 1, pregnancy per artificial insemination did
not differ between treatments (P ¼ 0.59) and was 57.3% (71/124)
and 60.5% (62/119) for the bST 0 and bST 500 treatments, respec-
tively. Concentrations of progesterone in plasma on D7, before
Table 1
Pregnancy per AI (P/AI) in Nelore cows subjected to fixed timed artificial insemi-
nation and treated with either 0, 250 or 500 mg of bovine somatotropin on Day 0 at
AI (Experiment 1) or treated with 0 or 500 mg of bovine somatotropin on Day 7
(Experiment 2).
Variable Pregnancy per AI (P/AI) P value
Experiment 1
bST 0 59.28% (115/194)
bST 250 58.38% (115/197)
bST 500 65.82% (129/196)
P ¼ 0.26
Experiment 2
bST 0 57.3% (71/124)
bST 500 60.5% (62/119)
P ¼ 0.59
184 N.P. Costa et al. / Theriogenology 141 (2020) 180e185
treatments were applied, did not differ between bST 0 or bST 500
(4.73 ± 0.33 ng/mL vs. 5.48 ± 0.38 ng/mL) treatments. Plasma pro-
gesterone concentrations on D16 were smaller than 1.0 ng/mL in 5
out of 96 cows evaluated (4 from bST 0 and 1 from bST 500 group),
indicating that the cows underwent luteolysis by D16. Therefore,
when considering only those cows with an active CL based on
concentration of progesterone in plasma greater than 1.0 ng/mL on
D16 were again greater (P ¼ 0.02) in the bST 500 treatment
(11.63 ± 0.84 ng/mL) than in the bST 0 treatment (9.83 ± 0.88 ng/
mL).
3.3. Experiment 3
As shown in Table 2, there were no differences between the bST
0 and the bST 500 treatments for the diameter and weight of the
ovary, diameter on the CL, and concentration of progesterone in
plasma. The bST 500 group had a heavier CL compared to the bST
0 treatment (P ¼ 0.04). There was no difference between the bST
0 and the bST 500 treatment for the percentage of SLC
(86.28 ± 1.39% vs. 91.40 ± 1.52%, respectively; P ¼ 0.07). However,
the percentage of LLC was lower in the bST 500 treatment than in
the bST 0 treatment (8.60 ± 1.52 vs. 13.72 ± 1.16%, respectively;
P ¼ 0.01). There were no significant differences between the bST
0 and bST 500 treatments in terms of the number, perimeter and
area of the superficial endometrial glands, deep endometrial glands
and the total endometrial glands.
4. Discussion
The present experiment evaluated the effects of a single dose of
bST during FTAI protocols on beef cattle. In experiments 1 and 2, the
P/AI did not differ between treatments. The use of bST in FTAI
protocols has contradictory results in the literature. Albuquerque
et al. [28] used a similar synchronization protocol in Nelore cows to
the one we used here, with an additional administration of 167 and
333 mg of bST on the day of the FTAI. These same authors found P/
AI of 37.4% in the 0 mg treatment, 45.4% in the 167 mg treatment,
and 46% in the 333 mg treatment; they found a significant treat-
ment effect but not a dose effect. In a second experiment, the same
authors used two administrations of 333 mg of bST the day of FTAI
and 11 days after. The P/AI was 48.0% in the control and 42.3% in the
treatment. This could indicate that bST improve the P/AI only when
the control group has very low results, meaning cows with low BCS
or nutritional deficiencies. In the current study, the bST 0 group
Table 2
Experiment 3: Means ± standard errors of the mean (SEM) of ovarian and luteal diameter and weight, luteal and endometrial microscopic characteristics and progesterone
concentrations in heifers who received 30 h after the last injection of estradiol benzoate, 0 or 500 mg bST, applied SC in the ischiorectal fossa, slaughtered on D15
(D0 ¼ ovulation day).
Variable bST 0 (n ¼ 9)
Ovarian diameter (cm) 2.06 ± 0.13
Ovarian weight (g) 5.61 ± 0.66
Luteal diameter (cm) 1.33 ± 0.06
Luteal weight (g) 2.25 ± 0.20
Large luteal cells (% of total luteal cells) 13.72 ± 1.16
Small luteal cells (% of total luteal cells) 86.28 ± 1.39
Number of superficial gland ducts 16.46 ± 3.14
Mean superficial gland duct perimeter (mm) 247.66 ± 18.23
Mean superficial gland duct area (mm2) 3955.52 ± 487.68
Number of deep gland ducts 28.75 ± 3.25
Mean deep gland duct perimeter (mm) 189.25 ± 7.53
Mean deep gland duct area (mm2) 2110.30 ± 114.14
Total gland duct density 46.22 ± 5.86
Mean total gland duct perimeter (mm) 456.96 ± 28.04
Mean total gland duct area (mm2) 6571. 59 ± 765.12
Progesterone concentrations (ng/mL) 11.06 ± 1.43
presented higher pregnancy per artificial insemination than those
reported for commercial herds, which leads to the assumption that
cows with low body condition scores may possibly be favored in the
use of bST.
In several studies, the use of bST was found to promote a gradual
increase in the plasma concentrations of GH and IGF-I during the
first 7 days, when the peak of these hormones is observed, with a
continued daily gradual decline [21,29]. Thus, it is assumed that the
application of bST at the time of artificial insemination promotes a
sustained increased in concentrations of GH and IGF-I during the
period coinciding with the beginning of embryonic development
through the seventh day. Kolle et al. [8] demonstrated that GH
receptors are present in the embryo beginning from the two-cell
stage, when it acts on glycogen metabolism and the transport of
lipids to the embryo. Spencer et al. [12] showed that the glandular
epithelium of the ovine uterus responded to the intrauterine
administration of GH by increasing the mRNA expression of the
proteins that constitute the histotroph; this condition favored a
higher production of IFN-t. However, when the administration of
bST is performed 7 days after FTAI, it is assumed that the greatest
effects would occur from the 7th to the 14th days of the develop-
ment of the conceptus, which would be the elongation stage.
Ribeiro et al. [21] observed that treating dairy cows with 350 mg of
bST on the day of artificial insemination did not alter the size of the
conceptus or fertility; however, administration of 350 mg of bST on
the day of AI and 14 days later increased the development of the
conceptus, reduced embryo mortality and increased fertility. The
authors found that the use of two doses increased the longitudinal
diameter of the amniotic vesicle and the size of the fetus at 34 and
48 days of pregnancy; however, there was no effect on the P/AI. The
increase of cellular proliferation and elongation of the conceptus
are extremely important in order to prevent luteolysis and main-
tain pregnancy [12]. Such associated events potentiate embryo
development, increasing the chances of embryo survival. Consid-
ering these reports, one would expect an increase in P/AI in the
present experiment; however, this did not hold true. Rossetti et al.
[30] also found that the application of 500 mg of bST 7 days after
FTAI did not increase the conception P/AI rates in Nelore cows,
which were 57.3% in placebo and 61.1% in the bST group. Even when
bST was associated with the application of flunixin meglumine and/
or human chorionic gonadotropin (hCG), 16 days after the FTAI,
there was no increase in fertility or the concentration of P4.
In Experiment 3, the use of a single dose of 500 mg of bST on D0
failed to promote changes in ovaries size and weight; however, bST
bST 500 (n ¼ 11) P value
2.27 ± 0.09 0.21
6.50 ± 0.48 0.28
1.54 ± 0.08 0.06
3.11 ± 0.32 0.04
8.60 ± 1.52 0.01
91.40 ± 1.52 0.07
17.34 ± 2.03 0.97
251.53 ± 17.34 0.88
3885.93 ± 476.58 0.92
29.94 ± 1.69 0.74
198.82 ± 10.83 0.50
2153.33 ± 196.11 0.86
47.25 ± 2.64 0.86
453.05 ± 19.63 0.97
6052.43 ± 555.09 0.58
9.92 ± 1.43 0.58
 N.P. Costa et al. / Theriogenology 141 (2020) 180e185
led to an increase in the weight of the CL and plasma progesterone
concentrations on D16 were greater in the bST 500 treatment than
in the bST 0 treatment (11.63 ± 0.84 ng/mL vs. 9.83 ± 0.88 ng/mL).
As noted in the present study, some in vivo and in vitro studies
have shown that bST favors the growth of luteal tissue. GH re-
ceptors (transcripts and proteins) have been previously found in
the CL and LLC [31]. The administration of bST also has been shown
to increase the weight of the CL [31], but it did not increase the
plasma concentrations of P4 in dairy cows [21]. Lucy et al. [31]
treated cows with 25 mg of bST daily for 16 days after estrus and
then, on day 17, cows were slaughtered to collect ovaries, uterus
and the conceptus; bST increased CL weight and the number of
large follicles (10e15 mm in diameter). In this study, note that
differences in ovulatory follicle diameter may have resulted in
differences in CL size and weight, as a function of the number of
granulosa and theca cells that were luteinized, which would not be
related to treatments. Later studies, however, have failed to show a
positive effect of bST on the CL during the peri-ovulatory period
[31]. In the present study, bST led to an increase in the weight of the
CL and a decrease in the percentage of LLC in heifers. Hence, bST
altered the cellular composition of the CL, but not the concentration
of P4. In fact, experiments that showed improvements in P/AI with
bST did not associate the positive effects on fertility to be mediated
by increased concentrations of P4 [21].
We conclude that the doses of bST used in this study do not
promote increased P/AI, although bST altered the cellular compo-
sition of the CL and increased the P4 concentration in the late
diestrus.
Acknowledgements
The authors wish to acknowledge the Coordination for the
Improvement of Higher Education Personnel (CAPES), the Devel-
opment Foundation of UNESP (FUNDUNESP) and MSD Animal
Health.
References
[1] Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows
using PGF2alpha and GnRH. Theriogenology 1995;44(7):915e23.
[2] Baruselli PS, Sales JNS, Sala RV, Vieira LM, Sa Filho MF. History, evolution and
perspectives of timed artificial insemination programs in Brazil. Anim Reprod
2012;9(3):139e52.
[3] Meneghetti M, Sa Filho OG, Peres RFG, Lamb GC, Vasconcelos JLM. Fixed-time
artificial insemination with estradiol and progesterone for Bos indicus cows I:
basis for development of protocols. Theriogenology 2009;72(2):179e89.
[4] Santos JE, Thatcher WW, Chebel RC, Cerri RL, Galvao KN. The effect of em-
bryonic death rates in cattle on the efficacy of estrus synchronization pro-
grams. Anim Reprod Sci 2004;82e83:513e35.
[5] Breuel KF, Lewis PE, Schrick FN, Lishman AW, Inskeep EK, Butcher RL. Factors
affecting fertility in the postpartum cow: role of the oocyte and follicle in
conception rate. Biol Reprod 1993;48(3):655e61.
[6] Binelli M, Thatcher WW, Mattos R, Baruselli PS. Antiluteolytic strategies to
improve fertility in cattle. Theriogenology 2001;56(9):1451e63.
[7] Hansen P. Embryonic mortality in cattle from the embryo's perspective.
J Anim Sci 2002;80(E-Suppl 2):E33e44.
[8] Kolle S, Stojkovic M, Prelle K, Waters M, Wolf E, Sinowatz F. Growth hormone
(GH)/GH receptor expression and GH-mediated effects during early bovine
embryogenesis. Biol Reprod 2001;64(6):1826e34.
[9] Yaseen MA, Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. Changes in
the relative abundance of mRNA transcripts for insulin-like growth factor
(IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplan-
tation bovine embryos derived from different in vitro systems. Reproduction
185
2001;122(4):601e10.
[10] Rhoads ML, Meyer JP, Kolath SJ, Lamberson WR, Lucy MC. Growth hormone
receptor, insulin-like growth factor (IGF)-1, and IGF-binding protein-2
expression in the reproductive tissues of early postpartum dairy cows. J Dairy
Sci 2008;91(5):1802e13.
[11] Wathes DC, Reynolds TS, Robinson RS, Stevenson KR. Role of the insulin-like
growth factor system in uterine function and placental development in ru-
minants. J Dairy Sci 1998;81(6):1778e89.
[12] Spencer TE, Gray A, Johnson GA, Taylor KM, Gertler A, Gootwine E, et al. Ef-
fects of recombinant ovine interferon tau, placental lactogen, and growth
hormone on the ovine uterus. Biol Reprod 1999;61(6):1409e18.
[13] Kolle S, Stojkovic M, Boie G, Wolf E, Sinowatz F. Growth hormone inhibits
apoptosis in in vitro produced bovine embryos. Mol Reprod Dev 2002;61(2):
180e6.
[14] Markham KE, Kaye PL. Growth hormone, insulin-like growth factor I and cell
proliferation in the mouse blastocyst. Reproduction 2003;125(3):327e36.
[15] Bilby TR, Block J, do Amaral BC, Sa Filho O, Silvestre FT, Hansen PJ, et al. Effects
of dietary unsaturated fatty acids on oocyte quality and follicular develop-
ment in lactating dairy cows in summer. J Dairy Sci 2006;89(10):3891e903.
[16] Hartnell GF, Franson SE, Bauman DE, Head HH, Huber JT, Lamb RC, et al.
Evaluation of sometribove in a prolonged-release system in lactating dairy
cows–production responses. J Dairy Sci 1991;74(8):2645e63.
[17] Bilby TR, Guzeloglu A, Kamimura S, Pancarci SM, Michel F, Head HH, et al.
Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian,
conceptus, and insulin-like growth factor system responses. J Dairy Sci
2004;87(10):3256e67.
[18] Cooke RF, Bohnert DW, Francisco CL, Marques RS, Mueller CJ, Keisler DH.
Effects of bovine somatotropin administration on growth, physiological, and
reproductive responses of replacement beef heifers. J Anim Sci 2013;91(6):
2894e901.
[19] Mercadante VR, Fontes PL, Ciriaco FM, Henry DD, Moriel P, Ealy AD, et al.
Effects of recombinant bovine somatotropin administration at breeding on
cow, conceptus, and subsequent offspring performance of beef cattle. J Anim
Sci 2016;94(5):2128e38.
[20] Rivera F, Narciso C, Oliveira R, Cerri RL, Correa-Calderon A, Chebel RC, et al.
Effect of bovine somatotropin (500 mg) administered at ten-day intervals on
ovulatory responses, expression of estrus, and fertility in dairy cows. J Dairy
Sci 2010;93(4):1500e10.
[21] Ribeiro ES, Bruno RG, Farias AM, Hernandez-Rivera JA, Gomes GC, Surjus R,
et al. Low doses of bovine somatotropin enhance conceptus development and
fertility in lactating dairy cows. Biol Reprod 2014;90(1):10.
[22] Moreira F, Badinga L, Burnley C, Thatcher WW. Bovine somatotropin increases
embryonic development in superovulated cows and improves post-transfer
pregnancy rates when given to lactating recipient cows. Theriogenology
2002;57(4):1371e87.
[23] Starbuck MJ, Inskeep EK, Dailey RA. Effect of a single growth hormone (rbST)
treatment at breeding on conception rates and pregnancy retention in dairy
and beef cattle. Anim Reprod Sci 2006;93(3e4):349e59.
[24] Oosthuizen N, Fontes PLP, Henry DD, Ciriaco FM, Sanford CD, Canal LB, et al.
Administration of recombinant bovine somatotropin prior to fixed-time
artificial insemination and the effects on fertility, embryo, and fetal size in
beef heifers. J Anim Sci 2018;96(5):1894e902.
[25] Cooke RF, Cappellozza BI, Reis MM, Bohnert DW, Vasconcelos JL. Plasma
progesterone concentration in beef heifers receiving exogenous glucose, in-
sulin, or bovine somatotropin. J Anim Sci 2012;90(9):3266e73.
[26] Hansel W, Alila HW, Dowd JP, Yang XZ. Control of steroidogenesis in small and
large bovine luteal cells. Aust J Biol Sci 1987;40(3):331e47.
[27] Lei ZM, Chegini N, Rao CV. Quantitative cell composition of human and bovine
corpora lutea from various reproductive states. Biol Reprod 1991;44(6):
1148e56.
[28] Albuquerque J, Dias H, Bueno I, Rodrigues A, Pereira M, Carvalho E, et al. Uso
da somatotropina bovina (bST) associada a protocolos de inseminaça ~o artifi-
cial em tempo fixo, (IATF) em vacas de corte. In: Anais da XXVI Reunia ~o Anual
da Sociedade Brasileira de Tecnologia de Embrio ~es; 2012. p. 360.
[29] Aboin A, Cooke RF, Vieira F, Leiva T, Vasconcelos JLM. Effects of bovine so-
matotropin injection on serum concentrations of progesterone in non-
lactating dairy cows. Livest Sci 2013;154(1e3):240e5.
[30] Rossetti RC, Perdigao A, Mesquita FS, Sa Filho M, Nogueira GP, Machado R,
et al. Effects of flunixin meglumine, recombinant bovine somatotropin and/or
human chorionic gonadotropin on pregnancy rates in Nelore cows. Ther-
iogenology 2011;76(4):751e8.
[31] Lucy MC, Thatcher WW, Collier RJ, Simmen FA, Ko Y, Savio JD, et al. Effects of
somatotropin on the conceptus, uterus, and ovary during maternal recogni-
tion of pregnancy in cattle. Domest Anim Endocrinol 1995;12(1):73e82.