Accepted Manuscript Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and ICSI Lorena Fernandez-Gonzalez , Romy Hribal , Julia Stagegaard , Jennifer Zahmel , Katarina Jewgenow PII:
To appear in:
Received Date: 16 July 2014 Revised Date:
27 November 2014
Accepted Date: 29 November 2014
Please cite this article as: Fernandez-Gonzalez L, Hribal R, Stagegaard J, Zahmel J, Jewgenow K, Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and ICSI, Theriogenology (2015), doi: 10.1016/j.theriogenology.2014.11.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and ICSI
Lorena Fernandez-Gonzalez1, Romy Hribal1, Julia Stagegaard2, Jennifer Zahmel1, Katarina
10324 Berlin, Germany
Leibniz Institute for Zoo and Wildlife Research, Department Reproduction Biology, PF 700430,
Corresponding author: Lorena Fernandez-Gonzalez, Email: [email protected]
Institute for Zoo and Wildlife Research, Department Reproduction Biology, PF 700430, 10324
Berlin, Germany. Telephone: 0049305168619.
Assisted reproductive techniques (ARTs) are becoming widely applied to the breeding of
endangered species, but establishing reliable protocols for the production of embryos in vitro is
challenging because of the scarcity of sample material. In our study, we applied an ART protocol
for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes
retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours post-mortem. In total,
68 cumulus-oocyte complexes (COCs) of good quality were randomly distributed and cultured for
32 to 34 hours into two different maturation culture media, consisting of Medium 199 with Earle's
salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate,
0.15 mg/mL L-glutamine and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was
0.02 IU/mL FSH and 0.05 IU/mL LH, IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH and 1
µg/mL 17ß-Estradiol. Differences in hormonal supplementation did not produce significant
differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured
oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage
rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in
IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in
IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onwards, and
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Ree Park Safari, Stubbe Søvej 15, 8400 Ebeltoft, Denmark
blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison to
domestic cats. This is the first report of in vitro produced blastocysts using ICSI in the lion, and the
results demonstrate that IVM and ICSI can be successfully performed with COCs retrieved from
ovaries after eight hours of shipping, obtaining competent embryos in culture.
Panthera leo, assisted reproductive technology (ART), ICSI, blastocyst
Assisted reproductive technologies (ARTs) are now strongly recommended to be applied towards
the breeding of endangered species, because they provide a helpful tool to link different populations
that are separated in space (in situ and ex situ) and time (gamete banking).
The Felidae family is a broadly endangered group. Among them, the African lion (Panthera leo)
was recently highlighted because its population in the wild is decreasing very rapidly [1, 2]. The
fact that the groups of these animals in the wild are becoming constantly more fragmented and
smaller supports the urgent need for ways to preserve the genetic variation of individuals, both in
the wild and in captivity.
The establishment of protocols, e.g., for AI or IVF, is challenging because of the scarcity of sample
materials. Therefore, numerous methods have been developed in a model species, the domestic cat
(Felis catus) [3-6]. Some ARTs have already been successfully applied in wild felids, such as the
leopard cat (Felis bengalensis) , tiger (Panthera tigris) , cheetah (Acinomyx jubatus)  and
the black-footed cat (Felis nigripes) . In the lion, embryo retrieval succeeded after the induction
of ovulation and non-surgical AI [11, 12]. Furthermore, the preservation of ovarian cortex samples
by slow freezing followed by xenotransplantation of thawed tissue proved suitable for obtaining
growing follicles . Successful IVM of oocytes has been reported with success rates of 73.1% of
26 cumulus-oocyte complexes (COCs) obtained from one lioness, in which the oocytes reached
telophase I or metaphase II . In another study, metaphase II oocytes were retrieved
laparoscopically from three lionesses, followed by IVF of 19 oocytes , resulting in 30% of the
cleaved oocytes developing to blastocysts. The combination of both IVM and IVF was performed in
two studies. The first report resulted in a 22.9% oocyte maturation rate from 108 COCs recovered
from two lionesses, and 11.4% successful fertilization . The second study resulted in 77.2% of
matured oocytes from 57 COCs retrieved from three lionesses and 31.6% embryos cleaved beyond
the 8-cell stage . However, to our knowledge, there are no reports available on the successful
production of blastocysts after combined IVM and intracytoplasmic sperm injection (ICSI).
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Sample materials from wild felids are usually obtained from animals undergoing castration or
medically-indicated euthanasia in captivity, or because of death either in captivity or in the field.
Therefore, all methods of oocyte retrieval and gamete preservation developed in non-domestic
felids are considered as basic steps towards gamete rescue programs .
In our study, the aim was to test a full ART protocol combining oocyte retrieval, IVM and ICSI,
previously developed in the domestic cat [17, 18], that could be established as a standardized
embryo production approach in Panthera leo. Also, we compared two IVM culture media for their
ability to support oocyte maturation as judged by embryo cleavage and blastocyst formation after
2. Materials and methods
All chemicals were purchased from Sigma Aldrich (Taufkirchen, Germany) unless stated otherwise.
Ovaries were obtained from four 2-year-old lionesses after euthanasia for population management
in Ebeltoft Ree Park (Ebeltoft, Denmark). They were transported in Dulbecco’s PBS in a cooling
box with ice packs, carefully avoiding direct contact of the ovaries with the frozen packs. After
transportation lasting about eight hours, ovaries were processed immediately upon arrival at the
laboratory. One half of each ovary was assigned for oocyte retrieval by slicing the outer surface in
Washing Medium (WM), prepared from Medium 199 (M-199) with Earle's salts, supplemented
with 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL HEPES, 0.25 mg/mL sodium pyruvate, 0.6
mg/mL sodium lactate, 0.15 mg/mL L-glutamine and 0.055 mg/mL gentamicin. The remaining
halves of the ovaries were used for ovarian cortex preservation for another experiment. This
preparation was performed in Dulbecco’s PBS supplemented with 0.3% BSA . For the present
study, after cortex dissection COCs were directly collected from this solution and combined with
the other oocytes in WM. Both types of oocyte retrieval were performed in 6 cm Nunc Petri Dishes
(VWR International, Germany) and collection and quality assessment of COCs was performed
under a stereomicroscope. Only oocytes of good quality, with dark, homogeneous cytoplasm and
several granulosa cell layers were processed further. IVM was performed in WM supplemented
with two different established hormone regimens: IVM_1: 0.02 IU/mL oFSH and 0.05 IU/mL oLH
(ovine origin, Sioux Biochemical Inc., City, IA, USA)  and IVM_2: 1.64 IU/mL pFSH, 1.06
IU/mL pLH (porcine origin, both from the National Hormone and Pituitary Program, Rockville,
MD, USA) and 1 µg/mL 17ß-Estradiol [17, 19]. Our previous trials with lion oocytes (unpublished)
indicated that maturation might take longer in this species than in the domestic cat. Therefore,
maturation culture was performed for 32 to 34 hours at 38.5ºC in 5% CO2 in a humidified air
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The lion sperm used for fertilization was from a 12-year-old male, which was also euthanized in
2012 for management reasons in the Givskud Zoo (Givskud, Denmark) that was already stored in
our felid gametes bank. Prior to cryopreservation, the estimated fresh motility was 30%, and
freezing was performed according to a modified protocol from Lengwinat and Blottner . For
thawing the sample, a cryo tube was rapidly plunged into a warm bath at 38°C, and the spermatozoa
were washed in Sperm Medium, prepared from Tyrode's Salts Solution supplemented with
6 mg/mL BSA, 1.2 mg/mL HEPES, 1.1 mg/mL sodium lactate, 0.15 mg /mL L-glutamine, and
0.1 mg/mL sodium pyruvate. Finally, the spermatozoa were resuspended in the same medium.
For ICSI, a 60 cm petri dish was prepared with two 10µL droplets of polyvinylpyrrolidone (PVP)
diluted 1:2 (v:v) in WM, where sperm were placed, and ten 5 µL droplets of WM supplemented
with 0.5 mg/mL HEPES, where the oocytes were transferred after being stripped of cumulus cells
by gently pipetting with a micropipette (The Stripper®, BioTipp, Waterford, Ireland). All drops
were covered with mineral oil. Each oocyte was assessed for morphology and extrusion of the first
polar body as a sign of metaphase II, under 200 X magnification on an inverted microscope
(Axiovert 100, Carl Zeiss, Jena, Germany). ICSI was performed as previously described for the
domestic cat [6, 18]. In brief, under the inverted microscope, a sperm cell was immobilized and
caught with an injection pipette, the oocyte was held in place with a holding pipette with the polar
body between 11 to 1 or 5 to 7 o’clock, and the spermatozoon was pushed inside head first from the
3 o’clock position.
After ICSI, all oocytes were placed into 400 µL drops of embryo culture medium, consisting of
Ham's F-10 supplemented with 5% fetal calf serum (FCS), 0.11 mg/mL sodium pyruvate, 0.075
mg/mL L-glutamine, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Assessment of embryo
development was performed every 24 hours. The medium was not changed during embryo culture.
In total, 68 COCs were recovered from eight ovaries. The number of COCs obtained from the parts
that were used for ovarian cortex preservation was lower than the COCs retrieved by surface cutting
(12 vs. 56, respectively). Before maturation, all COCs were combined and then randomly divided
into two maturation groups (IVM_1: n = 33; IVM_2: n = 35). After 32 to 34 hours of culture, 13
oocytes from the IVM_1 group (39.4%) and 12 oocytes from IVM_2 group (34.3%), clearly
showed a first polar body (Figure 1a). All these mature oocytes were microinjected with frozen-
thawed lion sperm cells. After sperm thawing, only around 10% of the spermatozoa were motile, so
injection was performed using only cells with good progressive motility and normal morphology.
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The cleavage rate, as assessed 30 hours post-ICSI, reached 31.8% (four of 13) in the IVM_1 group
and 58.3% (seven out of 12) in the IVM_2 group (Figure 1b). Half of the cleaved embryos in the
IVM_1 group reached advanced embryo stages (50%, two of four cleaved), and 28.6% (two of
seven cleaved) in the IVM_2 group (Figure 1c). All the embryos that developed into morulae also
reached the blastocyst stage (Figure 1d), independently of IVM group. Blastocysts were observed
between Days 9 and 11 after ICSI. All results are presented in Table 1.
Here we describe for the first time the successful production of blastocysts after IVM and ICSI
procedures in the lion.
As already shown for the domestic cat, strong morphological criteria to characterize good quality
oocytes are also very important to achieve good rates of maturation and development in other felid
species. Although working with scarce experimental material from a non-domestic species is
difficult, attempts to increase oocyte numbers by collecting oocytes of suboptimal quality does not
benefit the eventual outcome of the approach. In the domestic cat, high quality COCs were
designated as those with uniformly dark cytoplasm and with several layers of cumulus cells
surrounding the entire oocyte [5, 21]. When adopting these criteria, IVM rates in the domestic cat
are between 50 to 80% [3, 16, 19, 21]. These criteria were also valid for lion oocytes [4, 14, 16].
When the same guidelines were applied to the present experiment, from eight lion ovaries (four
animals), 68 high quality COCs were obtained (~7 COCs per ovary). This rate is in accordance with
or even slightly higher than the average of 3 to 4 good COCs per ovary recovered in the domestic
cat [17, 19, 21] and other felids [4, 16]. However, fewer oocytes were retrieved from the solution
where the ovary halves assigned for ovarian cortex preservation were prepared. One possible
explanation for the higher retrieval of oocytes in the present study might be the young age of the
lions. In other species, such as cattle, it was shown that the number of good quality COCs recovered
per ovary is significantly higher in prepubertal females [22, 23], but at the expense of the oocyte
maturation outcome. To overcome the deficient IVM competence of prepubertal COCs, higher
gonadotropin levels in the maturation culture media were proposed [24, 25].
Different maturation media have been tested in the domestic cat. The most pronounced differences
were divergent levels of gonadotropins. We compared a medium with low FSH/ LH content [6, 26]
with one having an 80-fold and 20-fold higher gonadotropin content for FSH and LH and 1 µg/mL
17ß-Estradiol, respectively . In the cat, the first protocol resulted in a blastocyst formation rate
of 25% of cleaved embryos and 43% with the second treatment [6, 17, 19]. However, in the current
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experiment, no significant difference in the maturation rate between the two protocols was evident.
The maturation rate was 39% in the low gonadotropin medium (IVM_1) compared to 34% in the
high gonadotropin medium (IVM_2). The overall lower rate (compared to the domestic cat) may be
explained by the fact that the protocol used is not yet specific enough for the lion. Even in the
domestic cat it has been suggested that different media compositions could influence the time
needed for meiotic maturation and thus increase the proportions of matured oocytes .
Despite previous studies indicating that IVM in lions can be attained in 24 hours and that
maturation rates are comparable to those in the domestic cat [4, 14], recent trials in our laboratory
(results not published) demonstrated that extrusion of the first polar body took longer than in the
domestic cat. Since this stage of maturation is required for ICSI, we established the culture time for
maturation at 32 to 34 hours, instead of the usual 24 hours indicated for Felis catus.
Another point that could have a negative influence on the oocyte competence, was the time required
for transporting ovaries from the zoo to the laboratory, which took about eight hours in the present
study. Prolonged storage of ovaries after excision clearly affects the competence of the collected
ovarian oocytes . Furthermore, lionesses enter puberty around three years of age, and usually
have their first litter at around four years [29, 30] but the lionesses in the present study were only
two years old and therefore, probably were not yet fully mature. This may play a role in the
maturation competence of the oocytes. In other species, it was shown that oocytes retrieved from
prepuberal animals are less meiotically competent than oocytes from adult females [31, 32]. In the
domestic cat, however, ovaries are usually obtained from local animal clinics, and the queens are
often ovariectomized before their first heat, thus they are also prepuberal, as with the lions in our
study. The impact of donor animals’ ages on oocyte developmental competence needs to be
considered in future studies.
Successful cryopreservation of feline spermatozoa has been reported many times, achieving rates
higher than 50% of motility post-thawing [20, 33]. Nevertheless, poor sperm quality is a limiting
factor in non-domestic felid ARTs, due to both lower quantity and quality of samples obtained from
wild individuals [34, 35]. Because of this, ICSI has been suggested as a solution to increase embryo
production by avoiding this male factor. Microinjection of fresh or frozen-thawed sperm cells into
in vivo and in vitro matured oocytes has been successful in producing competent feline embryos
[18, 27] and has led to the birth of live kittens [3, 36]. In this study, no sham injection was
performed because of the limited number of COCs finally retrieved and the intrinsic value of the
samples. We showed in another study that sham injection did not produce any parthenogenetic
cleavage under our conditions . Furthermore, while previous studies reported cleavage of cat
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oocytes after sham injections, no parthenote was able to develop to the blastocyst stage [36-39].
Also, it is known that the cell cycle is faster in parthenogenetic oocytes  which contrasts with
the developmental speed of the embryos obtained here.
The chronology of domestic cat embryo development has been established, comparing in vivo and
in vitro fertilization and development . At least until the morula stage, embryos derived from
these two approaches have similar developmental timings. The morula stage is reached around Day
4 post-fertilization. Also with both ICSI and IVF, embryos develop at similar speeds . Thus, the
blastocyst stage was reached on Day 7 after IVM and ICSI, with around 41% cleavage and 19%
blastocyst formation . In the lion, embryo development seems to be retarded in comparison to the
pattern observed with domestic cats. At the first examination 30 hours post-fertilization, lion
embryos reached the 2-cell stage, while on Day 4 we found 8- to 16-cell embryos. Morulae were
observed only from Day 6 onwards, with all of them developing into blastocysts between Days 9
and 11. Since no information about the timing of lion embryo development has been available until
now, we cannot assess whether this “retarded” embryo development is an artifact of in vitro
production (caused by prolonged transportation of ovaries at low temperature, suboptimal culture
media or some other origin) or if it reflects a species-specific feature. However, the embryos in our
study were competent to develop up to blastocyst stages (Figure 1d), overcoming the developmental
block which is a typical problem of in vitro cultured feline embryos [26, 41]. In addition, the fact
that all morulae were competent to develop to the blastocyst stage indicates the good quality and
competence of these embryos.
In summary, we showed that in the lion (Panthera leo), IVM and ICSI can be successfully
performed using COCs, which were collected from ovaries of two year old females after 8 hours’
transportation at cold temperatures. The in vitro produced embryos required at least nine days for
development to the blastocyst stage, in contrast to our experiences in other felid species.
We thank Kim Rasmussen from the Givskud Zoo, in Denmark, for providing lion testes. Also we
want to acknowledge Susanne Eder, Christiane Franz and Karin Müller for preparation and freezing
of the sperm. For shipping the ovaries used in this experiment, we thank Susanne Pribbenow and
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Tables and figures
Table 1: In vitro maturation, fertilization and embryo development of Panthera leo oocytes.
Figure 1: Different stages of in vitro development of (a) a lion oocyte after in vitro maturation, and
ICSI with homologous frozen-thawed sperm, (b) 2-cell embryo on Day 2, (c) morula on Day 6 and
(d) blastocyst on Day 9.
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1. Lion blastocysts can be obtained after in vitro maturation of the cumulus-oocyte complexes and ICSI with homologous sperm. 2. In Panthera leo, a slower embryo developmental speed in comparison to the model species, domestic cat (Felis catus), was observed. 3. After eight hours of shipping at cold temperatures, the oocytes obtained from the ovaries were
4. Different hormonal supplementation in the media did not produce significant differences in the outcome of the maturation.
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5. In vitro maturation took longer than the usual time frame in Felis catus.