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

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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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

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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.

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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.
References
Dissen, G.A., Dees, W.L., Ojeda, S.R., 1993. Neural and neurotrophic con-
trol of ovarian development. In: Adashi, E.Y., Leung, P.C.K. (Eds.), The
Ovary. Raven Press, New York, pp. 1–19.
Dolan, S., Evans, N.P., Richter, T.A., Nolan, A.M., 2003. Expression of
gonadotropin-releasing hormone and gonadotropin-releasing hor-
mone receptor in sheep spinal cord. Neurosci. Lett. 346, 120–122.
Espey, L.L., 1978. Ovarian contractility and its relationship to ovulation: a
review. Biol. Reprod. 19, 540–551.

Author's personal copy
22 R. Annalisa et al. / Animal Reproduction Science 126 (2011) 19–22
Gajewski, Z., Thun, R., Faundez, R., Pawlinski, B., 2006. The influence of
␣-adrenergic receptors stimulator and blockers and beta-blocker on
the ovary and endocrinological activity in heifers during ovulation. J.
Physiol. Pharmacol. 57 (8), 173–188.
Gümen, A., Wiltbank, M.C., 2002. An alteration in the hypothalamic action
of estradiol due to lack of progesterone exposure can cause follicular
cysts in cattle. Biol. Reprod. 66, 1689–1695.
Gümen, A., Wiltbank, M.C., 2005. Length of progesterone exposure needed
to resolve large follicle anovular condition in dairy cows. Theriogenol-
ogy 63 (1), 202–218.
Johnson, R.A., Lopez, M.J., Hendrickson, D.A., Kruse-Elliot, K.T., 1996.
Cephalad distribution of three differing volumes of new methylene
blue injected into the epidural space in adult goats. Vet. Surg. 25 (5),
448–451.
Jones, S.W., 1987. Luteinizing hormone-releasing hormone as a neuro-
transmitter in bullfrog sympathetic ganglia. Ann. N.Y. Acad. Sci. 519,
310–332.
Kim, I.H., Kim, U.H., 2007. Comparison of the effect of estradiol benzoate
plus progesterone and GnRH on the follicular wave emergence and
subsequent follicular development in CIDR-treated, lactating dairy
cows with follicular cysts. Anim. Reprod. Sci. 98 (3–4), 197–203.
Lee, I., Yamagishi, N., Oboshi, K., Ayukawa, Y., Sasaki, N., Yamada, H., 2005.
Distribution of new methylene blue injected into the caudal epidural
space in cattle. Vet. J. 169, 257–261.
Lievaart, J.J., Parlevliet, J.M., Dieleman, S.J., Rientjes, S., Bosman, E., Vos, P.L.,
2006. Transvaginal aspiration as first treatment of ovarian follicular
cysts in dairy cattle under field circumstances. Tijdschr. Diergeneeskd.
131 (12), 438–442.
Lopez, M.J., Johnson, R., Hendrickson, D.A., Kruse-Elliot, K.T., 1997. Cranial
migration of differing doses of new methylene blue injected in the
epidural space after death of calves and juvenile pigs. Am. J. Vet. Res.
58 (7), 786–790.
Morimoto, K., Okamura, H., Kanzaki, H., Okuda, Y., Takenaka, A., Nishimura,
T., 1981. The adrenergic nerve supply to bovine ovarian follicles. Int.
J. Fertil. 26 (1), 14–19.
Niswender, G.D., Juengel, J.L., Silva, P.J., Rollyson, M.K., MCIntush, E.W.,
2000. Mechanisms controlling the function and life span of the corpus
luteum. Physiol. Rev. 80 (1), 1–29.
Palomar, M.M., Acosta, J.C., Salvetti, N.R., Barberis, F., Beldomenico, P.M.,
Garnero, O., Ortega, H.H., 2008. Treatment of cystic ovarian disease
with Naloxone in high production dairy cows. J. Vet. Pharmacol. Ther.
31, 184–186.
Pelagalli, G.V., Botte, V., 2003. Anatomia veterinaria sistematica e com-
parata. Edi-Ermes, Milan.
Peter, A.T., 2000. Managing postpartum health and cystic ovarian dis-
ease. In: Proceedings of Eighteenth Annual Western Canadian Dairy
Seminar: Advances in Dairy Technology , Alberta, Canada, pp. 85–99.
Peter, A.T., 2004. An update on cystic ovarian degeneration in cattle.
Reprod. Domest. Anim. 39, 1–7.
Ramakrishnappa, N., Merwe, G.K.V.D., Rajamahendran, R., 2001.
Gonadotropin-releasing hormone receptor messenger ribonucleic
acid expression in bovine ovary. Biol. Reprod. 64 (Suppl.), 229
(Abstract).
Ramakrishnappa, N., Rajamahendran, R., Lin, Y.-M., Leung, P.C.K., 2005.
GnRH in non-hypothalamic reproductive tissues. Anim. Reprod. Sci.
88, 95–113.
Ribadu, A.Y., Nakada, K., Moriyoshi, M., Zhang, W.C., Tanaka, Y., Nakao,
T., 2000. The role of LH pulse frequency in ACTH-induced ovarian
follicular cysts in heifers. Anim. Reprod. Sci. 64, 21–31.
Rizzo, A., Cosola, C., Mutinati, M., Spedicato, M., Minoia, G., Sciorsci, R.L.,
2010. Bovine ovarian follicular cysts: in vitro effects of lecirelin, a
GnRH analogue. Theriogenology 74, 1559–1569.
Rizzo, A., Minoia, G., Trisolini, C., Mutinati, M., Spedicato, M., Manca, R.,
Sciorsci, R.L., 2009a. Renin and ovarian vascularization in cows with
follicular cysts after epidural administration of a GnRH analogue.
Anim. Reprod. Sci. 116, 226–232.
Rizzo, A., Minoia, G., Trisolini, C., Mutinati, M., Spedicato, M., Jirillo,
F., Sciorsci, R.L., 2009b. Reactive oxygen species (ROS): involv-
ment in bovine follicular cysts etiopathogenesis. Immunopharmacol.
Immunotoxicol. 31 (4), 631–635.
Sazelova, P., Kasicka, V., Solinova, V., Koval, D., 2006. Determination of
purity degree and counter-ion content in lecirelin by capillary zone
electrophoresis and capillary isotachophoresis. J. Chromatogr. B 841,
145–151.
Schneider, F., Tomek, W., Grundker, C., 2006. Gonadotropin-releasing hor-
mone (GnRH) and its natural analogues: a review. Theriogenology 66,
691–709.
Silvia, W.J., Hatler, T.B., Nugent, A.M., da Fonseca, L.F., 2002. Ovarian follic-
ular cysts in dairy cows: an abnormality in folliculogenesis. Domest.
Anim. Endocrinol. 23, 167–177.
Skinner, D.C., Albertson, A.J., Navratil, A., Smith, A., Mignot, M., Talbott,
H., Scanlan-Blake, N., 2009. GnRH effects outside the hypothalamo-
pituitary-reproductive axis. J. Neuroendocrinol. 21 (4), 282–292.
Thurmon, J.C., Tranquilli, W.J., Benson, G.J., 1996. Lumb and Jones’ Veteri-
nary Anesthesia, 3rd ed. Lea & Febiger, Baltimore, MD, pp. 115–147.
Todoroki, J., Yamakuchi, H., Mizoshita, K., Kubota, N., Tabara, N., Noguchi, J.,
Kikuchi, K., Watanabe, G., Taya, K., Kaneko, H., 2001. Restoring ovula-
tion in beef donor cows with ovarian cysts by progesterone-releasing
intravaginal silastic devices. Theriogenology 55, 1919–1932.
Vanholder, T., Opsomer, G., de Kruif, A., 2006. Aetiology and pathogenesis
of cystic ovarian follicles in dairy cattle: a review. Reprod. Nutr. Dev.
46, 105–119.
Xu, C., Roepke, T.A., Zhang, C., Ronnekleiv, O.K., Kelly, M.J., 2008.
Gonadotropin-releasing hormone (GnRH) activates the M-current in
GnRH neurons: an autoregulatory negative feedback mechanism?
Endocrinology 149 (5), 2459–2466.
Zulu, V.C., Nakao, T., Yamada, K., Moriyoshi, M., Nakada, K., Sawamukai,
Y., 2003. Clinical response of ovarian cysts in dairy cows after PRID
treatment. J. Vet. Med. Sci. 63, 57–62.
Zulu, V.C., Penny, C., 1998. Risk factors of cystic ovarian disease in dairy
cattle. J. Reprod. Physiol. 44, 191–195.