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Epidural vs intramuscular administration of lecirelin, a GnRH analogue, for the resolution of follicular cysts in dairy cows

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Abstract

Bovine follicular cysts are an ovarian disorder of dairy cows associated with abnormal estrous behaviour and infertility. The treatment of choice is intramuscular administration of a GnRH analogue, which acts by triggering pituitary release of LH. However, the presence of GnRH and GnRH receptors on spinal cord and ovary in some species, and the kind of innervation of the ovary, let us hypothesize that GnRH and its analogues may also act when administered by epidural route, as happens for other drugs. Therefore the aim of this study was to compare the effects of epidural vs intramuscular administration of lecirelin (a GnRH analogue) on FC regression, estrus detection and pregnancy outcomes. The study was conducted on 220 Friesian cows affected by follicular cysts, divided among 4 groups: Group L(epid) and Group L(im) received, respectively 50 μg of lecirelin in the epidural space and intramuscular; Group C(epid) and Group C(im) were used as control groups. In Group L(epid), estrus induction and pregnancy rates were significantly higher than in Group L(im). The results of this study show that the epidural administration of lecirelin promoted the remission of follicular cysts and an improvement of reproductive parameters compared to intramuscular administration. Thus, an alternative therapeutical approach is available for FC treatment, in order to obtain an easier restoration of the ovarian activity, especially in those cases refractory to classical therapeutic approaches.
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Author's personal copy
Animal Reproduction Science 126 (2011) 19–22
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Epidural vs intramuscular administration of lecirelin, a GnRH
analogue, for the resolution of follicular cysts in dairy cows
Rizzo Annalisa, Campanile Debora, Mutinati Maddalena, Minoia Giuseppe,
Spedicato Massimo, Sciorsci Raffaele Luigi
Department of Animal Production, Faculty of Veterinary Medicine, University of Bari Aldo Moro, Strada Prov. le per Casamassima, Km 3, 70010 – Valenzano
(Bari), Italy
article info
Article history:
Received 15 November 2010
Received in revised form 11 April 2011
Accepted 20 April 2011
Available online 29 April 2011
Keywords:
Cow
Follicular cysts
GnRH
Epidural administration
Intramuscular administration
abstract
Bovine follicular cysts are an ovarian disorder of dairy cows associated with abnormal
estrous behaviour and infertility. The treatment of choice is intramuscular administration of
a GnRH analogue, which acts by triggering pituitary release of LH. However, the presence
of GnRH and GnRH receptors on spinal cord and ovary in some species, and the kind of
innervation of the ovary, let us hypothesize that GnRH and its analogues may also act when
administered by epidural route, as happens for other drugs. Therefore the aim of this study
was to compare the effects of epidural vs intramuscular administration of lecirelin (a GnRH
analogue) on FC regression, estrus detection and pregnancy outcomes.
The study was conducted on 220 Friesian cows affected by follicular cysts, divided among
4 groups: Group Lepid and Group Lim received, respectively 50 g of lecirelin in the epidural
space and intramuscular; Group Cepid and Group Cim were used as control groups. In Group
Lepid, estrus induction and pregnancy rates were significantly higher than in Group Lim .
The results of this study show that the epidural administration of lecirelin promoted the
remission of follicular cysts and an improvement of reproductive parameters compared to
intramuscular administration.
Thus, an alternative therapeutical approach is available for FC treatment, in order to
obtain an easier restoration of the ovarian activity, especially in those cases refractory to
classical therapeutic approaches.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Bovine follicular cysts (FC) are an important disorder
often encountered in dairy cows, and is associated with
abnormal estrous behaviour and infertility (Peter, 2004).
Follicular cysts are defined as anovulatory follicles with a
diameter of at least 17 mm, presenting a thin wall and per-
sisting on one or both ovaries for more than 6 days (Silvia
et al., 2002). The estimated prevalence of the disorder in
dairy herds varies from 5% to 30% and may lead to eco-
Corresponding author. Tel.: +39 0805443882; fax: +39 0805443883.
E-mail address: r.sciorsci@veterinaria.uniba.it (S.R. Luigi).
nomic loss due to increased days open, veterinary costs,
and culling (Peter, 2004; Vanholder et al., 2006).
Notwithstanding that FC have been studied from an
endocrine, histological, molecular and clinical aspect, the
etiopathogenesis of FC is not fully understood (Zulu and
Penny, 1998; Ribadu et al., 2000; Peter, 2004; Palomar
et al., 2008; Rizzo et al., 2009a, 2009b), although aetiol-
ogy is now proposed to be multifactorial. A dysfunction
of the hypothalamus-pituitary-ovary axis, leading to an
insufficient GnRH release, and/or an inadequate recepto-
rial responsiveness, is considered the primary cause (Silvia
et al., 2002; Peter, 2004).
In accordance with its multifaceted aspect, many thera-
peutical approaches have been attempted for the treatment
0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2011.04.013
Author's personal copy
20 R. Annalisa et al. / Animal Reproduction Science 126 (2011) 19–22
of FC (Peter, 2000; Todoroki et al., 2001; Gümen and
Wiltbank, 2002; Zulu et al., 2003; Gümen and Wiltbank,
2005; Lievaart et al., 2006; Kim and Kim, 2007), even if the
treatment of choice is the intramuscular administration of
a GnRH analogue (Peter, 2000, 2004).
Exogenous GnRH analogues act by activating pituitary
GnRH receptors to trigger a sharp secretion of LH leading
to luteinisation (Schneider et al., 2006).
Expression of receptors for GnRH has been demon-
strated also in many mammalian extrapituitary tissues
(reviewed by Ramakrishnappa et al., 2005). Particularly,
Ramakrishnappa et al. (2001) reported the presence of
GnRH-receptor mRNA in bovine ovary, in both follicle and
corpus luteum tissue. Moreover, GnRH receptors are also
expressed at all levels of the mammalian spinal cord (Dolan
et al., 2003). Moreover, a very recent in vitro study by our
group (Rizzo et al., 2010) has indirectly demonstrated the
presence of GnRH-receptors on the wall of follicular cysts.
These premises highlight the likelihood that GnRH ana-
logues may trigger their effects by action not only on
pituitary receptors, but also on extrapituitary ones.
In veterinary medicine, many pharmacological agents,
above all local anesthetic and analgesic drugs, are admin-
istered into the epidural space (Thurmon et al., 1996).
This route can also be used for administering hormones
and analogues, achieving a selective local pharmacological
response. As the bovine ovaries are innervated by sympa-
thetic neurons derived from the ovarian plexus nerve and
the hypogastric nerve (Dissen et al., 1993; Pelagalli and
Botte, 2003), the epidural administration could indirectly
influence ovaries. Therefore the aim was to compare the
effects of epidural vs intramuscular administration of GnRH
on FC regression.
2. Materials and methods
2.1. Ethical policy
All the following procedures were conducted according
to the Italian D. L.vo 116/92 and the statute of the University
of Bari concerning to the use of client-owned animals under
clinical investigation.
2.2. Animals
The study was conducted on commercial dairy farms in
Puglia (Italia) on 220 pluriparous Friesian cows affected by
follicular cysts (3–5 years old, mean weight 600 kg, aver-
age milk yield per lactation 7500 kg), in post-partum from
60 to 120 days. All cows were tethered in tie stalls and
fed hay, concentrate, and minerals. Water was available
ad libitum. All animals were diagnosed free from com-
mon parasites (coccidian and strongyloides) and declared
free from bovine diarrhoea, brucellosis, bovine leucosis and
tuberculosis. Moreover, the anamnesis of the cows revealed
neither previous obstetrical problems nor local or systemic
pharmacological treatments in the 30 days preceding the
enrolment in the study.
Preliminary diagnosis of FC was performed by clinical
examination, transrectal palpation of ovaries, transrectal
ultrasonography (Scanner 480, linear probe, 5 MHz, Pie
Medical, The Netherlands) and determination of plasma
progesterone (P4) concentration (Enzyme-linked fluo-
rescent assay, VIDAS®Progesterone, BioMerieux®, Lyon,
France: sensibility: 0.25–80 ng/mL; specificity: 100%). All
the procedures were repeated 10 days later, to confirm the
diagnosis.
For progesterone determination blood samples were
collected, by venipuncture of the coccygeal vein into vacu-
tainer tubes containing EDTA. Once in the laboratory, the
tubes were centrifuged at 1620 ×gfor 15min at +4 C, and
the decanted plasma was stored at 20 C.
Concentration of P4 <1 ng/mL, the presence of follicle-
like structures with diameters larger than 17 mm and
persisting for 10 days, with a wall thinner than 3 mm, as
determined by ultrasonography, let us diagnose FC.
2.3. Experimental protocol
Soon after diagnosis, cows were randomly allocated, via
coin toss, into one of the following groups:
- Group Lepid: 60 cows administered 50 g of lecirelin (cor-
responding to 2 mL of Dalmarelin®, Fatro, Italy) per head
in the sacrococcygeal epidural space.
- Group Cepid: 50 cows administered 2 mL of normal saline
solution (0.9% NaCl) per head in the sacrococcygeal
epidural space.
- Group Lim: 60 cows administered 50 g of lecirelin (cor-
responding to 2 mL of Dalmarelin®, Fatro, Italy) per head
im.
- Group Cim: 50 cows administered 2 mL of normal saline
solution (0.9% NaCl) per head im.
Lecirelin was the drug of choice because of its high
lipophilicity.
Every 2 days from 5 to 20 days post-treatment, all
animals underwent a clinical examination and transrectal
ultrasonography to detect the eventual regression of FC and
first estrus appearence. Estrus was detected observing typi-
cal oestrus signs (oedema of the vulva, clear mucous vaginal
discharge, standing to be mounted) and by the detection of
a preovulatory follicle on transrectal palpation.
Cows found to be in estrus and without genital disor-
ders were inseminated with semen of bulls with proven
fertility by a skilled veterinarian. After insemination, cows
were further observed to detect an eventual return to heat
and so allow a second AI.
Forty days after insemination, pregnancy was diagnosed
by transrectal palpation and ultrasonography. Thus, preg-
nancy rate at first and second AI were recorded. The results
concerning estrus induction and pregnancy rate were ana-
lyzed statistically using a 2test.
3. Results
Epidural administration was accomplished very easily
in all animals. No side effects were noted, and cows did
not show any sign of pain or discomfort either soon after
administration, at the doses we employed, or at follow up.
Results concerning reproductive parameters are presented
in Table 1.
Author's personal copy
R. Annalisa et al. / Animal Reproduction Science 126 (2011) 19–22 21
Table 1
Reproductive parameters (estrus, pregnant cows after 1st and 2nd AI, and total pregnancies) in Group Lepid (cows administered lecirelin by epidural route),
Group Cepid (cows administered saline solution by epidural route); Group Lim (cows administered lecirelin by intramuscular route) and Group Cim (cows
administered saline solution by intramuscular route).
No. of cows Estrus Pregnant cows after 1st AI Pregnant cows after 2nd AI Total pregnancies
Group Lepid 60 45 (75%) a 32 (71%) 10 (77%) 42 (93%) a
Group Cepid 50 0 (0) 0 (0) 0 (0) 0 (0)
Group Lim 60 34 (57%) b 18 (53%) 8 (50%) 26 (76%) b
Group Cim 50 0 (0%) 0 (0%) 0 (0%) 0 (0%)
In the columns: a vs b: p< 0.05.
In Group Lepid, 75% of cows showed estrus within
15 ±3 d, compared to 57% of Group Lim. Statistical analysis
revealed that lecirelin administered in the epidural space
was significantly more efficacious than lecirelin injected
intramuscular (p< 0.05). Total pregnancy rate was higher
in Lepid group than in Lim group (p< 0.05).
None of the cows of the control groups recovered from
FC.
4. Discussion and conclusions
To the best of our knowledge, this is the first in depth
report comparing epidural vs intramuscular administra-
tion of lecirelin, a GnRH analogue, for the treatment of
bovine follicular cysts. The experiment shows that epidural
administration of lecirelin is a better and more efficacious
alternative to intramuscular injection. Epidural injection
triggered the remission of FC and return to estrus in a higher
percentage of cows, thus proving its efficacy when admin-
istered by this route.
In veterinary medicine, analgesic drugs, such as local
anesthetics, opioids, 2 agonists and ketamine are usually
administered directly into the epidural space (Thurmon
et al., 1996). It is now evident that hormones and, par-
ticularly lecirelin, can be epidurally administered without
inducing any side effect.
The result obtained in this study is likely to be due to a
synergistic action between lecirelin systemically absorbed,
and the effect of lecirelin on sites other than hypophysis,
such as spinal cord and/or ovary. In fact, lecirelin have acted
on the spinal cord and/or directly on the ovary, as well as via
a systemic diffusion and consequent action on the hypoph-
ysis; or it may have reached the pituitary gland through the
epidural canal.
The pharmacokinetics of drugs administered in the
epidural space depends on many factors, such as the vol-
ume injected, the speed of inoculation, lipophilicity and
molecular weight (Johnson et al., 1996; Thurmon et al.,
1996; Lopez et al., 1997; Lee et al., 2005).
Lecirelin has a glycine substituted by an ethylamide
group, at the aminoterminal side (pGlu-His-Trp-Ser-Tyr-d-
Tle-Leu-Arg-Pro-NH-Et), which makes the molecule more
lipophylic than the parent compound (Sazelova et al.,
2006). This characteristic allows the molecule to cross
the lipidic content of the epidural space and meninges
and reach the spinal cord. Dolan et al. (2003) demon-
strated the presence of GnRH receptors in ovine spinal
cord, and our group has recently demonstrated the pres-
ence of GnRH receptors also in bovine spinal cord (data not
published). Thus, we can speculate that lecirelin, acting on
spinal GnRH receptors, may have caused a neuronal influx
of Ca2+ (Schneider et al., 2006), triggering norepinephrine
release from the sympathetic terminals of nerves innervat-
ing ovary (Dissen et al., 1993). The sympathetic innervation
plays an important role in follicular maturation, steroid
secretion and ovulation (Morimoto et al., 1981; Gajewski
et al., 2006) and, moreover, norepinephrine is able to
enhance follicular wall contractility, an event useful for
rearrangement of post-ovulatory granulation tissue and
subsequent development of luteal tissue (Espey, 1978;
Niswender et al., 2000).
Furthermore, when administered epidurally, lecirelin
could reach, vehicled by cerebrospinal fluid, the central
nervous system, and thus hypothalamus and hypophysis
(Dolan et al., 2003; Xu et al., 2008; Skinner et al., 2009).
Thus, we can hypothesize that, when epidurally admin-
istered, lecirelin is thought to activate GnRH receptors also
on sites other than the hypohysis and, to achieve a better
response (functional synergism of action).
In conclusion, lecirelin, can certainly reach the blood-
stream when administered by an epidural route, but it is
also possible that a more direct action occurred on the
spinal cord and ovary, leading to the resolution of FC by
local mechanisms, beyond LH surge triggering.
This hypothesis is intriguing and strengthens the
possibility that GnRH may have a role as neurotrans-
mitter/neuromodulator in the central nervous system,
as already demonstrated in other animals (Jones, 1987;
Skinner et al., 2009).
The present study is a clinical one, and can be imple-
mented by molecular and/or pharmacological studies, in
order to better understand the pharmacokinetics and phar-
macodynamics of lecirelin injected by epidural route. It
could be interesting to detect the presence of lecirelin into
neurons upon epidural administration or the expression of
GnRH receptors on FC and sympathetic nerves.
Given our results yhis study provides the basis to a
new therapeutical approach for FC treatment, based on the
epidural administration of a GnRH agonist, with a view to
obtain an easier restoration of ovarian activity, especially in
those cases refractory to classical therapeutic approaches.
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... Lecirelin is a synthetic analogue of GnRH obtained through the substitution of a glycine by an ethylamide group at its terminal site (Baruselli et al., 2001) which has been used in many animal species (Sazelova et al., 2006;Carluccio et al., 2007;Kaya et al., 2008;Zapletal and Pavlik, 2008;Anjum et al., 2009;Fries et al., 2010). Administered at the time of insemination, Lecirelin improves the reproductive performance of dairy cattle (Martin et al., 2006) and positive results have been obtained also in follicular cysts affected cows (Robbe et al., 2002;Rizzo et al., 2009Rizzo et al., , 2011. ...
... The substitution of the glycine with the ethylamide group provides for a higher lipophilicity and a lower enzymatic degradability of the molecule thus may have enhanced the absorption of Lecirelin, through the epidural fat, into the cerebrospinal fluid and also through the phospholipidic membrane of the nerve endings (Barron et al., 1982;Sazelova et al., 2006). Previous studies provided evidence for direct actions of GnRH agonists on the gonads and both GnRH-like peptides (Ireland et al., 1988) and GnRH receptors have been found in gonadal tissues (Whitelaw et al., 1995;Skinner et al., 2009) as well as within mammalian spinal cord (Dolan et al., 2003;Rizzo et al., 2011). ...
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Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD.
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Cystic ovarian (OC) condition is an important ovarian dysfunction and a major cause of reproductive failure in dairy cattle. Previously defined as enlarged anovulatory follicle like structures (<2.5 cm) and persisting for 10 or more days OC in dairy cows are currently defined as cystic ovarian follicular structures of at least 17 mm that persist for more than 6 days in the absence of corpus luteum (CL). Past evaluations of OC using trans-rectal palpations have increased, and accurate diagnosis currently employs a combination of trans-rectal palpation, trans-rectal ultrasonography and plasma progesterone assay. Previously the manual rupture of OC was advised, but during the past several years single or combination hCG, GnRH, progesterone and prostaglandins have been frequently practice. Other therapies include clomiphene citrate and trans-vaginal ultrasound guided cystic follicle aspiration. Among the various therapies the Ov-Synch treatment appears to be the most logical approach, yet the pregnancy rates with timed inseminations following therapy with the OvSynch treatment are low, as with other hormonal treatments. It can be concluded that OC can be diagnosed easily yet in spite of many therapeutic options the establishment of pregnancy in cows with OC requires a longer time.
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Cystic ovarian (OC) condition is an important ovarian dysfunction and a major cause of reproductive failure in dairy cattle. Previously defined as enlarged anovulatory follicle like structures (<2.5 cm) and persisting for 10 or more days OC in dairy cows are currently defined as cystic ovarian follicular structures of at least 17 mm that persist for more than 6 days in the absence of corpus luteum (CL). Past evaluations of OC using trans-rectal palpations have increased, and accurate diagnosis currently employs a combination of trans-rectal palpation, trans-rectal ultrasonography and plasma progesterone assay. Previously the manual rupture of OC was advised, but during the past several years single or combination hCG, GnRH, progesterone and prostaglandins have been frequently practice. Other therapies include clomiphene citrate and trans-vaginal ultrasound guided cystic follicle aspiration. Among the various therapies the Ov-Synch treatment appears to be the most logical approach, yet the pregnancy rates with timed inseminations following therapy with the OvSynch treatment are low, as with other hormonal treatments. It can be concluded that OC can be diagnosed easily yet in spite of many therapeutic options the establishment of pregnancy in cows with OC requires a longer time.
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Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1β, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1β, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1β and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1β and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.
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This chapter discusses the involvement of the ovarian innervation and neurotrophic factors in the control of follicular development and ovulation, and provides a succinct account of relevant aspects of early follicular development, the distribution of ovarian nerves, and some of the important features that define the neurotrophins (NTs) family and its receptors. The mammalian ovary is a prominent terminal field for sympathetic and sensory neurons. The neurons projecting to the ovary innervate the different structural components of the gland, including the vasculature, interstitial tissue, and developing follicles, with various degrees of complexity. Before the 1990s, several reports appeared documenting the distribution of intraovarian nerve fibers, their presence in different species, and their neurotransmitter identity. During the early 1990s, the availability of simplified culture procedures and new biochemical and molecular techniques provided insights into the specific contribution that ovarian nerves may have to the regulation of ovarian function. Thus, a number of reports appeared indicating an involvement of neurotransmitters contained in ovarian nerves, such as norepinephrine (NE) and vasoactive intestinal peptide (VIP), in the control of follicular steroidogenesis and suggesting the facilitatory participation of the sympathetic innervation in follicular development.
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