World Rabbit Science Association

12th World Rabbit Congress - November 3-5 2021 - Nantes, France, Communication R-00 (Invited paper), 10 pp

PHYSIOLOGY AND MODULATION FACTORS OF OVULATION IN

RABBIT REPRODUCTION MANAGEMENT

Mattioli S.1*, Maranesi M.2, Castellini C.1, Dal Bosco A.1, Arias-Alvarez M. 3, Lorenzo P.L.4,
Rebollar P.G.5, Garcia-Garcia R.M.4
1
Dept. of Agricultural, Food and Environmental Science, University of Perugia, Borgo 20 Giugno 74, 06123, Perugia, Italy.
2
Dept. Veterinary Medicine, University of Perugia, via San Costanzo, 4, 06126, Perugia, Italy.
3
Dept. of Animal Production, Veterinary Faculty, Complutense University of Madrid, Avda. Puerta de Hierro s/n,
28040 Madrid, Spain.
4
Dept. of Physiology, Veterinary Faculty, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040
Madrid, Spain.
5
Dept. of Agrarian Production. Agronomic, Alimentary and Biosystems, Polytechnic University of Madrid, Ciudad
Universitaria s/n 28040 Madrid, Spain
*
Corresponding author: simona.mattioli@hotmail.it

ABSTRACT

Rabbit is an induced ovulatory species, so ovulation takes place after mating. Traditionally, exogenous
and synthetic hormonal factors (administrated by intramuscular and intravaginal via) such as GnRH
and analogues, or different physical procedures (i.e. stimulation by intravaginal cannula) have been
used to induce ovulation in females when artificial insemination is applied in rabbit farms. Restriction
and public rejection in the use of hormones is leading to the study of the seminal plasma components
with potential action on ovulation induction. The objective of the present review is to collect and
summarize the strategies used, during the last years, to trigger ovulation and improve rabbit fertility
management with respect to more animal-friendly manipulation methods. Furthermore, special
attention has been paid to the use of a semen component (as endogen molecule) such as beta nerve
growth factor (β-NGF) in male and female rabbit reproductive physiology. This neurotrophin and their
receptors (TrKA and p75NTR) are abundantly distributed in both male and female rabbit reproductive
tract, and it seems to have an important physiological role in sperm maturation and behaviour
(velocity, apoptosis and capacitation), as well as a modulatory factor of ovulation. Endogen β-NGF is
diluted in the seminal doses with the extenders; hence it could be considered an innovative and
alternative strategy to avoid the current hormonal exogenous (by intramuscular route) and stressful
treatments used in ovulation induction. Their addition in seminal dose could be more physiological
and improve animal welfare in rabbit farms.

Key words: Rabbit, ovulation, seminal plasma, β-NGF, GnRH, reproduction.

INTRODUCTION

In the last 30 years, the average productivity of rabbit farms has increased and become more homogeneous
through the use of good management practices, such as the artificial insemination (AI; Rebollar et al., 1994;
Castellini, 1996). The optimization of reproductive performance is one of the main facts that assure high
productivity on rabbit farms; however, some managements can cause several problems related to the
welfare of does such as intensive reproductive rhythms, high mortality and sub-fertility (Cardinali et al.,
2008; Castellini et al., 2010). This requires that the new management practices in rabbitries take into
account the physiology, reproductive behaviour and welfare of animals.
Considering that using the AI procedure did not provoke LH peak like stimulation by coitus, exogen
gonadotropin-releasing hormone (GnRH) or its analogues, such as Lecirelin (Dal Bosco et al., 2014) and
Buserelin (Rebollar et al., 2012; Viudes-de-Castro et al., 2014) must be administered intramuscularly or
intravaginally to the rabbit does. Nowadays, the most disseminate practice for ovulation induction in

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12th World Rabbit Congress - November 3-5 2021 - Nantes, France, Communication R-00 (Invited paper), 10 pp

rabbit commercial farms is the intramuscular injection of GnRH analogues. However, this practice is
considered an additional stressful situation for the animal and a further work for the farm operators, then
it seems not well accepted from the general public (due to the issue animal welfare-related).
Furthermore, the use of any exogenous hormones did not meet a good people's consent.
In this context, recent studies demonstrated the presence of an endogen molecule contained in the
seminal plasma (SP) of numerous animal species that produce ovulation in camelids. Specifically, this
molecule, whose identity was not initially known, has been called for many years ovulation-inducing
factor (OIF) and only in 2014, biochemical studies identified it as the beta nerve growth factor (β-
NGF; Ratto et al., 2014). In fact, this protein was firstly identified in the SP of Bactrian camels (Chen
et al., 1985), and later in the SP of other induced ovulatory animals, such as rabbits (Silva et al., 2011),
koalas (Johnston et al., 2004), alpacas, llamas and other camelids (Silva et al., 2014; Ulloa-Leal et al.,
2014; Berland et al., 2016; El Allali et al., 2017; Silva et al., 2020), and even in the SP of species with
spontaneous ovulation, such as cattle, pigs, and horses (Adams et al., 2016).
In the light of these reports, the objective of the present review is to collect and summarize the main
strategies used to modulate the physiology of ovulation with the aim to improve rabbit fertility using
animal-friendly manipulation methods: i.e. use of endogen β-NGF. The action mechanism of β-NGF
in female and male has also been discussed. The development of innovative molecular biology
techniques in the live system and the increasing attention of rabbit production actors (farm rabbit
employers, consumers, genetic centers, etc.) to the respect of animal welfare (Directive 2010/63/EU)
favour the developments of a “more precision management” and “less impactful strategies” to improve
the rabbit’s reproduction performances.

PHYSIOLOGY OF OVULATION: A BRIEF UPDATE

Rabbit is an induced (reflex) ovulatory species. Conversely to the spontaneous ovulatory (human, dog,
cow, etc.) which display a defined oestrous cycle, rabbits (such as ferrets, cats, camelids) need
copulation to trigger GnRH secretion from the hypothalamus into the hypophyseal portal system.
Females use to display oestrous behaviour each 4-6 days (Milligan, 1982). The receptive sexual
behaviour is induced by estrogen, which acts on the brain (Bakker and Baum, 2000), producing
different signals in the rabbit females: i.e. lordosis at the male presence (Theau-Clément et al., 2005)
or red/purple vulva colour (Ubilla and Rebollar, 1995). When the follicles degenerate, the oestrogen
secretion decreases, and rabbits enter into a non-receptive period (Harcout-Brown, 2002). If mating
occurs, genital somatosensory receptors are stimulated (Bakker and Baum, 2000) evoking signals
funnel, via neural pathways (Lin and Ramirez, 1991); as consequence, a release of GnRH peaking 1–2
h after mating and followed by a pre-ovulatory release of luteinizing hormone (LH), from the anterior
pituitary, leads to ovulation 11–12 h after mating (Bakker and Baum, 2000). This suggests that female
rabbit reproduction requires tuned coordination of the hypothalamic-pituitary-ovarian axis in order to
control folliculogenesis, oestrous cycle, sexual behaviour, and ovulation via a series of complex
neuroendocrine feedback mechanisms (Boiti et al., 2006; Garcia-Garcia et al., 2020).
As in other mammals, fertilization occurs in the ampulla about 14 hours after mating or AI. If a non-
fertile copulation occurs, pseudopregnancy takes place in the rabbit female after ovulation. The
pseudopregnancy period is characterized by corpora lutea development and progesterone production.
The corpora lutea reached maximum size 10-12 days after mating and serum progesterone
concentration raised at Day 14 and then luteal regression starts; this process is completed around Day
18, when progesterone concentrations decline to basal values (Browning et al., 1980). The functional
and structural luteolysis is mainly related to prostaglandin F2α (PGF2α) secretion by the uterine
endometrium reaching its highest level at Day 17 of pseudopregnancy (Lytton and Poyser 1982).

CURRENT METHODOLOGIES FOR OVULATION INDUCTION

Considering the routinely use of AI in European rabbitry, many strategies to control or induce
ovulation have been developed to increase the rabbits’ reproductive performances and optimize human

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12th World Rabbit Congress - November 3-5 2021 - Nantes, France, Communication R-00 (Invited paper), 10 pp

resources. The main strategies currently used are: standardization of physical procedures and/or
improvement of hormonal treatments with exogenous molecules.

Physical procedures
In rabbit does, an important factor responsible for ovulation induction seems to be the genital
somatosensory stimuli during the copulation because most of females are able to ovulate only with
mechanical stimulation (Rebollar et al., 2012; Viudes de Castro et al., 2017; Maranesi et al., 2018).
Then, AI cannulas mimicking this effect has been studied.

- Cannula types. The type of AI cannula used affected the ovulation induction of the female rabbits: a
short and flexible cannula showed higher values (64.0±8.0 %) than a long and rigid one (30.0±6.8 %).
Authors concluded that the first cannula mimicked better the stimulation associated with the mating of
the male to provoke ovulation induction (Viudes de Castro et al., 2017). However, when the authors
compared these two types of cannulas inseminating does with a semen extender containing hormones
(GnRH analogues, as reported below) no differences in does fertility and/or prolificacy were found.
Further studies with cannulas able to adapt to female rabbit tract’s physiology could be interesting.

Exogenous hormonal treatments

Different hormonal treatments with exogenous hormones have been used in AI over the years:

- hCG or LH. The first treatment used consisted of an ovulation induction procedure with human
chorionic gonadotropin (hCG) or LH administration (Adams, 1961). Their activities in rabbit does are
the release of the oocyte from the follicle, but also initiate the conversion of the residual follicle into
a corpus luteum that, in turn, produces progesterone to prepare the endometrium for a
possible implantation (Theau-Clément, 2007). Although the pharmacological actions of hCG and LH
are similar, their bioavailability is different because LH has a shorter half life than hCG (Simmon et
al., 1988). Furthermore, the action of LH in the follicle is more selective: it induces earlier ovulation
and improves the oocyte and embryo quality (after 24 and 48 h, respectively), due to the secretion of
high levels of estradiol and progesterone (Molina et al., 1991). However, these types of treatments
induce antibody formation and then, triggers an immune response when using repeatedly (Lebas et al.,
1996; Boiti et al., 1995), increasing the number of haemorrhagic follicles by about 20 h after ovulation
induction (Rebollar et al., 2012). For this reason, the utilization of these hormones has been discarded
in the AI procedure.

- injection of parenteral GnRH analogues. Intramuscular (i.m.) administration of GnRH or analogues

is the most reliable method used nowadays because they can be used for repeated treatments and do
not induce antibodies formation, in contrast to the LH or hCG administration. These molecules have
the same biological activity as the natural one in the physiology of ovulation. Several different GnRH
analogues have been studied: i.e. gonadorelin (Fertagyl, Intervet; effective dose: 20-40 µg/AI dose),
lecirelin (Dalmarelin, Faltro; 5 µg/AI dose), buserelin (Receptal, Hoechst AG; effective dose: 1-2
µg/AI dose) and many others (Dal Bosco et al., 2011). They showed similar results to those obtained
by natural mating (Theau-Clément, 2007), and triggered a LH peak 2 h after i.m. injection; then the AI
should be performed during this period. Furthermore, their efficacy widely depends on the quantity
used with a concentration range from 1 to 20 µg/AI dose (Ubilla and Rebollar, 1995; Rebollar et al.,
2012; Dal Bosco et al., 2014). However, in most rabbit farms, GnRH is usually administered by the
farmer himself, with the risk of calibration mistake and an increase in the time spent for each AI
(Quintela et al., 2004).

- intravaginal administration of GnRH analogues. Alternative use of GnRH analogues and a less-
impactful strategy for animals is the intravaginal administration of such hormones. Their addition in
the semen extender allows reducing the time consumed by farmers in AI procedure (Dal Bosco et al.,
2011). However, the success of this method depends on the enzymes (proteases, namely
aminopeptidases) present in the SP, the status of the vaginal mucosa, the extender composition and the
type of analogue used (Dal Bosco et al., 2014).

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12th World Rabbit Congress - November 3-5 2021 - Nantes, France, Communication R-00 (Invited paper), 10 pp

It is well known that the vagina has a great potential for systemic delivery because of its large surface
area, rich blood supply and permeability to a wide range of compounds, including GnRH analogues
(Benziger and Edelson, 1983). On the other hand, due to the high presence of proteases in the vaginal
tract, many of the molecules introduced can be degraded. Viudes-de-Castro et al. (2014) demonstrated
that positive results in the reproductive performances were obtained when GnRH analogues (10
µg/mL of buserelin acetate) were added to 1:20 diluted semen than 1:5 dilution rate, during the AI
practice, although variations due to the genetic lines tested were found.
Unfortunately, to achieve fertility results similar to those with i.m. GnRH administration, the
intravaginal hormone concentration must be double the amount administered with the first method
(Rebollar et al., 2012; Dal Bosco et al., 2014). As an alternative, the introduction of inhibitors of
proteases like ethylenediaminetetraacetic acid (EDTA) or bestatin in the AI dose (Casares-Crespo et
al., 2015), acting as hormonal protectors, has been studied. In this sense, Casares-Crespo et al. (2015)
demonstrated that the aminopeptidases activity of SP was 55.5 % lower than the control with no
inhibitors, when EDTA or betastin were added to the AI extender. Furthermore, no negative effects in
semen characteristics have been reported, when these molecules were added to the AI dose
(Fernández‐Serrano et al., 2017). A promising and innovative strategy tested is the use of chitosan-
dextran sulfate nanoparticles as GnRH protector for intravaginal application in rabbit AI (Casares-
Crespo et al., 2018). This can allow the reduction of hormone (buserelin acetate) concentration used in
extenders (4-5 µg/AI dose), supplemented with bestatin and EDTA, without affecting the fertility and
prolificacy of rabbit females. In addition, the effect induced by intravaginal administration GnRH
analogues (4 µg/AI dose) and chitosan-dextran sulfate nanoparticles was similar to that of i.m.
injection of the same molecules, and consist of an earlier LH-surge than that observed in does treted
with 0.8 µg i.m. of GnRH (90 vs 120 min; Hassaneim et al., 2021).

Alternatives to the use of GnRH analogues

Restriction in the use of synthetic and/or exogenous hormones is leading to the study of some
molecules naturally present in the SP with potential action on ovulation induction. This would be more
animal-friendly methods for applying animal AI. One of this seminal component is the β-NGF. It is a
member of the neurotrophin family, and it was first discovered for its effects on neuronal survival and
differentiation (Levi‐Montalcini and Hamburger, 1951).

- SP β-NGF. Recent studies have demonstrated that the rabbit SP contains a β-NGF amount ranged
from 0,002 to 150 µg/mL (Maranesi et al., 2015, 2018, Casares-Crespo et al., 2018, García-García et
al., 2018a, Castellini et al., 2019, 2020a). However, concentration changes depending on the
individual variation, the age of rabbit bucks, the collection rhythm and other not clearly defined
factors (Castellini et al., 2020a). Among these factors, the season seems to be very important. In this
sense, Casares-Crespo et al., (2018) reported that the β-NGF content in rabbit SP during winter
decreases 4-times compared with the other seasons. In agreement, Schneidgenova et al. (2011) found
that during the winter season, sperm motility and concentration is lower than in other seasons,
reflecting the natural fluctuation of breeding capability during the year.

Regarding the systemic role of β-NGF, it is reported that it plays a multi-physiological role in rabbit
reproduction, acting by an endocrine, autocrine and paracrine way in female rabbits (Maranesi et al.,
2018; Garcia-Garcia et al., 2020). However, no clear evidence exists on the specific role of β-NGF on
the ovulation induction in this species: it is supposed that it collaborates with the sensory stimulation
exerted by coitus, which is considered the main activator of LH release (Garcia-Garcia et al., 2020).
As a consequence, some groups of researchers have focused on the study of the molecular mechanisms
by which this molecule could affect the rabbit ovulation to improve rabbit reproduction. However,
some doubts remain on whether the β-NGF is able to trigger the ovulation in rabbits or if the nervous
system is the main stimulus in this species and β-NGF has only a modulatory role on rabbit ovulation.
Silva et al. (2011) did not detect ovulation in rabbits after i.m. injections of rabbit SP at different
doses, although the same procedure induced ovulation in llamas. Conversely, Cervantes et al. (2015)
reported that i.m. injection of rabbit SP induced ovulation in group-housed, but not in individually

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housed rabbits. An experiment conducted by Rebollar et al. (2012) confirmed ovulation in 75 % of

rabbits after intravaginal administration of raw semen without treatment with a GnRH analogue,
probably due to the semen components effect (e.g. β-NGF). Quite surprisingly, however, there was no
(Rebollar et al., 2012) or a reduced percentage of ovulation (17%, Maranesi et al., 2018) in does
inseminated with raw semen after lumbar intra-epidural anaesthesia.

A recent study (García-García et al., 2018b) reported that only 1 of 6 female rabbits treated
intramuscularly with 24 µg of murine β-NGF, ovulated, and no preovulatory elevation in blood plasma
LH was detected. In the same study, mechanical stimulation of the vagina increased the ovulation rate
by 50 % in β-NGF-treated females, suggesting that besides the β-NGF-induced ovulation these
females needed a physical stimulation. Similarly, Maranesi et al. (2018) reported ovulation rates of 67
% and 17 % in female rabbits inseminated with raw semen in non-anesthetized and lumbar-
anesthetized females, respectively; in all these females, ovulation was preceded by an increase in
plasma LH concentration during the 2 h after AI. Lumbar anaesthesia before AI blocked the increase
in plasma LH concentration, attenuated the systemic blood rise of β-NGF and reduced the ovulation
rate. The authors postulated that other SP cytokines, beside β-NGF, might contribute to local
stimulation of the female reproductive tract and ovulation in the rabbit (Maranesi et al., 2022). Based
on these findings, the same authors proposed a novel paracrine mechanism driven by raw semen OIF,
likely β-NGF, in the uterus/cervix, which reinforces the neuroendocrine reflex provoked by vaginal
stimuli during natural mating.

This novel mechanism could be summarized as follows: (a) semen-derived β-NGF stimulates de novo
synthesis of β-NGF in the uterine wall, (b) both seminal and uterine β-NGF are absorbed into the
bloodstream and act directly on the ovary, (c) semen derived and locally synthesized β-NGF stimulate
uterine/cervix sensory neurons, which trigger GnRH neurons in the hypothalamus.
From a cellular viewpoint, the β-NGF exerts its own biological functions binding two cell surface
receptors: TrKA, a high-affinity receptor, and p75 neurotrophin receptor (p75NTR), a low-affinity
receptor (Holgado-Madruga et al., 1997). Interactions among TrKA and p75NTR pathways are critical
for the final biological effects of β-NGF in the different cell types. Indeed, β-NGF is produced and
acted both in male and female reproductive tissues (Maranesi et al., 2016, 2018; Castellini et al.,
2020a; García-García, 2018b; Figure 1).

In rabbit female, the presence of the β-NGF and its receptors in several types of ovarian cells (theca,
granulosa and cumulus), corpus luteum (Zerani et al., 2021) and in uterus and oviduct (Maranesi et al.,
2018, García-García, 2018b) involves important roles in this reproductive system, such probably in
folliculogenesis and ovulation (Garcia-Garcia et al., 2020), as well as in embryo development (Pei,
2010). In addition, the β-NGF could be a powerful stimulator of prostaglandins biosynthesis by the
rabbit uterus, because induces the in vitro synthesis secretion of both PGF2α and PGE2 (Maranesi et
al., 2016). This type of effect suggests that β-NGF may also represent a link between the immune,
endocrine, and nervous system (Tometten et al., 2005; El Allali et al., 2017).
Indeed, prostaglandins and/or nitric oxide are synthesized by the uterus after β-NGF binding to its
receptors in the sensory neuron stimulation. β-NGF-induced prostaglandins secretion would then
stimulate directly or indirectly (via local chemical mediators) uterine/cervix neurons that reach, via
spinal cord afferent pathways, the hypothalamic centres responsible for the LH surge that induces
ovulation). Even, an influence of both estradiol and NGF, on gonadotrophin secretion in other induced
ovulators as camelids has been reported (Carrasco et al., 2021). However, in the case of rabbits, only a
luteotrophic effect of estradiol has been evidenced by preventing apoptosis in luteal cells (Godman et.,
1998), also demonstrated by the decreased expression of luteal type 1 estrogen receptor in PGF2α-
induced luteolysis (Maranesi et al., 2010).

In rabbit male, the β-NGF and its receptors have been identified in several parts of the reproductive
system (testes, epididymis and accessory glands; Maranesi et al., 2016; Sanchez-Rodriguez et al.,
2018, 2019a) and in epididymal and ejaculated sperm, with a high variation rate depending on the
physiological status of sperm (Castellini et al., 2020a). The different distribution and abundance of

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12th World Rabbit Congress - November 3-5 2021 - Nantes, France, Communication R-00 (Invited paper), 10 pp

receptors in raw sperm of rabbit modulates the role of β-NGF in sperm behaviour and then, their
efficacy when it is added to semen extender (Castellini et al., 2020a, b).

Recently, it has been demonstrated that the β-NGF affected the sperm speed, apoptosis and
capacitation, mainly binding the p75NTR receptor. In contrast, motile, live cells, necrosis and
acrosome reaction were modulated via TrKA (Castellini et al., 2019, Figure 1).

Figure 1: Summary of the β-NGF activity in female and male rabbit reproductive physiology. The
upper section describes the systemic role of β-NGF (nervous, endocrine and paracrine pathways); the
downer section describes the physiological role of β-NGF in relation to the TrKA/P75NTR receptors-
binding and the cellular localization of the receptors in reproductive tissues.

Such outcomes are justified by the different distribution of receptors on spermatozoa: in ejaculated
sperm p75NTR is mainly located in the midpiece and tail, whereas TrKA resides in the head and
acrosome (in agreement with other animal species: Li et al., 2010; Sari et al. 2018). Furthermore, the
p75NTR receptors abundance on spermatozoa membrane seems to increase with the storage-period (8-
12 hours), while TrKA remained unchanged (Castellini et al., 2020b). These findings suggest that in in
vitro condition, the β-NGF exerts its “pro-survival” effect only within 8 hours, hence, when the β-NGF
is used in the AI extender, the AI practice must not exceed this time, in order to avoid that the β-NGF
act as “pro-death” factor for sperm (due to the physiological increasing of p75NTR receptors on cell
surface. Such finding is in agreement with the physiological destiny of sperm during the fertilization
process: after mating, sperm need about 6-8 hours to reach the egg in tubes (Harper, 1970); after this
time, the sperm that were not able to fertilizised the egg undergo to programmed death (apoptosis) and
as a consequence, they were eliminated. The present approach could also be exploited in semen
conservation and then improve assisted reproduction techniques. In agreement, Sari et al. (2020)
demonstrated in llama, that the addition of 10 or 100 ng/ml of human β-NGF in refrigeration sperm
promoted motility and vigour, while viability and mitochondrial activity are maintained.

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- rrβ-NGF. Recently, it was produced a recombinant NGF from rabbit prostate tissue and its effects
were tested on sperm parameters and does ovulation rate (Sanchez-Rodriguez et al., 2019b). Authors
demonstrated that the in vitro addition of rrβ-NGF to the ejaculated semen (1 µg/mL) did not affect
sperm viability, whereas sperm motility parameters were enhanced. Addition of this same
concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced
ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery.
Afterwards, the same Authors (Sanchez-Rodriguez et al., 2020), demonstrated that such modulation of
male and female reproductive parameters was dose-depending. The authors found an intermediate
ovulation rate (OR, 30, 60 and 42.9%, respectively), at concentrations of 20 ng/ml, 1 mg/ml and 20
mg/ml of rrb-NGF, respect to the highest (100% OR) ovulation rate obtained with the GnRH i.m.
administration, whereas other doses tested (100 ng/mL and 100 mg/mL) had the lowest OR (20 and
14.3%, respectively). The present features open up new perspectives in the use of this molecule in the
practice of AI although improvements should be done to obtain reproductive rates similar to those of
GnRH doses, probably by nanoprotection of the molecule

FURTHER RABBIT REPRODUCTION STRATEGIES

Due to the present context of the refuse of hormonal treatments in animal production and the
improvement of animal welfare with a more physiological approaching, the focus on β-NGF
application in rabbit reproduction is of growing interest. It could be considered an alternative and
innovative strategy to avoid exogenous hormonal treatments in ovulation induction and to improve
animal welfare in rabbits avoiding i.m. injection. Such a way, a dual using approach could be
provided:
i. The exogenous addition of recombinant β-NGF to the semen extender could be beneficial for
successful refrigeration of sperm and related assisted reproduction techniques.
ii. The development of strategies to use NGF by intravaginal via probably byprotection of this
factor to avoid its degradation, considering that all growth factors have a short half-life
when they are in vivo administered.

In any case, studies about the efficiency in rabbit ovulation rate and on the effect on sperm
characteristics (e.g. dose, incubation time, storage condition) when β-NGF is used must be continued,
and methods including mechanical stimulus and β-NGF could be considered.

ACKNOWLEDGEMENTS

Authors wish to thank Ms. Beatriz Velasco, Mr. Giovanni Migni and Mr. Osvaldo Mandoloni for their
contribute in animal handling and Dr. Alessandra Pistilli, Dr. Anna Maria Stabile, Prof. Mario Rende,
Dr. Francesca Mancuso, Prof. Massimo Zerani, Prof. Cristiano Boiti, Dr. Linda Petrucci, Prof.
Gabriele Brecchia and Dr. Ana Sanchez-Rodriguez for their contribution to the achievement of the
different experiments necessary to increase knowledge on β-NGF role in rabbit reproduction.

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