Theriogenology 92 (2017) 30e35

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Theriogenology
journal homepage: www.theriojournal.com

Embryo production in heifers with low or high dry matter intake

submitted to superovulation
Marcos R. Mollo a, Pedro L.J. Monteiro Jr. b, Ricardo S. Surjus b, Aline C. Martins c,
Alexandre F. Ramos d, Gerson B. Moura ~o b, Luiz H.D. Carrijo e, Glaucio Lopes Jr. f,
g h b, *
Rodolfo Rumpf , Milo C. Wiltbank , Roberto Sartori
a
National Water Agency (ANA), Brasília, Federal District, Brazil
b
Department of Animal Science, ESALQ, University of Sa ~o Paulo, Piracicaba, Sa
~o Paulo, Brazil
c s, Brazil
Rio Verde University, Jataí, Goia
d
Embrapa Genetic Resources and Biotechnology, Brasília, Federal District, Brazil
e
Integral Animal Nutrition, Goia^nia, Goias, Brazil
f
Alta Genetics Inc, Watertown, WI, USA
g
Geneal Genetics and Animal Biotechnology, Uberaba, Minas Gerais, Brazil
h
Department of Dairy Science, University of Wisconsin-Madison, Madison, WI, USA

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

Article history: This study investigated the influence of feed intake on superovulatory response and embryo production
Received 29 October 2016 of Nelore heifers. Pubertal heifers were kept in a feedlot and were submitted to the same diets, but with
Received in revised form different levels of feed consumption: High (1.7 M; n ¼ 20) or Low (0.7 M; n ¼ 19) feed intake. Heifers in
6 January 2017
the 1.7 M treatment consumed 170% (2.6% of body weight [BW] in dry matter) and the 0.7 M heifers ate
Accepted 7 January 2017
70% (1.1% of BW in dry matter) of a maintenance diet. After 7 wk on these diets, heifers were treated with
Available online 8 January 2017
eight decreasing doses of follicle-stimulating hormone (FSH) given every 12 h, totaling 133 mg Folltropin
(Folltropin-V; Bioniche Animal Health, Canada) per heifer. Seven d after AI, heifers had their uteri flushed
Keywords:
Feed intake
and embryos were recovered and graded according to the International Embryo Technology Society
Insulin standards. Data were analyzed using the GLIMMIX procedure of SAS and results are presented as least-
Embryo squares means ± SEM (P < 0.05). At the onset of the FSH treatment (Day 0 of the protocol), 1.7 M heifers
Multiple ovulation had greater body condition score (BCS), BW and serum insulin concentrations than 0.7 M heifers
Bos indicus (4.1 ± 0.1 vs. 3.0 ± 0.1; 462.5 ± 10.1 vs. 382.7 ± 10.4 kg; and 14.3 ± 1.7 vs. 3.5 ± 0.8 mIU/mL, respectively).
The 0.7 M heifers had more follicles 6 mm at the time of the last FSH (Day 7; 47.9 ± 6.4 vs. 23.5 ± 4.3
follicles), related to a better follicle superstimulatory response to FSH. Similarly, 0.7 M heifers had more
corpora lutea at the time of embryo collection (33.6 ± 1.4 vs. 15.7 ± 0.9) than the 1.7 M heifers, which
resulted in greater number of recovered embryos and ova (9.9 ± 0.7 vs. 6.7 ± 0.6) and viable embryos
(5.3 ± 0.5 vs. 3.8 ± 0.4), despite having similar proportions of viable embryos (~62%). A negative cor-
relation between circulating insulin and follicle superstimulatory response to FSH was observed
(r ¼ 0.68). Therefore, we conclude that high feed intake, for a long period of time, compromised the
superovulatory response and embryo production potential of Bos indicus heifers possibly related to the
elevation in circulating insulin.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction major limitation to the profitable and efficient implementation of

There continues to be substantial variability in the super-
ovulatory response of cattle to FSH protocols and this represents a
* Corresponding author.
E-mail address: robertosartori@usp.br (R. Sartori).
embryo technology in cattle [1e3]. Approximately 20% of cows
produce most of the viable embryos after multiple ovulation
treatments [4]. Among the factors that influence the multiple
ovulatory response and embryo production in cattle, nutritional
management has a substantial impact [5,6]. Commonly, embryo
donors are overweight and overfed due to inappropriate nutritional
management, which may result in poor oocyte/embryo quality [7].

http://dx.doi.org/10.1016/j.theriogenology.2017.01.015
0093-691X/© 2017 Elsevier Inc. All rights reserved.
 M.R. Mollo et al. / Theriogenology 92 (2017) 30e35
The antral follicle count (AFC) at the beginning of a follicle-
stimulating hormone (FSH) treatment is the most important fac-
tor affecting embryo production in cattle. The AFC has a high pos-
itive correlation with multiple ovulatory response [8e10] and
embryo yield [10,11]. Nutritional flushing prior to multiple ovula-
tion treatments has been related to increased number of small
follicles in the ovaries, apparently through an increase in circulating
insulin-like growth factor 1 (IGF1) [8,12] and insulin. It has been
reported that these factors increase the sensitivity of granulosa
cells to FSH [13,14]. However, high blood insulin concentration is
also related to compromised AFC in cattle [15], lower multiple
ovulatory responses [16], and poor embryo quality [15].
Data on the effect of dietary energy levels, or dry matter intake
(DMI), on the multiple ovulatory response are controversial.
Although some research has shown positive effects of DMI on
multiple ovulatory response [8,12], most studies have shown
negative effects [17e20]. Moreover, high-energy diets compro-
mised in vitro and in vivo embryo production and changed
expression of important genes related to embryo development
[6,21,22].
Therefore, the objective of this study was to evaluate the chronic
effect (9 wk) of distinct levels of DMI on multiple ovulatory
response, and the quantity and quality of embryos produced in Bos
indicus heifers.
2. Materials and methods
2.1. Animals and experimental diets
Thirty nine pubertal Nelore heifers, age ranging from 24 to
36 mo, BW of 390 ± 6.8 kg (ranging from 330 to 459 kg) and BCS of
3.5 ± 0.04 (ranging from 3.0 to 3.8; on a 1 to 5 scale; [23]) were kept
on a feedlot system with ad libitum access to water and mineral
mix. The experiment was performed at the Sucupira Experimental
Station of Embrapa Genetic Resources and Biotechnology in Brasí-
lia, DF, Brazil.
Before the start of the experiment, heifers were kept together
and were fed a maintenance diet for 21 d according to the National
Research Council [24]. After, this 21-d period, heifers were
randomly divided into two treatment groups: 1.7 M, in which they
consumed 170% of the necessary maintenance diet (2.62% of BW in
DM) or 0.7 M, in which heifers received and ate 70% of the neces-
sary maintenance diet (1.08% of BW in DM). Groups were fed once a
day at 7:00 a.m. on the same schedule in two pens (one for each
treatment group with a feeding area with 70 cm of length per heifer
for a period of 9 wk (Table 1). Body weight was evaluated weekly
and the data were used to adjust the diet. The BCS was also eval-
uated weekly by two experienced technicians, and the mean was
calculated.
Table 1
Diet composition and percentages of dry matter (DM), total digestible nutrients
(TDN) and crude protein (CP).
% of diet in DM
Coast-cross hay 40.8
Corn silage 51.9
Energy and protein supplementa 7.3
Total of digestible nutrients 62.4
Crude protein 11.5
a
Boi~
ao PPU, Integral Animal Nutrition, Goi^ ania, GO, Brazil (Quantities per kg of
product): antioxidant (320.0 mg), calcium (39.4 g), cobalt (32.0 mg), copper
(240.0 mg), sulfur (14.4 g), iron (320.0 mg), fluorine (max; 542.0 mg), phosphorus
(32.8 g), iodine (48.0 mg), magnesium (99.6 g), manganese (160.0 mg), Non-
protein nitrogen (NPN; 80.9 g), NPN equiv. protein (max; 50.5%), flavor agent
(200.0 g), selenium (4.8 mg), sodium (118.8 g), vitamin A (4000.0 IU), vitamin E
(40.0 IU), zinc (960.0 mg).
31
2.2. Multiple ovulation protocol and embryo collection
After 7 wk on treatment diets, heifers were submitted to a su-
perovulation treatment using the following protocol (Fig. 1): On
Day 0, all embryo donors received an intravaginal device (IVD)
impregnated with 1 g of progesterone (DIB, Syntex S.A., Argentina)
and 2 mg of estradiol benzoate (EB; Ric-Be, Syntex S.A., Argentina)
for synchronization of follicle wave emergence. Although EB is used
in South American and other countries, we acknowledge there are
countries that prohibit its use. On Days 4e7, heifers received eight
decreasing doses of FSH (133 mg total, i.m., Folltropin-V, Bioniche,
Ontario, Canada), and concomitant with the sixth treatment of FSH
(Day 6), all heifers received d-cloprostenol (PGF2a, 0.150 mg, i.m.,
Prolise, ARSA S.L.R., Argentina) and the IVD was removed. Estrus
detection was observed twice-daily for 1 h and heifers detected in
estrus were inseminated twice 12 and 24 h later by the same
technician using semen from a single sire of proven fertility. Em-
bryo collection was performed 7 d after estrus using the double
uterine flushing technique [25]. All embryos were classified for
quality (1 ¼ good, 2 ¼ fair, 3 ¼ poor and 4 ¼ degenerate) according
to the International Embryo Technology Society (IETS) guidelines
[26]. Embryos grading 1 and 2 were defined as viable embryos.
2.3. Ultrasound evaluations
Ovarian structures were evaluated using a 7.5-MHz linear-array
transducer (Aloka SSD-500 V; Corometrics Medical Systems Inc.,
Wallingford, CT, USA). On Day 0, the ovaries were evaluated and
structures recorded. On the first d of treatment with FSH (Day 4) all
follicles  3 mm were counted. Similarly, on the fourth d of treat-
ment with FSH (Day 7) all follicles  6 mm were counted. The
follicle size of 6 mm in diameter was chosen because follicle de-
viation occurs when the largest growing follicle of the wave reaches
between 5.7 and 7.0 mm in diameter and becomes dominant in Bos
indicus cattle [27]. Follicle superstimulatory response to FSH was
defined and calculated by subtracting the number of antral follicles
 6 mm at the time of the last FSH treatment (Day 7 of the protocol)
from the number of antral follicles  3 mm at the time of the first
FSH treatment (Day 4 of the protocol) in each heifer. The super-
stimulatory response was also evaluated by dividing the number of
antral follicles  6 mm at the time of the last FSH treatment by the
number of antral follicles  3 mm at the time of the first FSH
treatment in each heifer. On Day 10, all follicles  6 mm still present
in the ovaries were considered anovulatory follicles. On Day 15, the
number of CL were evaluated by ultrasound to estimate the
superovulatory response.
2.4. Insulin assay
Blood samples were collected on Day 4 of the protocol from the
coccygeal vein or artery into evacuated tubes (Becton Dickinson Co.,
Franklin Lakes, NJ, USA), stored at 4  C for 24 h, and then centri-
fuged at 1700  g for 15 min. The overnight blood storage before
centrifugation was done to minimize post-centrifugation fibrin in
serum samples [R. Sartori, personal observation] without
compromising circulating insulin concentrations measurement
[28]. Serum was kept at 20  C until the hormone assay was per-
formed. Insulin concentrations were determined using a single
solid-phase radioimmunoassay (Coat-a-Count; Diagnostic Products
Corporation, Los Angeles, CA, USA). The intra-assay CV was 1.9%.
2.5. Statistical analyses
The GLIMMIX procedure (ANOVA) of SAS was used with
generalized linear models methodology. For BW, BCS and
32 M.R. Mollo et al. / Theriogenology 92 (2017) 30e35

Fig. 1. Schematic illustration of the superovulation protocol in Nelore embryo donors. Three weeks before onset of feeding experimental diets, heifers were fed a maintenance (M)
diet. At wk 0 (W0) heifers started to eat either 0.7 M (low) or 1.7 M (high). On the seventh wk heifers were submitted to a superovulation protocol. At a random stage of the estrous
cycle (D0), a progesterone-releasing intravaginal device (P4) was inserted and 2 mg, i.m., estradiol benzoate (EB) was administered. Four d later (D4), heifers were treated with
133 mg pFSH administered twice daily in eight decreasing doses over a 4-d period (40%, 30%, 20%, and 10% from D4 to D7, respectively). On D6, 0.150 mg cloprostenol sodium
(PGF2a) was administered and P4 device was withdrawn. Estrus was observed for 1 h twice a day and heifers were inseminated twice at 10e14 h and 22e26 h after estrus detection,
with frozen/thawed semen from a proven sire. Ova/embryos were collected 7 d after estrus (D15).

circulating concentrations of insulin, a Gaussian distribution using
the identify link function was used, for count variables, a Poisson
distribution using the logarithmic link function was used, and for
proportional variables, a binomial distribution with the logit link
function was employed. The variables were analyzed using a
mathematical model that included the effects of treatment. The
Satterthwaite method, for the BW, BCS, circulating concentrations
of insulin, and AFC, and Residual method, for data related to embryo
production, were used to calculate the denominators degrees of
freedom to approximate the F tests in the mixed models. The
Pearson correlations of the circulating concentrations of insulin
with the superstimulatory response to FSH or with ovulation rate
were analyzed using the PROC CORR procedure.
All statistical comparisons were done using means adjusted by
the least squares means method and written results are presented
as least squares means ± standard error of the mean (SEM) with the
distributions shown in the original scales to aid interpretation.
Differences with P  0.05 were considered significant and those
with 0.05 < P  0.10 were considered tendencies.
3. Results
Underfed (0.7 M) heifers lost weight (372 g/d) and decreased
BCS (from 3.5 to 3.0) as opposed to 1.7 M heifers, which gained
1164 g/d (Fig. 2A) and increased BCS (3.5e4.1, Fig. 2B) from
experimental week 0 to week 7. Differences between groups in BW
and BCS were identified as early as at the second and first experi-
mental week, respectively (P < 0.05) and continued significant until
the end of the study. At the onset of the treatment with FSH (Day 4;
Fig. 1), 1.7 M heifers had more BW, greater BCS, and greater serum
concentrations of insulin (Table 2).
There was a tendency (P ¼ 0.07) for a difference between diets
in number of follicles  3 mm in diameter at the time of the first
FSH treatment (Table 3). Moreover, 0.7 M heifers had twice as many
(P < 0.01) follicles  6 mm in diameter at the time of the last FSH
treatment, more (P < 0.01) CL at embryo flushing, as well as a
greater numbers of ova and embryos, and viable embryos than
1.7 M heifers (P < 0.01; Table 3). The follicle superstimulatory
response to FSH was lower (P < 0.01) for the 0.7 M heifers than for
the 1.7 M group (Table 3; Fig. 3A).
Irrespective of treatment, greater circulating insulin on Day 4
was associated with a compromised follicle superstimulatory
response to FSH. There was a negative correlation (0.68;
P < 0.001) between circulating insulin on Day 4 of the protocol and
the relative difference between the number of the
follicles  6 mm at the last FSH and the number of
follicles  3 mm at the first FSH treatment (last d/first d of FSH;
Fig. 3A).
Although, high DMI, and the associated high circulating insulin,
reduced the follicle superstimulatory response, it did not affect
ovulation rate (Fig. 3B), or embryo quality, as the percentage of
viable embryos, and the number or percentage of degenerate em-
bryos were similar (P > 0.10) between groups. Although 0.7 M
heifers had (P < 0.01) a greater number of unfertilized oocytes, the
percentage of fertilized oocytes did not differ (P ¼ 0.36) between
treatments (Table 3).
4. Discussion
This study was designed to evaluate the effect of a relatively long

Fig. 2. A) Body weight (mean ± SEM) and B) body condition score (mean ± SEM) of Nelore heifers with low (0.7 M; n ¼ 19) or high (1.7 M; n ¼ 20) dry matter intake. *P < 0.05.
 M.R. Mollo et al. / Theriogenology 92 (2017) 30e35 33

Table 2
Results (least squares means ± SEM) of body weight, body condition score and circulating insulin at the time of the first FSH treatment for superovulation in Nelore
heifers that consumed 70% (1.1% of body weight [BW] in dry matter; 0.7 M) or ate 170% (2.6% of BW in dry matter; 1.7 M); 1.7 M) of a maintenance diet.

0.7 M (n ¼ 19) 1.7 M (n ¼ 20) P

Body weight, kg 382.7 ± 10.4 462.5 ± 10.1 <0.001

Body condition score, 1 to 5 3.0 ± 0.07 4.1 ± 0.06 <0.001
Serum insulin concentration, mIU/mL 3.5 ± 0.8 14.3 ± 1.7 <0.001

Table 3
Least squares means ± SEM of antral follicle count, superstimulatory and superovulatory responses, and embryo production of Nelore heifers that consumed 70% (0.7 M) or
170% (1.7 M) in dry matter of a maintenance diet.

0.7 M (n ¼ 19) 1.7 M (n ¼ 20) P

Follicles  3 mm at first FSH; n 46.2 ± 6.5 34.3 ± 2.8 0.07

Follicles  6 mm at last FSH; n 47.9 ± 6.4 23.5 ± 4.3 0.003
Difference between follicles  3 mm at first and follicles  6 mm at last FSH; n 1.7 ± 3.1 10.8 ± 3.0 0.006
Corpora lutea at embryo collection; n 33.6 ± 1.4 15.7 ± 0.9 <0.001
Total ova/embryos; n 9.9 ± 0.7 6.7 ± 0.6 0.001
Degenerate embryos; n 2.9 ± 0.4 2.2 ± 0.3 0.161
Degenerate embryos; % 27.1 ± 10.8 28.2 ± 10.6 0.945
Viable embryos; n 5.3 ± 0.5 3.8 ± 0.4 0.028
Viable embryos; % 58.2 ± 12.0 65.2 ± 11.2 0.670
Unfertilized oocytes; n 1.6 ± 0.3 0.6 ± 0.2 0.004
Fertilization; % 85.3 ± 8.6 95.0 ± 5.1 0.360
Viable embryos from fertilized; % 67.8 ± 11.3 68.4 ± 11.0 0.972

period with differences in dry matter intake on the super-
stimulatory and superovulatory response, and embryo production
in Bos indicus heifers. The experimental design employed two
Fig. 3. A) Relationship between circulating concentrations of insulin on Day 4 of the
superovulation treatment and the superstimulatory response to FSH (relative differ-
ence between follicles  6 mm at last FSH (Day 7) and follicles  3 mm at first (Day 4)
FSH (last d/first d of FSH); B) Relationship between circulating concentrations of in-
sulin on Day 4 of the superovulation treatment and ovulation rate per heifer (relative
difference between the number of the corpora lutea 7 d after estrus and number of
follicles  6 mm at last (Day 7) FSH. Open circles represent 0.7 M heifers (n ¼ 13) and
solid circles represent 1.7 M heifers (n ¼ 17).
contrasting treatment groups, with extremes of feed allowance. As
seen in Fig. 2, the design was successful because by experimental
week 7 (at the time of onset of superstimulation treatments) there
were extremely large differences in BW and BCS between heifers
due to experimental treatments.
Due to conflicting reports in the literature as well as our own
data from other experiments, we did not formulate any precon-
ceived hypothesis. In reality, the observed results while extremely
dramatic were somewhat unexpected and unprecedented.
It has been reported that the AFC at the beginning of ovarian
multiple ovulation treatment with gonadotropins is one of the
main factors affecting embryo production [8,11]. Although some
studies have shown an increase in AFC associated with nutritional
flushing for short periods of time [8,12,29], especially in Bos taurus
breeds, this association was not observed in most of the studies
with Bos indicus cattle [30]. In our study, we did not detect a sta-
tistical difference for number of follicles at the start of the multiple
ovulation protocol (Day 4) between 0.7 M and 1.7 M heifers.
Similarly, in the study by Adamiak et al. [15], the AFC was similar
between Bos taurus heifers fed a maintenance diet vs heifers fed
twice the maintenance requirement (13.0 vs 12.7, respectively).
However, when circulating insulin concentrations were considered
by Adamiak et al. [15], hyperinsulinemic heifers had fewer follicles
as compared to heifers with normal insulin concentrations
(12.0 ± 0.6 vs.16.0 ± 0.9).
Despite having similar AFC at the time of the first FSH treat-
ment (Day 4), the follicle superstimulatory response to FSH and
the CL number at the time of embryo collection were lower for the
1.7 M than that of the 0.7 M group. Our results agree with those
from several studies, which reported that high feed intake
compromised the superovulatory response in beef cows and
heifers [17,19,20]. For example, Nolan et al. [17] detected a greater
number of stimulated follicles after FSH treatment in heifers with
low feed intake as compared with those on a diet of ad libitum
feed intake (13.5 ± 2.4, n ¼ 14 vs 9.6 ± 1.2, n ¼ 13). In our study,
the follicle superstimulatory response to FSH was negatively
associated with high circulating insulin (Fig. 3), as the 1.7 M group
had a substantial decrease in AFC from Day 4 to Day 7 (43.3%
decrease), whereas the 0.7 M heifers had almost no change in AFC
(1.2% increase). Corroborating our data, a Latin-square study [31]
34 M.R. Mollo et al. / Theriogenology 92 (2017) 30e35
with 32 non-lactating Nelore cows fed different diets for 32 d
before starting a superovulation program showed a negative cor-
relation between circulating insulin on Day 4 and CL number
(r ¼ 0.32; P < 0.05) as a result of a greater drop in follicle number
between Day 4 and Day 7 of the protocol in cows with greater
circulating insulin. The greater reduction in AFC reported in the
present experiment is probably due to the relatively long time
cows were exposed to a high-energy diet (65 d). This negative
relationship between circulating insulin and follicle stimulatory
response to FSH suggests that hyperinsulinemic females may be
less sensitive to FSH treatment, possibly due to an insulin resistant
state in overfed animals, as suggested by others [15,32e35].
Interestingly, although circulating insulin affected the super-
stimulatory response in our study, there was no relationship be-
tween circulating insulin and ovulation rate (Fig. 3B), i.e. as long as
the follicles developed to an ovulatory size at Day 7 of the pro-
tocol, there was no correlation between circulating insulin and
ovulation rate. Moreover, despite significant differences in circu-
lating insulin between 0.7 M and 1.7 M heifers, ovulation rate was
not different between treatments (68% vs. 65% for 0.7 M and 1.7 M,
respectively; P ¼ 0.65).
The total number of embryos was greater for the 0.7 M heifers as
compared to the 1.7 M heifers, although the percentage of viable
embryos was similar between groups. Several articles reported a
deleterious effect of high DMI on quantity and/or quality of em-
bryos [17,19,36,37]. Similar to our study, Siddiqui et al. [20] found
that high BCS and high nutritional level compromised response to
multiple ovulation treatments. Adamiak et al. [15] reported that the
effect of over-nutrition on oocyte and embryo quality was depen-
dent on the BCS of the animal at the start of the experiment. Yaakub
et al. [19] reported that high feed intake compromised both mul-
tiple ovulatory response ((12.3 ± 1.4 vs 15.5 ± 1.6 CL)) and the
number of viable embryos (2.8 ± 0.4 vs 4.8 ± 0.7) in heifers fed ad
libitum concentrate diets compared to heifers receiving only 3 kg of
concentrate. In addition, heifers offered citrus/beet pulp had more
transferable embryos (4.8 ± 0.7) than heifers offered only barley
(2.9 ± 0.5), possibly due to greater acetate vs. propionate produc-
tion in the rumen with these two diets. A similar stimulatory effect
of the citrus/beet pulp diet was also observed for the super-
ovulatory response (15.5 ± 1.6 vs 12.3 ± 1.4 CL) [18,19]. Adamiak
et al. [15] related the coefficient of blastocyst yield in an in vitro
embryo production study to a decrease in insulin (0.024 ± 0.016 for
first week) throughout the experiment (0.71 ± 0.312 last week),
speculating that there was a deleterious effect of chronic high
circulating insulin concentrations. Our results did not provide evi-
dence for a direct effect of a prolonged elevation in feed intake and
extremely elevated circulating insulin on fertilization rate or early
embryo development, as measured by morphological embryo
quality. However, our results provide strong and dramatic support
for an effect of prolonged elevations in insulin, due to high feed
intake, producing reduced responsiveness of follicles to FSH
stimulation.
In conclusion, high feed intake for more than 7 wk compromised
the superstimulatory and superovulatory responses of Bos indicus
heifers, probably due to a lower ovarian sensitivity to FSH associ-
ated with hyperinsulinemia.
Acknowledgments
This project was funded by a grant from the Support Research of
the Federal District Foundation (FAP-DF) and scholarships from the
Coordination for the Improvement of Higher Education Personnel
and the National Council for Scientific and Technological Devel-
opment of Brazil.
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