Use of antioxidants to improve the pregnancy rate in beef cattle submitted to a synchronization protocol ...

477

DOI:10.5216/cab.v11i3.3848

USE OF ANTIOXIDANTS TO IMPROVE THE PREGNANCY RATE IN BEEF

CATTLE SUBMITTED TO A SYNCHRONIZATION PROTOCOL WITH
PROGESTERONE (P4)
Ricardo Zanella,1 Carlos Bondan,2 Julio Cesar Mendes Soares,2 Eraldo Lourenso Zanella2 e
Marina Ragagnin de Lima3
1. Veterinary Doctor, doctorate student at Washington State University, Department of Animal Science Pullman WA, 99163.
E-mail: zanellar@wsu.edu
2. Veterinary Doctors, professors of Veterinary Medicine at Faculty of Agronomy and Veterinary Medicine, Universidade de Passo Fundo
3. Veterinary Doctor, Caapava do Sul, RS.

ABSTRACT

Estrus synchronization with progesterone is commonly used in farms with better reproductive control. Free
radicals production, i.e. oxidative stress, is associated with
progesterone levels. The oxidative stress is responsible
for aggression to the cellular membrane, leading to a lise
and lipoperoxide formation. In this work, the antioxidant
compensatory effect (Vitamins C and E) associated with
the exogenous progesterone implant (P4), used in estrus
synchronization protocols in cattle, was evaluated. Twentyfive cows were randomly selected in 5 different groups:

1) control without P4, 2) control with P4, 3) P4 + vitamin

C and E, 4) P4 + vitamin E, 5) P4 + vitamin C. The lipid
lipoperoxidation was measured through thiobarbituric acid
reactive substances (TBA-RS) and glutathione peroxidase
enzyme (GSHpx) at days 0 and 7 of the estrus synchronization protocol. The use of vitamin E, in this experiment,
showed a better pregnancy rate, however, the results must
be validated before orienting the use of the vitamin in cows
synchronized with P4.

KEYWORDS: Antioxidants, cows, oxidative stress, pregnancy rate, progesterone.

RESUMO
USO DE ANTIOXIDANTES PARA MELHORAR A EFICINCIA REPRODUTIVA DE REBANHO BOVINO SUBMETIDO A
PROTOCOLO DE SINCRONIZAO COM PROGESTERONA (P4)
Progestgenos para a sincronizao de estro tm sido
utilizados como rotina em propriedades com melhor controle
reprodutivo. A produo de radicais livres, estresse oxidativo,
est relacionada com os nveis de progesterona circulante. O estresse oxidativo responsvel pela agresso membrana celular,
causando lise e a formao de lipoperxidos. Neste trabalho, buscou-se avaliar o efeito compensatrio de antioxidantes (vitaminas C
e E) associados ao implante de progesterona exgena (P4), utilizada
em protocolos de sincronizao de estro em fmeas bovinas. Vinte
e cinco fmeas bovinas foram divididas aleatoriamente em cinco

grupos: 1) controle sem implante de P4; 2) implante de P4; 3)

implante de P4 + vitamina C e E; 4) implante de P4 + vitamina E;
5) implante de P4 + vitamina C. Avaliaram-se a lipoperoxidao
lipdica, atravs da mensurao de substncias reativas ao cido tiobarbitrico (TBA-RS), e a enzima glutationa peroxidase
(GSHpx), nos dias zero e sete do protocolo de sincronizao de
estro. A utilizao de vitamina E, neste experimento, apresentou
uma maior taxa de prenhez; entretanto, existe a necessidade de
validar esses resultados, antes que seu uso seja preconizado em
fmeas bovinas sincronizadas com P4.

PALAVRAS-CHAVES: Antioxidantes, estresse oxidativo, progesterona, taxa de prenhez, vacas.

Ci. Anim. Bras., Goinia, v. 11, n. 3, p. 477-481, jul./set. 2010

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ZANELLA, R. et al.

INTRODUCTION
The development and use of reproductive biotechniques, synchronization and estrus induction in
beef cattle are fundamental for the Brazilian production system. The concentration of mating activities
with good conception rates allows females to show a
good parturition rate at the most appropriate moment
of the year. (BAYARD et al., 2002). Researches have
demonstrated that the levels of success of estrus synchronization varies according to the protocol used,
but they are usually effective. However, the level of
animal fertility is reduced with the use of exogenous
progesterone implants, as described by AINSWORTH & SHRESTHA (1983). Recent studies of estrus
synchronization presented pregnancy rates between
37.68 and 57.6% (CMARA et al., 2006; BORGES
et al., 2009).
In bovine culture, management flaws, such as
estrus misidentification, are one of the great problems
which lead to low pregnancy rate in the herd. Because
of that, the use of hormone therapy, with the intent
of synchronizing estrus, and of fixed-time artificial
insemination (FTAI) became important (BARROS
et al., 1995). Exogenous progesterone (P4), estrogen
(E2), equine chorionic gonadotropin (eCG) and GnRH
have been broadly used in estrus synchronization and
ovulation protocols, including in animals in anestrus
(ROCHE et al., 1992; JOLLY et al., 1995; MORAES
& JAUME, 1997; CMARA et al., 2006; BORGES
et al., 2009). The purpose of utilizing these hormones is to mimic estrous cycle, as well as stimulate
the hypothalamus-hypophyseal-gonadal axle of the
females and the cells of such structure (GONZALEZPADILHA et al., 1975; TROXEL et al., 1993; KESLER
& FAVERO, 1996; WILTBANK et al., 1996; BAYARD
et al., 2002). LUCY et al. (2001) mention that the
pregnancy rate of cows synchronized with vaginal
device presented higher indices than the control group,
without the device. SIMONETTI et al. (1999), in an
experiment with ovines, obtained 92.86% of estrus,
but only 50.43% of pregnancy. The low reproductive
efficiency indices were related to the use of progestans as estrus modelling; however, the reasons are not
Ci. Anim. Bras., Goinia, v. 11, n. 3, p. 477-481, jul./set. 2010

entirely known yet.

The increase of the P4 levels circulating is directly related to the increase/elevation of the levels thiobarbituric acid (TBA-RS) (MRQUEZ et al., 1997).
On the other hand, E2 has a contrary effect to the ones
of P4, inhibiting the oxidative stress process (EJIMA et
al., 1999; CHENG et al., 2006). The levels of TBA-RS
indicate damages to the cellular membrane due to the
production of reactive oxygen species (ROS), which
may have a deleterious effect on eggs and embryos.
MILLER & BRZEZINSKA-SLEBODZINSKA (1993)
documented the negative effect of the reactive oxygen
species (ROS) on metabolism and health of dairy
cows. Nevertheless, such effect may be neutralized or
reduced by enzymatic and non-enzymatic antioxidative
mechanisms.
In this study, an ovulation synchronization
protocol, with intravaginal devices with medroxiprogesteron (MPA), associated with intramuscular
administration of estradiol benzoate, prostaglandin
and equine chorionic gonadotropin (eCG), was used
for the synchronization of ovulation and estrus. At the
same time, antioxidative vitamins were administrated
aiming at evaluating their effect on the oxidation level
and pregnancy rate in beef cattle. The hypothesis of
this study is that the utilization of exogenous P4 as a
synchronization method increases the lipid peroxidation in preovulatory follicles, leading to problems in the
oocyte development, causing, hence, low pregnancy
rate.

MATERIAL AND METHODS

Crossbred Nellore X Angus cows (n=25), from
Agropecuria Zanella (Zanella Livestock), municipality of Paim Filho, North of Rio Grande do Sul State,
were used in this experiment. The animals were classified according to their body condition score (BCS)
from one to five, being one considered thin and five
obese (GRAHAM, 1982). The animals were randomly
divided into five different groups, with five animals
each: (1) control group without P4 implant; (2) P4
implant; (3) P4 implant with vitamins C and E administration; (4) P4 implant with vitamin E administration;

Use of antioxidants to improve the pregnancy rate in beef cattle submitted to a synchronization protocol ...

(5) P4 implant with vitamin C administration. Estrus

synchronization protocol was carried out according to
description by BAYARD et al. (2002), with the application of medroxiprogesteron (MAP 250mg) by an intravaginal device for seven days. An injection of 2.5 mg
estradiol benzoate, 3.0 ml of vitamin C (150 mg/mL;
BRAVET Laboratories, RJ) and 5.0 ml of vitamin E
(100 mg/mL; BRAVET laboratories, RJ) was applied
intramuscularly (IM) on day zero (d0). On day six,
eCG (100mg) was applied intramuscularly (IM) and
PGF2a (25 mg) via vulvar mucosa. All the procedures
were carried out according to the ethic principles for
animals experimentation recommended by the Colgio
Brasileiro de Experimentao Animal (COBEA, Brazilian School of Animal Experimentation).
Blood samples were collected by venipuncture
of the jugular vein after antisepsis, using disposable
needles and sterilized sample tubes. The collected
heparinized blood was conserved under refrigeration
at 4C and used for analysis right after the collection
for measuring the levels of lipoperoxide on days zero
and seven of the synchronization protocol.
The quantification of the cellular peroxidation
was carried out by the thiobarbituric acid reactive
substances (TBA-RS) measurement technique. The
heparinized samples were centrifuged at 2000 g for
five minutes and the supernatant was discarded and
replaced by a 0.9% NaCl physiological solution with
same volume. This procedure was carried out twice,
and, after that, the blood cells concentrate was resuspended with an hematocrit of 50%. After erythrocytes
washing with NaCl 150 mM, the samples were precipitated with acetic acid 40% in a 1:2 proportion
and centrifuged at 2000 g for the separation of the
supernatant. Lipid peroxidation was determined according to the method of thiobarbituric acid reactive
substances (TBA-RS) formation, used by OHKAWA
et al. (1979), and non-proteic sulfydril groupings
(NPSH), by ELLMANS (1959). Sixty days after the
insemination of the animals, pregnancy was detected
by transrectal palpation. The values of TBA-RS and
GSHpx production were submitted to ANOVA test,
followed by Tukey test, considering the differences
were significant when p<0.05.

479

RESULTS AND DISCUSSION

Hormone therapy is an indispensable tool for
manipulation and improvement of the use of biotechnology in beef cattle. The greatest challenge for the
use of such biotechnology is the obtainment of better
reproductive rates in livestock production. Different
alternatives have been utilized, such as the P4, to try
to concentrate the estrus and ovulation synchronization
activities and, hence, parturition period in a small interval. However, when this method is used, pregnancy
rate is under the expectation, considering the increase
of the cost with animal production.
In this experiment, on the general, pregnancy
rate was 52%. This index is compatible with the results
observed by BORGES et al. (2009), who obtained
57.6% of pregnancy rate. It should be considered that
in this research non-suckling cows were used, that
is, cows without the calves, whereas in the research
mentioned suckling cows were used, with the calves
removal for four days. Besides, the number of caws
in this experiment was small when compared with the
other work.
The pregnancy rate in the control groups (with
and without P4) was 40%. The production levels of
TBA-RS in the control group with synchronization
protocol with and without P4 were higher on day seven
than on day zero (p<0.0002) in the same group, confirming, thus, the activity of exogenous and endogenous
progesterone with the production of ROS, already
described by Mrquez et al. (1997). Consequently,
there was a proportional increase of the production of
GSHpx enzyme, suggesting a compensatory mechanism to inhibit ROS activity (p<0.0002).
The pregnancy rate in the group treated with
vitamin C was 40%. In this group, the production
levels of TBA-RS were smaller on day seven than on
day zero (p<0.05), what is likely to suggest whether
an antioxidative effect of vitamin C or that the animals
were in estrous period between days seven and eighteen
of the first collect, when there is a greater production
of endogenous progesterone. This result corresponds to
the description by Mrquez et al. (1997), in which high
levels of progesterone are found in the presence of a
Ci. Anim. Bras., Goinia, v. 11, n. 3, p. 477-481, jul./set. 2010

480

functional and mature corpus luteum, what is directly

related to the increase of the levels of thiobarbituric
acid reactive substances (TBA-RS). There is a significant increase of oxidative stress with the mature corpus
luteum than with luteal regression, showing low levels
of TBA-RS and serum progesterone (MRQUEZ et
al., 1997). Even the reduced levels of TBA-RS on
day seven, GSHpx levels were not higher than on
day zero. Although this increase was not significant,
it may suggest a compensatory mechanism against
ROS production.
The association of vitamins C and E caused an
increase of TBA-RS production on day seven compared to day zero, suggesting a pro-oxidative effect of
vitamins C and E. In this case, the GSHpx enzyme had
also a compensatory effect to keep ROS production
balanced. Nevertheless, even with the pro-oxidative
effect the pregnancy rate was not different (40%),
comparing to control-groups and treated with vitamin
C and vitamins C and E. High doses of antioxidative
vitamins may have a deleterious effect on the animals
organism. For instance, ascorbic acid (vitamin C) might act as a pro-oxidative, leading to the production
of radical hidroxila, which is highly reactive, causing
oxidative damages to the cellular membrane.
In the group treated with vitamin E, the levels
of TBA-RS and GSHpx were higher on day seven
than on day zero, demonstrating, thus, the compensatory effect this enzyme hs on the production of
ROS. Pregnancy rate of the group with vitamin E was
100%, demonstrating the efficiency of associating this
enzyme to the protocol (p<0.05). The use of vitamin E
as an antioxidative resulted in higher pregnancy rates.
These data are in accordance with the ones previously
published by LAFLAMME& HIDIROGLOU (1991)
and RICHARDSON et al. (2008), when a significant
increase of pregnancy rates in cows and heifers, supplemented with vitamin E, was observed.
The interaction mechanism between antioxidative vitamins and the reduction of embryonary losses
is still unknown. Nevertheless, associations between
vitamin E and uterine health have been observed.
Such results confirm the ones reported by EVANS &
BISHOP (1922), who administered vitamin E in femaCi. Anim. Bras., Goinia, v. 11, n. 3, p. 477-481, jul./set. 2010

ZANELLA, R. et al.

le rats and observed reduction of embryonary losses

and fetal absorption. The findings of this experiment
support the hypotheses of the inhibiting association of
antioxidative vitamins and the levels of lipidic peroxidation. It can be concluded, thus, that the association of
vitamin E to the estrus synchronization protocol with
the use of P4 is beneficial for the obtainment of good
reproductive results. The carry-out of further experiments with a higher number of animals is suggested
for a better evaluation of such results.

ACKNOWLEDGEMENTS
To the owners of Agropecuria Zanella (Zanella
Livestock), Mr. Ipenor Zanella and Mrs. Nair Zuleika
Zanella, for supplying the animals for this study..
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Submitted on April 30, 2008. Accepted on November 23, 2009

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