Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 August 2020 | 12(11): 16469–16477
ISSN 0974-7907 (Online) | ISSN
0974-7893 (Print)
doi: https://doi.org/10.11609/jott.5803.12.11.16469-16477
#5803 | Received 25 February 2020
| Final received 19 August 2020 | Finally accepted 21 August 2020
Suppression of ovarian activity in a captive African
Lion Panthera leo
after deslorelin treatment
Daniela Paes de Almeida Ferreira Braga 1,
Cristiane Schilbach Pizzutto
2, Derek Andrew Rosenfield 3, Priscila Viau
Furtado 4, Cláudio A. Oliveira 5,
Sandra Helena Ramiro Corrêa 6 , Pedro Nacib Jorge-Neto 7 &
Marcelo Alcindo de Barros Vaz
Guimarães 8
1 Fertility Medical Group / Av Brigadeiro Luis Antonio, 4545, 01401-002, São Paulo, SP,
Brazil.
2, 3, 4, 5, 7, 8 Department of Animal
Reproduction, Faculty of Veterinary Medicine and Animal Science, University of
São Paulo (USP) / Av. Prof. Dr.
Orlando Marques de Paiva, 87 - Cidade Universitária, São Paulo / SP, 05508-270, Brazil.
6 Faculty of Veterinary Medicine
and Animal Science, Federal University of Mato Grosso (UFMT), Av. Fernando Corrêa da Costa - Coxipó, Cuiabá
- MT, 78060-900, Brazil.
1 dbraga@fertility.com.br
(corresponding author), 2 cspizzutto@yahoo.com.br, 3 dro@usp.br,
4 priviau@usp.br, 5 cadolive@usp.br,
6 correasandrahelena@gmail.com, 7
pepovet@usp.br
Editor: Rajeshkumar G. Jani, Anand Agricultural University, Anand, India. Date of publication:
26 August 2020 (online & print)
Citation: D.P. de A.F. Braga, C.S. Pizzutto, D.A. Rosenfield, P.V. Furtado, C.A. Oliveira,
S.H.R. Corrêa, P.N. Jorge-Neto,
& M.A. de B.V. Guimarães (2020). Suppression of ovarian activity
in a captive African lion (Panthera leo) after deslorelin treatment. Journal of Threatened Taxa 12(11): 16469–16477. https://doi.org/10.11609/jott.5803.12.11.16469-16477
Copyright: © Braga et al. 2020. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: This work
was supported by FAPESP
(Grant no. 04/14135-6; MABV Guimarães).
Competing interests: The authors declare no
competing interests.
Portuguese abstract and Author
contribution see end of this article.
Author details: Daniela Paes de
Almeida Ferreira Braga, DMV, PhD. Head of Research at the Fertility
Medical Group. Cristiane Schilbach Pizzutto, DMV,
MSc, PhD, Postdoc. Assistant Professor at School of Veterinary Medicine and
Animal Science of University of São Paulo. Chairman of the Animal Welfare
Committee of CRMV - SP. Member of the International Environmental Enrichment
Conference Committee and of REPROCON research group. Derek Andrew Rosenfield, DMV, MSc, PhD.
Specialist in non-lethal wildlife population control methods. Priscila Viau
Furtado, DMV, MSc, PhD. Specialist in charge of the Laboratory of
Hormonal Dosages of School of Veterinary Medicine and Animal Science of
University of São Paulo. Cláudio A. Oliveira, DMV, MSc, PhD, Postdoc.
Full Professor at School of Veterinary Medicine and Animal Science of
University of São Paulo. Sandra Helena
Ramiro Corrêa, DMV, MSc, PhD. Assistant
Professor at Federal University of Mato Grosso (UFMT) and Manager of the Wild
Animal Medicine and Research Center of UFMT. Pedro
Nacib Jorge-Neto,DVM, MBA, MSc.
Actually, PhD student (PPGRA-FMVZ / USP) and Technical-Commercial Director of
IMV Technologies Brazil. Member of REPROCON research group. Marcelo Alcindo de
Barros Vaz Guimarães
(in memorian), DMV, MSc, PhD. Associate Professor at
School of Veterinary Medicine and Animal Science of University of São Paulo.
Acknowledgements: The authors
acknowledge the following institutions and persons: Fundação Parque Zoológico de São Paulo; and Professor Marcelo A. de Barros Vaz Guimarães (in memorian) for the friendship and teachings transmitted in
the reproduction of wild animals.
Abstract: With the intent to evaluate the efficiency of a
contraceptive treatment for cyclic ovarian suppression in African Lionesses Panthera leo using
a Gonadotrophin-Releasing Hormone (GnRH) agonist bioimplant, noninvasive fecal steroid assay
associated with the observation of the behavioral estrus were employed for a period of 36 months. Five captive adult females, maintained with a
vasectomized male, subcutaneously received a 9.4mg deslorelin
acetate implant. The treatment initially
stimulated behavioral estrus
along with ovarian activity, demonstrated by an estrogen
increase in two lionesses. A rise in
progesterone concentration in two other animals suggested possible
treatment-induced ovulation. After the
initial period, deslorelin prevented ovarian activity
for at least 22 months. Two females
exhibited signs of behavioral estrus
after 22 and 31 months. A third lioness
with an increased estrogen concentration did not
exhibit behavioral estrus
signs or a consequent progesterone surge until 33 months after implantation,
suggesting a possible resumption of ovarian activity. One female did not exhibit any behavioral estrus signs nor a
rise in steroid levels after the “treatment-induced” estrus
throughout the entire experiment (36 months).
One lioness died after 15 months without exhibiting signs of estrus or an increased progesterone level, however, the estrogen concentration increased 12 months
post-implantation, suggesting resumed ovarian activity. The study showed that long-term treatment
with a GnRH agonist can be extremely effective as a contraceptive treatment in
African lionesses, however, the duration of contraception may vary among individuals
and may bear the risk of permanent loss of normal ovarian activity.
Keywords: African Lion, contraception, estrus behavior, fecal assay, GnRH agonist.
Introduction
The reproduction of wild
animals in captivity is an important tool for ex situ conservation of
endangered species (Jorge Neto et al. 2018b). Some species such as the African Lion Panthera leo,
however, can adapt to captivity, and thus, are capable of reproducing in
such an environment. The abundant
reproduction of large carnivores is associated with low adult mortality and
increased longevity in captivity. This
creates a number of complications as the physical space and financial resources
available for their maintenance is limited (Woodroffe & Frank 2005).
The objective of the
present study was to use the noninvasive fecal steroid assay associated with
behavioral estrus to evaluate the efficiency of chronic treatments with the
Gonadotrophin-Releasing Hormone (GnRH) agonist bioimplants to suppress cyclic
ovarian activity in African Lionesses.
Materials and Methods
Experimental Design
Five adult African
Lionesses (L1, L2, L3, L4 and L5) were maintained in captivity with a
vasectomized male at the Zoological Park of São Paulo. All females had at least one confirmed
pregnancy with a live birth, and none of them had been previously submitted to
any kind of contraceptive management, except for physical separation from male
lions and time with vasectomized males.
L1 (13 years old), L2 (6 y/o) and L4 (6 y/o) were born in the São Paulo
Zoo, while L3 (7 y/o) and L5 (7 y/o) came from another captive facility when
they were six months old.
The five lionesses
received a 9.4mg deslorelin acetate implant
subcutaneously. The efficiency of the
implant as a contraceptive was evaluated non-invasively using a fecal steroid
assay and through observation of the behavioral estrus. The study was approved by the University’s
Ethics Committee for Use of Animals in Research (CEUAVET-USP).
Gonadotrophin-Releasing Hormone Agonist Bioimplant Formulation and
Implantation
The GnRH agonist
bioimplants used in the present experiments were supplied by Peptech Animal Health Pty Limited, Australia (Suprelorin 9.4 mg; No. 978; Batch DR023). Each implant contained 9.4mg of GnRH agonist deslorelin acetate (C64H83N17O12). Implants were placed subcutaneously under
aseptic conditions using a commercial implanting device.
Sample Collection, Hormone Extraction, and Dosage
During the experiment,
two fecal samples were collected twice weekly, sealed in plastic bags, labeled
with the individual’s name/date, and stored at -20°C. From 45 days before to 36 months after
implant, fecal aliquots were extracted to quantify estrogen and progestogen
metabolites. Fecal hormone metabolites
were extracted from the samples, as previously described (Brown et al.
1994). Briefly, each fecal sample was
lyophilized, pulverized, and 0.18–0.2 g of dry fecal powder was boiled in 5mL
of 90% ethanol for 20min. During
boiling, 100% ethanol was added as needed, to maintain approximate pre-boil
volumes.
After centrifugation
(500g, 20min.), the supernatant was recovered, and the pellet re-suspended in
5mL of 90% ethanol, vortexed for 30 sec, and re-centrifuged (500g,
15min.). The first and second
supernatants were combined, air dried, and reconstituted in 1mL methanol. Methanol extracts were vortexed briefly and
placed in a sonicator for 15min. Each extract was diluted 1:10 in a steroid
dilution buffer and stored in polypropylene tubes at -20°C until further use.
Subsequently, each
sample extract was assayed for estradiol and progesterone metabolites following
RIA. Estradiol Coat-a-Count RIA kits
(Diagnostic Products, Los Angeles, CA, USA) were used to measure the estradiol
metabolites, while Progesterone DSL-3900® RIA kits (Diagnostic System
Laboratories Inc., Webster, USA) were used to measure the progesterone
metabolites. Samples were analyzed in
duplicate, and those with a coefficient variation of more than 15% were either
re-analyzed (if there was enough sample volume for re-analysis) or discarded.
Estrus Behavior Observation
Animals were observed
for 30 min periods twice each day (during the morning and the afternoon), three
times a week. The following estrus
behavioral patterns were recorded (Schaller 1972): vocalization, restlessness,
increased frequency and intensity of rolling, lordosis, male attraction, mating
acceptance, and copulation.
Results
Before implant
placements, all animals had normal ovarian activity, as confirmed by fecal
hormone metabolites dosages (figs. 1–5) and behavioral estrus signs, such as
vocalization, restlessness, increased frequency and intensity of rolling,
lordosis, male attraction, mating acceptance, and copulation. The average estrus length was 5.8 ± 2.2
days. Treatment with deslorelin
initially stimulated a behavioral estrus along with ovarian activity, as
demonstrated by increases in the estrogen concentration in two lionesses (L1
and L3, Figs. 1 and 3). We also noted a
rise in progesterone concentration in two other females (L2 and L5, Figs. 2 and
5), which suggests possible treatment-induced ovulation (Table 1). After this period, the GnRH agonist prevented
ovarian activity for at least 22 months.
Two lionesses exhibited
behavioral estrus signs 22 and 31 months after implantation, respectively (L2
and L3, Figs. 2 and 3, respectively). In
a third lioness (L4, Fig. 4), behavioral estrus signs and increases in estrogen
concentration, as well as a consequent surge in progesterone level was noted 33
months after implant use. The lioness L5
(Fig. 5) did not exhibit any signs of behavioral estrus. Moreover, she only experienced a rise in
female sex steroids levels (estrogen and progesterone) after the
“treatment-induced” estrus the end of the experiment (36 months). The lioness L1 (Fig. 1) died 15 months after
experiment initiation, without demonstrating any estrus signs, nor a rise in
progesterone level, however, her estrogen concentration increased 12 months
after the placed implant (Table 1).
Discussion
In the face of the large
loss of habitat due to human encroachment and fragmentation, some species
become overabundant through human ineptitude.
Indeed, humans often attempt to create conditions that favor the
proliferation of one species over their competitors. Protected parks and reserves provide animals
with an environment that is abundant in resources and predator-free, conditions
that allow for unchecked reproduction.
As a result, endangered species undergo a localized population explosion
that can have detrimental effects on the flora and fauna of the reserve,
putting other species at risk; thus, affecting the ecosystem in the same manner
as do invasive species (Grandy & Rutberg 2002; Jewgenow
et al. 2006).
Wildlife population
control by means of contraception has become extremely important, especially
for a number of wild carnivores.
Population management and alternative noninvasive contraceptive methods
have been studied extensively over the last two decades (Rosenfield 2016). Whereas ovariohysterectomy or ovariectomy
alone has been the method of choice for most domestic cats (Munson 2006), for
reproductive management of threatened or endangered species like the African
Lion, a reversible method is desired.
While lions can reach high densities inside reserves (Packer et al.
2013), they tend to fare poorly outside protected areas, where they are often
the first large carnivore species to disappear (Woodroffe 2001).
The GnRH analog deslorelin, a long-acting biocompatible subcutaneous
implant that suppresses specific pituitary functions, has been recommended as
reversible contraception (D’Occhio et al. 2002). The increased release of GnRH into the portal
vessels which connect the hypophysis to the pituitary gland results in an
increased secretion of the follicle stimulating hormone (FSH) and luteinizing
hormone (LH), which, in turn, regulate gonadal functions (Conn & Crowley
1994). With continuous exposure to high
concentrations of GnRH, the number of cell surface receptors at the portion of
the adenohypophysis – responsible for FSH/LH synthesis and release – gradually
decreases (Melson et al. 1986) with a concomitant desensibilization effect of gonadotroph cells on GnRH (D’Occhio & Kinder 1995). By this type of mechanism, known as receptor
down-regulation, chronic treatment with a GnRH agonist prevents the pulsatile
release of FSH, as well as LH (Gong et al. 1995) and the pre-ovulatory surge of
LH secretion (D’Occhio & Kinder 1995).
The absence of
surge-releases of LH in females treated with a GnRH agonist have led to studies
being conducted on the potential long-acting contraceptive effects of the GnRH
agonist bioimplant by preventing follicular development and ovulation, and
consequently, pregnancies (D’Occhio & Kinder
1995). In addition, the development of a
noninvasive fecal steroid assay for assessing the ovarian function of felid
species in combination with behavioral studies makes it possible to
systematically study various aspects of reproduction (Brown et al. 1994, 2001;
Graham et al. 2006). Therefore, the goal
of the present study is to use the noninvasive fecal steroid assay associated
with behavioral estrus to evaluate the efficiency of chronic treatments with
the GnRH agonist bioimplants to suppress cyclic ovarian activity in African
lionesses.
The inhibitory effects
of ovarian activities, such as the arrest of ovulation caused by desensibilization to endogenous GnRH, provide opportunities
to evaluate a GnRH agonist bioimplant as a potential antifertility agent in
mammals. In the present experiment, seven lionesses were implanted with a 9.4
mg deslorelin to monitor ovarian function for 36
months. Fecal steroid assay and estrus
behavioral observation were the monitoring methods used. Our findings suggest
that the GnRH agonist deslorelin suppresses ovarian
activity in African lionesses for prolonged periods of time. In fact, no behavioral estrus was noted until
22 months post-implantation. In the 22nd,
31st, and 33rd month, behavioral estrus was noted in
three of the lionesses, while the fourth lioness exhibited increased estrogen
concentrations and a consequent surge in progesterone level that corresponded
to the resumption of ovarian activity, including ovulation, in addition to
behavioral estrus.
One lioness died 15
months after the beginning of the experiment without demonstrating any estrus
signs nor a rise in progesterone level.
On the other hand, the estrogen concentration increased 12 months after
the implantation, indicating that the ovarian activity may have re-started.
Surprisingly, in a single female, neither estrus behavior nor a rise in fecal
progesterone concentration was noted up to the end of the experiment.
Various behavioral
activities that characterize estrus in lions appear to be common in several
feline species, such as the domestic cat (Graham et al. 2000; Pelican et al.
2005), Jaguar (Wildt et al. 1979; Jorge-Neto et al. 2018a), Siberian Tiger (Seal et al. 1987), Snow
Leopard (Schmidt et al. 1993), and Cheetah (Wielebnowski
& Brown 1998), possibly serving as indicators of physiological estrus in
these animals (Umapathy et al. 2007). It, however, remains unclear why behavioral
estrus was observed in two of the lionesses without a rise in fecal estrogen
and progesterone metabolites concentration.
Ovulation in Panthera genus species is
triggered by copulation or sensorial stimulation (Jorge-Neto
et al. 2020). Therefore, the lack of
ovulation observed during this study may demonstrate a estrus detection failure
or a compromised ovarian function. It
could also be hypothesized that, in these cases, ovarian activity may have
re-started and estradiol concentration increased, resulting in the stimulation
of behavioral estrus, although, not enough to trigger the cascade of events to
reach ovulation.
The fact that neither
estrus behavior nor a rise in fecal progesterone concentration was noted in one
of the lionesses up to the end of the experiment raises concern. For contraception to be successful for
population control, especially in endangered animals, it must not only be safe,
effective, and long-acting but also reversible (Castle & Dean 1996).
To date, deslorelin has been used in captive-held wild felines, such
as cheetahs (Bertschinger et al. 2001), leopards (Bertschinger et al. 2002) and lions (Bertschinger
et al. 2008), without showing any adverse effects. Conversely, in domestic cats, a 6mg implant
has been shown to suppress ovarian follicular activity for between four and 14
months, however, until the end of the study period, eight out of ten cats did
not fully return to normal ovarian cyclicity (Munson et al. 2001). Moreover, dosages of 12 or 15 mg deslorelin induced contraceptive effects for 12–18 months (Bertschinger et al. 2002).
The implant used in this study (9.4mg) has a matrix without
sodium acetate anhydrous, that allows slow liberation of the deslorelin, maintaining contraceptive effects for much
longer periods, making it impossible to compare the effectiveness of this
dosage in relation to the duration of previous products. It has been reported that the effectiveness
duration of Suprelorin in wild felids is, on average,
twice that prescribed by the manufacturer in dogs, which means that the 9.4mg
implant with a minimum effectiveness of 12 months is generally effective for
approximately 24 months (Asa et al. 2012).
Our findings show a ceasing of ovarian activity of 28.67 ± 5.86 months,
which corroborates those found by Bertschinger et al.
(2008), in which implants were effectively in lionesses for a period of ~30
months or longer. The reversal time (or
duration of efficacy) is variable between species and individuals, probably due
to the singularity in the metabolism of deslorelin or
the ability to recover from down-regulation (Asa et al. 2012). The findings suggest that long-term treatment
with deslorelin may have variability regarding the
duration of contraception among individuals due to several factors, including
drug/matrix used; genetic and/or environmental influences. The disadvantage of using Suprelorin
is the inability to safely predict the duration of effectiveness and the return
of ovarian activity, being a problem when there is interest in using these
females in conservation programs.
An extensive study using
140 implants (Suprelorin) on 14 species of wild
felids, including 59 lionesses, was conducted by the North American Association
of Zoos and Aquariums (AZA) and showed no side effects of deslorelin
treatment (Asa et al. 2012). Bertschinger et al. (2008) used deslorelin
treatment in 23 captive and 40 free-ranging lionesses (P. leo) and four captive tigers (P. tigris)
in South Africa and did not observe any side effects in any females, including
some treated four or five times for 5–8 years period. In domestic cat females the use of Suprelorin appears to be a convenient, efficient and safe
contraception method, demonstrating female fertile matting after approximately
two years post-treatment and no side effects (Fontaine 2015).
Prior to the occurrence
of a GnRH agonist antifertility effect, there is an acute phase (D’Occhio et al. 2002; Rosenfield 2016) in which the
secretion of LH and FSH increase sharply (Gong et al. 1995, 1996), leading to a
corresponding estrus response (Wright et al. 2001). In the present study, shortly after placing
the implant, two lionesses exhibited behavioral estrus, and an upsurge of
ovarian activity was observed, as demonstrated by increases in the estrogen
concentration. A rise in progesterone
concentration was noted in two other females.
As noted, the treatment-induced behavioral estrus signs without the
accompanying rise in progesterone, observed in the first two females could be
attributed to a copulation failure rather than compromised ovarian
function. As reported in other works,
after an initial GnRH treatment, lionesses and cheetahs may exhibit signs of
estrus behavior and become attracted to males for a few days, although mating
may not occur (Bertschinger et al. 2002).
Conversely, in animals
in which a rise in progesterone concentration was noted but no behavioral
estrus signs could be observed, a failure in observing estrus signs, a
spontaneous ovulation – or sensorial stimuli štriggering
ovulation – may have occurred.
Spontaneous ovulation has been previously reported in some felines
including all Panthera species, such as the
Leopard (Schmidt et al. 1988), Snow Leopard (Brown et al. 1995), Tiger (Graham
et al. 2006), Jaguar (Barnes et al. 2016; Gonzalez et al. 2017) and African
Lion (Schramm et al. 1994) while sensorial stimulation has induced ovulation in
Jaguars (Jorge-Neto et al. 2020). In one lioness, shortly after placing the
implant, no behavioral estrus signs were observed, nor was there a rise in
progesterone levels. This may be due to
the presence of active luteal tissue from a previous follicular cycle and/or
due to individual variations.
Our results reinforce
the importance of using non-invasive monitoring as an alternative for hormonal
assessment, especially in wild animals.
Blood collection is not only a stressful event and can itself cause
changes in hormonal concentrations (Sheriff et al. 2011), but also does not
allow successive collections for longer studies, such as monitoring of ovarian
cyclicity (Sgai et al. 2015). Many studies in several species have been
developed and validated for the longitudinal measurement of hormonal
metabolites, both for glucocorticoids (Sinhorini et
al. 2020) and steroids, enabling effective reproductive monitoring with fecal
matrix (Monfort et al. 1997; Van Meter et al.
2008). These studies demonstrated efficient
results without the need to perform a serum endocrine evaluation.
In conclusion, long-term
treatment with a GnRH agonist has been shown to be extremely effective in
inhibiting the synthesis and liberation of FSH and LH from the pituitary, and
as a result, ceasing ovarian activity in female African lions for 28.67 ± 5.86
months. The duration of contraception,
however, may vary among individuals, with the added risk of some females not
returning to normal ovarian activity, rendering that female infertile. It is strongly suggested that further studies
investigate the long-term antifertility effects of GnRH agonists in this
species.
Table 1. Rise in fecal steroids concentration
and/or estrus behavior
shortly after implant placements, and period of contraception in African lions
treated long-term with GnRH agonist (deslorelin).
Lioness |
Estrus behavior shortly after implantation |
Rise in fecal progesterone shortly after
implantation |
Rise in fecal estrogen
after downregulation |
Estrus behavior after downregulation |
Rise in fecal progesterone after
downregulation |
L1 |
Yes |
No |
12 months |
Not observed* |
Not observed * |
L2 |
No |
Yes |
No |
22 months |
Not observed** |
L3 |
Yes |
No |
No |
31 months |
Not observed** |
L4 |
No |
No |
No |
33 months |
33 months |
L5 |
No |
Yes |
No |
Not observed** |
Not observed** |
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