Theriogenology 82 (2014) 1287–1295
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Theriogenology
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Modulatory effect of three antibiotics on uterus bovine
contractility in vitro and likely therapeutic approaches in
reproduction
M. Piccinno a, A. Rizzo a, M.A. Maselli b, M. Derosa c, R.L. Sciorsci a, *
a
Department of Emergency and Organ Transplantation, Section of Veterinary Medicine and Animal Production, University of Bari
Aldo Moro, Valenzano Bari, Italy
Experimental Pharmacology Laboratory, Scientiﬁc Institute of Gastroenterology IRCCS “S. de Bellis,” Castellana Grotte, Bari, Italy
c
ASL, Prefecture of Bari, Putignano, Bari, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2014
Received in revised form 18 August 2014
Accepted 19 August 2014
This in vitro study investigates the modulatory effect of three antibiotics (amoxicillin,
enroﬂoxacin, and rifaximin) on contractility of the bovine uterine tissue in follicular and
luteal phases. The effects of these antibiotics at three single doses (106, 105, and 104 M)
on their basal contractility were evaluated in isolated organ bath. The functionality of the
strip throughout the experiment was evaluated by a dose of carbachol (105 M); the obtained effect had to be repeatable (difference of 20%) that is comparable to that induced by
the previous administration of the same substance. The results demonstrate the different
modulatory activities of these antibiotics on uterine contractility in follicular and luteal
phases. The effects induced by amoxicillin and enroﬂoxacin are opposite: the ﬁrst relaxes
and the second increases the uterine contractility in both cycle phases. Instead, the activity of
rifaximin varies depending on the phase of estrous cycle: it increases in the follicular phase
and relaxes in the luteal phase. The obtained data provide the hypothesis of possible implications of these drugs in the pharmacologic modulation of uterine contractions. Their
action at this level, associated with their speciﬁc antimicrobial effects, could suggest using
these antibiotics for the treatment of diseases related to postpartum or infections that may
occur in pregnant cattle, by virtue of their effects on myometrial contractility too.
Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Amoxicillin
Enroﬂoxacin
Rifaximin
Uterus contractility
Bovine
1. Introduction
The postpartum is a particularly critical period for bovine
fertility, because of an increased susceptibility to recurrent
infections of the uterus, which may give rise to chronic
forms resulting in more or less prolonged infertility [1–3].
Several researches have been conducted on diseases
that affect the uterus during delicate process of uterine
involution [4–6]. In this period, the natural antibacterial
mechanisms of the uterus (local production of antibodies,
the phagocytic action of neutrophils and macrophages), the
* Corresponding author. Tel.: þ39 0805443882; fax: þ39 0805443883.
E-mail address: [email protected] (R.L. Sciorsci).
0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2014.08.008
mechanical action of the secretions, and the contractile
activity of myometrium [7–11] perform an action of selfcleaning by allowing, in most cases, to limit the duration
and spread of infection [12].
However, sometimes the defense mechanisms are not
able to counteract the action of bacteria [13,14], resulting in
the development of pathologies, such as retained placenta
and metritis, that affect the genital tract and the fertility of
the subject [15,16]. These inﬂammatory processes may lead
to a prolongation of the time needed to complete uterine
involution and a delay in the resumption of ovarian activity
[17,18].
The therapy of postpartum pathologies involves the
local and systemic administration of antibiotics, alone or
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M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
associated with uterotonic substances [19–21]. The use of
antibiotics was justiﬁed by their antimicrobial activity and
for some of these (penicillins, quinolones, rifamycins, and
macrolides) by their anti-inﬂammatory action and stimulatory effect on the immune system ([22–30]; M. Albrizio
et al., unpublished data, 2014). Moreover, the antibiotics,
such as penicillins and quinolones, can also affect the
oxidative state of the organism and the production of
reactive oxygen species [30–34]. Several studies have reported that antibiotic therapy is able to directly modulate
the contractility of smooth muscles [35–39] also. However,
this effect is subject to changes attributed to the species,
tissue, or organ under examination [38–41]. For example,
the erythromycin is able to stimulate the contraction of
smooth muscle cells of the stomach and duodenum,
probably by acting on the receptors of motilin [42,43] and
moreover, seems to determine an increase in the amplitude
and frequency of contraction even in rat’s myometrium;
the effect probably mediated by histamine H1 receptors and
calcium channels [44]. On the contrary, other studies have
shown that erythromycin, as well as clarithromycin,
neomycin, gentamicin, clindamycin, and ceftriaxone, would
result in a relaxation of myometrial contractility in several
species (woman, rat, and cow) [45–48].
Caron et al. [49] have also shown that the administration of amoxicillin and clavulanic acid determines, in
humans, increased bowel activity, which could depend on
the intraluminal release of mediators of contractility (e.g.,
motilin) or by direct interaction of the b-lactam with
Gamma-AminoButyric Acid (GABA) receptors present at
the level of the myenteric plexus.
Other studies on smooth muscles of different organs and
animal species have reported that ﬂuoroquinolones act as antagonists of the GABA receptor by blocking the ATP-dependent
potassium channels [36–38,41,50–52]. In addition, ﬂuoroquinolones induce the release of PGF2a in guinea-pig ileum
[38], which are responsible for cholinergic transmission in the
myenteric plexus [53,54]. Moreover, in vitro studies on rat’s
myometrium have reported that danoﬂoxacin has a biphasic
dose-related effect on uterine contractility induced by
oxytocin. At low doses, the antibiotic induces an increase in the
frequency and amplitude, thereby blocking ATP-dependant
potassium channels, whereas at high doses, it decreases the
peak area, probably blocking hyperpolarization [41].
In in vivo studies, there are conﬂicting opinions about
the action of rifaximin on smooth muscles. Most authors
believed that this antibiotic is not able to inﬂuence either
the emptying rate or intestinal motility of mouse, rat, and
human, except presence of small intestinal bacterial overgrowth [29,55,56].
On the basis of these premises, the aim of this work was
to test the in vitro activity of amoxicillin, enroﬂoxacin, and
rifaximin commonly used in bovine reproduction on the
uterus contractility in this species.
2. Materials and methods
2.1. Uterine strip preparation
A total of 68 uteri were obtained from cows slaughtered at
a local abattoir. All uteri were found to be healthy and so were
considered in our study: 33 from cows in the follicular phase
and 35 from cows in the luteal phase. The estrus phase was
recognized by antemortem and postmortem examinations.
Antemortem, the phase of the estrous cycle and ovarian
activity were determined through clinical examination,
rectal palpation, and ultrasonography. At the same time,
blood samples were collected from the coccygeal vein of
each cow in prerefrigerated vacutainer glass tubes. After
transporting blood samples to the laboratory, they were
centrifuged at 1620 g for 10 minutes at 4 C. The sera
were subsequently frozen at 20 C for later analysis of
progesterone (P4), which was conducted with a competitive immunoenzymatic colorimetric method (Progesterone
EIA WELL; Radim SpA, Pomezia [Roma], Italy). The crossreactions between P4 and steroid hormones were reported as follows: P4 100%; 11-a OH-P4 18%; 17-a OH-P4
16%; 20-a OH-P4 1%; estradiol less than 1 102%; testosterone less than 1 102%; cortisol less than 1 103%; and
cholesterol less than 1 103%. The detection limit of the
assay was 0.05 ng/mL. The intra-assay and interassay precisions had coefﬁcient of variations of 2.9% and 4.8%,
respectively. Cutoff values for estrus and diestrus were set
at 1 ng/mL and greater than 2 ng/mL, respectively [57].
After stunning, the animals’ genital tract and functional
ovarian structures were visually examined for further
identiﬁcation of the phase of the estrous cycle and to
exclude any pathologic conditions [7].
The time from when the cows were slaughtered to when
the uteri were collected was about 20 10 minutes.
From each uterus, a single circular portion of the middle
part of the ipsilateral horn to the functional ovarian structure was excised and immediately placed in a ﬂask containing prerefrigerated and oxygenated Krebs solution
(NaCl 113 mM, KCl 4.8 mM, CaCl2$2H2O 2.2 mM, MgSO4
1.2 mM, NaH2PO4 1.2 mM, NaHCO3 25 mM, glucose 5.5 mM,
and sodium ascorbate 5.5 mM), which was prepared daily.
The ﬂask was then immediately transported to the laboratory in an insulated box. Mean transportation time was
about 15 5 minutes. Uterine circular full-thickness portions were cut into strips (10 mm 3 mm) parallel to the
longitudinal muscle ﬁbers.
2.2. Experimental design
The strips were immediately placed in an organ bath
(10 mL) (model 4050; Ugo Basile, Milan, Italy) containing
Krebs solution continuously bubbled with 95% O2 and 5%
CO2. The pH was kept at 7.4, and the temperature was
maintained at 37 C. A silk thread was used to tie the myometrial strips to an isometric transducer (FORT25; AD Instruments, Castle Hill, NSW, Australia). The contractile
activities were recorded using a PowerLab 4/35 (AD Instruments acquisition software). After 1 hour, stabilization
strips were placed under tension of 2 g for about 30 minutes.
Next followed the equilibration, carbachol (105 M), the
esteriﬁed form of acetylcholine, was added to the bath. This
concentration of carbachol had a selective and prolonged
contractant effect and was subsequently removed with the
wash (washout). Carbachol was repeated after 30 minutes,
time needed to ensure that the strip returned to the equilibrium condition. In the presence of a repeatable response
M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
with a deviation of 20% or less, calculated by the formula
(valuemaximum valueminimun/valuemaximum) 100, we
proceeded to the experimental protocol, otherwise carbachol at the same concentration (105 M) was again administered (Fig. 1). If the latter administration of carbachol was
not repeatable, the strip was discarded.
The experimental protocol included those strips that
were exposed to single concentrations of amoxicillin,
enroﬂoxacin, and rifaximin (106, 105, and 104 M).
Because there are no studies in this regard, the choice of
concentrations used in vitro was made starting from the
lowest concentration (106 M), and then ascending to reach
in vitro concentrations that are closest to the minimum
concentration of these antibiotics [58–60].
The rifaximin’s stock solution was made in ethanol,
whereas those of amoxicillin and enroﬂoxacin were made
in distilled water.
Previous studies from our group reported that
ethanol has no effect on in vitro uterine contractility of the
cow [61].
The three antibiotics at concentrations of 106, 105, and
4
10
M were left in the bath for 10 minutes and then
removed by washing. The time needed to obtain an effect of
antibiotics on myometrial contractility was 10 minutes.
Then, we proceeded to calculate the contractile activity
as average amplitude (grams) and average frequency
(number of contractions per minute), before and after the
administration of antibiotics.
For each concentration, the percentage increase from
baseline was evaluated using the following formula:
(T2 T1/T1) 100 [62].
1289
2.4. Statistical analysis
Amplitude and frequency values were expressed as the
mean standard error of the mean (SEM) and were subjected to statistical analysis with SPSS Statistics 19 (IBM,
New York, USA). Effects of different antibiotics in the two
phases of the cycle and on the contractile response were
analyzed with Student’s t test and one-way ANOVA and
post hoc least signiﬁcant difference test, respectively.
The values were considered statistically signiﬁcant at
P < 0.05.
3. Results
Spontaneous uterine contractility was observed in
27 strips of 33 uteri collected in the follicular phase and
32 strips of 35 uteri collected in the luteal phase. Nine strips
that not show any spontaneous or comparable responses to
carbachol (105 M) were discarded.
The representative tracings (Figs. 2–4), respectively,
show the effects induced by amoxicillin, enroﬂoxacin, and
2.3. Chemicals
Carbachol, amoxicillin, and enroﬂoxacin were purchased from Sigma-Aldrich, Milano, Italy and rifaximin was
a gift from Fatro, Italy. Other chemicals were obtained from
commercial sources.
Fig. 1. Representative tracing of the effect on uterine contractility induced
by carbachol (105 M). The presence of two contractile responses comparable (difference of 20%) (conﬁrmed) the functionality. The contraction
evoked by carbachol was the control of our experiments and was always
followed by its washout (w.o.). The amplitude (y-axis) is expressed in grams.
Fig. 2. Representative tracing of the effects induced by the highest concentration of amoxicillin (104 M) in the follicular (A) and luteal (B) phases
on uterine contractility. The amplitude (y-axis) is expressed in grams.
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M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
Fig. 4. Representative tracing of the effects induced by the highest concentration of rifaximin (104 M) in the follicular (A) and luteal (B) phases on
uterine contractility. The amplitude (y-axis) is expressed in grams.
Fig. 3. Representative tracing of the effects induced by the highest concentration of enroﬂoxacin (104 M) in the follicular (A) and luteal (B) phases
on uterine contractility. The amplitude (y-axis) is expressed in grams.
rifaximin at the highest concentration (104 M) tested in
this study, in the follicular (Figs. 2A, 3A, and 4A) and luteal
(Figs. 2B, 3B, and 4B) phases.
The mean values SEM of amplitude and frequency
before and after the administration of drugs (106, 105,
and 104 M) and the relative percentage index are reported
in Tables 1–6, respectively, for amoxicillin, enroﬂoxacin,
and rifaximin.
At all stages of the experiment, the amplitude and frequency of contraction baseline showed a statistically
signiﬁcant difference between the follicular and the luteal
phases (Tables 1–6), as reported by previous work [63,64].
The contractile activity induced by the drugs tested was
evaluated for a period of 10 minutes. This has allowed us to
identify the effect and the real range of action of the molecules, which amounted to 2.5 and 9 minutes for amoxicillin, enroﬂoxacin, and rifaximin, respectively.
The three antibiotics have modulated myometrial
contractility in a different manner for activity and duration
of action.
In particular, amoxicillin had a concentrationdependent relaxing effect delayed in time, which was
Table 1
Effect of single concentrations of amoxicillin on the amplitude contractions of bovine uterus contractions, during follicular and luteal phases.
Amoxicillin (amplitude, g)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
1.70
1.66
1.65
1.60
1.76
1.62
Mean SEM
0.08c
0.07a
0.07c
0.07c
0.13a
0.09a
Luteal phase
Percentage index (%)
Mean SEM
L2.35
1.00
0.98
0.92
0.90
1.06
1.03
L3.03
L7.95
0.18d
0.19b
0.17d
0.15d
0.17b
0.18b
Percentage index (%)
L2.00
L2.17
L2.83
Data are expressed as the mean SEM. The table also shows, in bold, the percentage of decrease in the average amplitude of contraction of the myometrium
bovine, after the administration of different concentrations of amoxicillin on basal value.
In row superscripts a, b indicate P < 0.05 and c, d indicate P < 0.01.
M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
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Table 2
Effect of single concentrations of amoxicillin on the frequency of contractions of bovine uterus, during follicular and luteal phases.
Amoxicillin (frequency number
of contractions/2.5 min)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
1.00
1.00
1.00
1.00
0.88
0.88
Mean SEM
0.13
0.13
0.14
0.17
0.15
0.19
Luteal phase
Percentage
index (%)
Mean SEM
0
0.67
0.67
0.80
0.80
0.80
0.80
0
0
Percentage
index (%)
0.08
0.13
0.13
0.13
0.12
0.12
0
0
0
Data are expressed as the mean SEM. The table also shows, in bold, the percentage index of the average frequency of bovine myometrium contraction, after
the administration of different concentrations of amoxicillin on basal value.
evident 1.5 minutes after the administration of the molecule, persisting for 2.5 minutes.
The tonic effect induced by enroﬂoxacin began 1 minute
after the administration and persisted for 3 minutes. This
activity was present in both phases of the cycle (Fig. 3A, B).
Even rifaximin had a delayed effect in time that occurred
1 minute after the administration and lasted until the
wahout (9 minutes). This antibiotic determined a high and
long-lasting contraction in the follicular phase (Fig. 4A),
whereas in the luteal phase (Fig. 4B) it induced a massive
and prolonged relaxation.
Regarding the comparison between concentrations,
statistically signiﬁcant differences were observed only for
the amplitude induced by rifaximin between 105 and
106 M in the follicular phase and between 104 and
106 M in the luteal phase, respectively (Table 5).
The frequency induced by increasing the concentration
of different antibiotics (106, 105, and 104 M) was not
signiﬁcantly different.
The activity of antibiotics was also evaluated by the
change in percentage index of contractility compared with
its baseline.
In both phases, amoxicillin caused a concentrationdependent decrease in the amplitude of contraction as
shown by the percentages in Table 1. Such an effect was
more evident in the follicular phase and, in particular, at a
concentration of 104 M (7.95%). The frequency of
contraction did not seem to be changed by amoxicillin
(Table 2).
Enroﬂoxacin had a tonic effect on myometrial contractility of the bovine species in both phases of the estrous
cycle, with the highest values in the luteal phase (3.30%,
4.69%, and 6.77% at concentrations of 106, 105, and
104 M, respectively) (Table 3). Like amoxicillin, this
antibiotic did not change the frequency of uterine
contraction (Table 4).
In the follicular phase, rifaximin determined an
impressive and long-lasting increase in the contractile tone
(12.45%, 21.25%, and 30.43% at concentrations of 106, 105,
and 104 M, respectively), whereas in the luteal phase, it
determined a relaxation of the strip by inducing a decrease
in the contractile tone (26.34%, 34.97%, and 53.01% at
concentrations of 106, 105, and 104 M, respectively)
(Table 5). As regards with the frequency, rifaximin determined a decrease in the contractile tone in both phases
with a greater decrease in the luteal phase at the concentration of 104 M (26.32%) (Table 6).
4. Discussion
Uterine contractility is crucial for the realization of
several reproductive events in both women and other
mammals [65–67]. Understanding the mechanisms involved in myometrial contractility and their possible
pharmacologic modulation is of great importance for the
purpose of human obstetrics and gynecology and veterinary medicine.
There are many studies that have evaluated the action of
certain antibiotics (erythromycin, clarithromycin, neomycin,
gentamicin, clindamycin, and ceftriaxone) on uterine
contractility of woman, rat, and bovine, bringing a relaxing
effect on myogenic activity and hormone induced [46–48,68].
These results support the hypothesis that antibiotics may act,
in the postpartum, as a potential obstacle in the physiological
process of self-cleaning, favoring the persistence of exudates
and microorganisms inside the uterus.
In our study, we tested for the ﬁrst time the in vitro effect of three antibiotics (amoxicillin, enroﬂoxacin, and
Table 3
Effect of single concentrations of enroﬂoxacin on the amplitude of contractions of bovine uterus, during follicular and luteal phases.
Enroﬂoxacin
(amplitude, g)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
2.04
2.05
2.07
2.13
2.00
2.12
Mean SEM
0.27a
0.28
0.18a
0.18a
0.23a
0.24a
Luteal phase
Percentage index (%)
Mean SEM
D0.49
1.21
1.25
1.28
1.34
1.33
1.42
D2.90
D6.00
0.21b
0.24
0.26b
0.26b
0.16b
0.16b
Percentage index (%)
D3.30
D4.69
D6.77
Data are the expressed as the mean SEM. The table also shows, in bold, the percentage of increase in the average amplitude of contraction of the
myometrium bovine, after the administration of different concentrations of enroﬂoxacin on basal value.
In row superscripts a, b indicate P < 0.05.
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M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
Table 4
Effect of single concentrations of enroﬂoxacin on the frequency of contractions of bovine uterus, during follicular and luteal phases.
Enroﬂoxacin (frequencydnumber
of contractions/3 min)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
1.33
1.33
1.20
1.20
1.25
1.25
Luteal phase
Mean SEM
0.19a
0.19a
0.11a
0.12a
0.08c
0.05e
Percentage
index (%)
Mean SEM
0
0.67
0.67
0.80
0.80
0.80
0.80
0
0
0.08b
0.13b
0.13b
0.13b
0.12d
0.12f
Percentage
index (%)
0
0
0
Data are expressed as the mean SEM. The table also shows, in bold, the percentage index of the average frequency of contraction of the myometrium
bovine, after the administration of different concentrations of enroﬂoxacin on basal value.
In row superscripts a, b indicate P < 0.05; c, d indicate P < 0.01; and e, f indicate P < 0.001.
rifaximin), normally used in bovine reproduction, on the
contractile activity of bovine uterus in the follicular and
luteal phases.
The results showed that all three antibiotics showed
modulatory activity on uterine contractility, with the
different effect and duration of action.
In particular, the amoxicillin has induced a relaxing
concentration-dependent effect on basal contractility in
both phases of the cycle, with a more striking effect in the
follicular phase.
In this study, we have not studied the mechanism of
action of the tested antibiotics; however, it is possible that
the muscle relaxant effect may be attributed to amoxicillininduced stimulation of mechanisms involved in physiological uterine relaxation: activation of adenylate cyclase or
the opening of potassium channels or to decreased levels of
intracellular free calcium. The effect of amoxicillin on
uterine contraction, associated with its antimicrobial effect
highly selective for the bacterial wall, could suggest the
using of this antibiotic for the treatment of diseases that
may occur in pregnancy as it is not toxic to the embryo and
fetus. Our analysis conﬁrms that the amoxicillin could be
used safely during pregnancy, as it may even assist and
enhance the quiescence induced by P4 [69]. On the contrary, the use of the same antibiotic in the postpartum is
counterproductive, as its effect on uterine contractility can
negatively affect the self-cleaning.
In our study, we also evaluated the effect exerted by
enroﬂoxacin on uterus bovine contractility.
This antibiotic has increased the spontaneous contractions of strips of bovine uterus in a concentrationdependent manner. In agreement with the previous
studies of other ﬂuoroquinolones [36,51], we believe that
enroﬂoxacin acts even in the uterus such as GABAA antagonist, blocking the ATP-dependent potassium channels
[36–38,41,50–52]. In support of this, the stimulatory action
of enroﬂoxacin on uterine contractility, measured in our
experiment, was higher in the luteal phase, the concentration of the GABAA receptor in uterine tissue [70]. On the
contrary, the increased presence of GABAB receptors in the
follicular phase [70] could explain the less effect of enroﬂoxacin on the uterine muscle measured by us at this stage
of the cycle.
In other studies conducted in the myenteric plexus, the
ability of ﬂuoroquinolones to stimulate the release of
PGF2a that are responsible for releasing of acetylcholine at
prejunctional cholinergic transmission and then inducing
muscle contractility [53,71] too was reported.
The excitatory effects on contractility highlighted in this
study allow us to consider the possibility of using enroﬂoxacin in the therapy of retained placenta and endometritis in cattle, because in addition to the already known
mechanisms of action, it may modulate the contractile activity facilitating cleaning and uterine involution. In
particular, the use of this drug for the treatment of all forms
of metritis, including chronic metritis, derives from the
absence of a conditioning dictated by steroid hormones. It
is possible that even in the uterus, as shown in the intestine
[53,71], this could lead ﬂuoroquinolones to induce the
release of PGF2a, which are responsible of myometrial
contractility and trigger the process of luteolysis, too.
Regarding the rifaximin, its modulatory action on the
contractility of the bovine uterus showed a twofold effect:
in the follicular phase it increases tonic activity of uterus,
Table 5
Effect of single concentrations of rifaximin on the amplitude of contractions, during follicular and luteal phases.
Rifaximin
(amplitude, g)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
2.49
2.80
2.87
3.48
2.30
3.00
Mean SEM
0.19a
0.22eB
0.15e
0.14eA
0.10a
0.13c
Luteal phase
Percentage
index (%)
Mean SEM
D12.45
1.86
1.37
1.83
1.19
1.83
0.86
D21.35
D30.43
0.12b
0.11fB
0.12f
0.17f
0.11b
0.12dA
Percentage
index (%)
L26.34
L34.97
L53.01
Data are expressed as the mean SEM. The table also shows, in bold, the percentage index of the average amplitude of contraction, after the administration
of different concentrations of rifaximin on basal value.
In row superscripts a, b indicate P < 0.05; c, d indicate P < 0.01; and e, f indicate P < 0.001. In column superscripts A, B indicate P < 0.05.
M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
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Table 6
Effect of single concentrations of rifaximin on the frequency of contractions, during both cycle phases.
Rifaximin (frequencydnumber
of contractions/9 min)
Follicular phase
Basal
106 M
Basal
105 M
Basal
104 M
1.06
1.03
1.25
1.18
1.11
1.02
Luteal phase
Mean SEM
0.15a
0.13a
0.10e
0.06e
0.05e
0.13c
Percentage
index (%)
Mean SEM
L2.83
0.64
0.58
0.50
0.40
0.57
0.42
L5.60
L8.11
Percentage
index (%)
0.07b
0.11b
0.07f
0.11f
0.08f
0.09d
L9.38
L20.00
L26.32
Data are expressed as the mean SEM. The table also shows, in bold, the percentage of decrease in the average frequency of contraction, after the
administration of different concentrations of rifaximin, during the follicular and luteal phases.
In row superscripts a, b indicate P < 0.05; c, d indicate P < 0.01; and e, f indicate P < 0.001.
whereas in the luteal phase it induces a relaxation of the
uterine musculature.
From these results, it is conceivable that the effect
induced by rifaximin is under the control of steroid hormones predominant at different stages of the cycle,
respectively, estrogen in the follicular phase and P4 in the
luteal phase.
The in vitro studies that illustrate the action of this
antibiotic on the smooth muscles are not present in the
literature, whereas in vivo studies conﬂicting opinions are
revealed.
However, it is known that this molecule is able to bind to
a nuclear receptor for steroids and xenobiotics [72–74]: the
pregnane-X-receptor (PXR) [75,76] involved, in turn, in the
modulation of myometrial contractility [77].
The binding of rifaximin with this receptor leads to an
increased expression of PXR [75,76,78], but the synthesis of
new receptors takes a long time [79], which is not covered
by the timing of our laboratory procedures. However, in
studies of the epithelial cells of the human colon knockout
for PXR, Mencarelli et al. [78] found that rifaximin acted as
an agonist of PXR too. Furthermore, Xue et al. [80] have
clearly shown that the PXRs can suffer from coactivation of
steroids and xenobiotics. In this sense, it is conceivable that
in the presence of rifaximin, the PXRs might have sustained
a hesitant coactivation potentiation of the contractant or
releasing action exercised by the predominant steroid in
the phase of the cycle in which it was the uterus (estrogen
in the follicular phase and P4 in the luteal phase).
In the light of what has been exposed, the increase in
the contractile tone induced by the rifaximin under the
inﬂuence of estrogen allows to consider the possibility of
using this molecule at the time of heat, for the treatment of
subacute metritis to facilitate the uterine cleaning.
On the other hand, the relaxation obtained in the luteal
phase, associated with the good selective toxicity given by
substantial structural difference between the RNA polymerase of the prokaryotic cell and that of the eukaryotic cell
[81,82], makes possible the use of rifaximin, if necessary,
even during pregnancy as it intensiﬁes uterine quiescence
induced by P4. For this reason, its use is not suitable for the
treatment of chronic metritis because the relaxing effect
induced by rifaximin would favor the persistence of organic
substances (liquid residues of childbirth, cellular debris,
blood, pus) able to inactivate its own action.
Further studies are, however, required to clarify the use
of this antibiotic for the treatment of acute metritis, in
which there is not a prevalent hormonal state, as the disease is realized before the functional reactivation of
reproductive axis.
4.1. Conclusions
This study shows, for the ﬁrst time, the modulatory
activity of three antibiotics (amoxicillin, enroﬂoxacin, and
rifaximin) on the bovine uterine contractility. The drugs
tested show different effects that allow to ascribe their
therapeutic use in accordance with their ability to modulate uterine contractility (Table 7) too.
From a clinical point of view, our study provides, for the
three antibiotics tested, a starting point for new directions
in the ﬁeld of bovine reproduction. The inhibitory effect of
amoxicillin on uterus contractility supports the hypothesis
that this drug can be used in therapy of infectious diseases
Table 7
Therapeutic indications and incorrect use of antibiotics tested in this study, according to their ability to modulate uterine contractility.
Antibiotic
Therapeutic indications
Do not use in case of
Class
Molecules
Period of use
Diseases
Period of use
Diseases
B-lactams
Amoxicillin
Pregnancy
Postpartum
Retained placenta
Metritis postpartum
Rifamycin
Rifaximin
Pregnancy
40–60 days postpartum
Chronic metritis
Fluoroquinolones
Enroﬂoxacin
14–40 days postpartum
Postpartum
Pregnancy
Each type of pathology
Mastitis
Respiratory system
Digestive system
Urinary tract
Skin and soft tissue
Mastitis
Digestive system
Subacute metritis
Metritis postpartum
1294
M. Piccinno et al. / Theriogenology 82 (2014) 1287–1295
in the course of pregnancy. To this end, it is possible that
the use of rifaximin under the inﬂuence of P4 is able to
determine a lasting uterus relaxation. The role of this
antibiotic, however, is twofold; if used in the presence of
estrogen, rifaximin may stimulate high contraction that
could justify its use during the heat for the treatment of
subacute metritis to facilitate the uterine cleaning.
On the contrary, enroﬂoxacin is not affected by hormonal changes; this would encourage the use of this drug
for the treatment of all forms of metritis, including chronic
metritis; thanks to the ability of this ﬂuoroquinolone to
induce the release of PGF2a as well.
Further studies are, however, needed to evaluate the
mechanism of action of the antibiotics tested and evaluate
their effect in combination with ecbolic substances normally used in the postpartum dairy cows.
[18]
[19]
[20]
[21]
[22]
[23]
[24]
Acknowledgments
[25]
Fatro, pharmaceutical industry, for supplying products
used in this research.
[26]
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