Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection

Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection

Accepted Manuscript Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and ICSI Lorena Fernandez-Gonzalez , Romy Hriba...

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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:

S0093-691X(14)00673-6

DOI:

10.1016/j.theriogenology.2014.11.037

Reference:

THE 13023

To appear in:

Theriogenology

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.

ACCEPTED MANUSCRIPT

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REVISED Title

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Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and ICSI

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Authors

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Lorena Fernandez-Gonzalez1, Romy Hribal1, Julia Stagegaard2, Jennifer Zahmel1, Katarina

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Jewgenow1

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Affiliations

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10324 Berlin, Germany

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Leibniz Institute for Zoo and Wildlife Research, Department Reproduction Biology, PF 700430,

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Corresponding author: Lorena Fernandez-Gonzalez, Email: [email protected] Leibniz-

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Institute for Zoo and Wildlife Research, Department Reproduction Biology, PF 700430, 10324

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Berlin, Germany. Telephone: 0049305168619.

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Abstract

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Assisted reproductive techniques (ARTs) are becoming widely applied to the breeding of

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endangered species, but establishing reliable protocols for the production of embryos in vitro is

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challenging because of the scarcity of sample material. In our study, we applied an ART protocol

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for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes

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retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours post-mortem. In total,

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68 cumulus-oocyte complexes (COCs) of good quality were randomly distributed and cultured for

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32 to 34 hours into two different maturation culture media, consisting of Medium 199 with Earle's

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salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate,

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0.15 mg/mL L-glutamine and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was

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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

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µg/mL 17ß-Estradiol. Differences in hormonal supplementation did not produce significant

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differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured

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oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage

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rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in

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IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in

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IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onwards, and

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blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison to

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domestic cats. This is the first report of in vitro produced blastocysts using ICSI in the lion, and the

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results demonstrate that IVM and ICSI can be successfully performed with COCs retrieved from

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ovaries after eight hours of shipping, obtaining competent embryos in culture.

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Keywords

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Panthera leo, assisted reproductive technology (ART), ICSI, blastocyst

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1. Introduction

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Assisted reproductive technologies (ARTs) are now strongly recommended to be applied towards

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the breeding of endangered species, because they provide a helpful tool to link different populations

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that are separated in space (in situ and ex situ) and time (gamete banking).

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The Felidae family is a broadly endangered group. Among them, the African lion (Panthera leo)

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was recently highlighted because its population in the wild is decreasing very rapidly [1, 2]. The

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fact that the groups of these animals in the wild are becoming constantly more fragmented and

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smaller supports the urgent need for ways to preserve the genetic variation of individuals, both in

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the wild and in captivity.

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The establishment of protocols, e.g., for AI or IVF, is challenging because of the scarcity of sample

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materials. Therefore, numerous methods have been developed in a model species, the domestic cat

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(Felis catus) [3-6]. Some ARTs have already been successfully applied in wild felids, such as the

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leopard cat (Felis bengalensis) [7], tiger (Panthera tigris) [8], cheetah (Acinomyx jubatus) [9] and

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the black-footed cat (Felis nigripes) [10]. In the lion, embryo retrieval succeeded after the induction

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of ovulation and non-surgical AI [11, 12]. Furthermore, the preservation of ovarian cortex samples

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by slow freezing followed by xenotransplantation of thawed tissue proved suitable for obtaining

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growing follicles [13]. Successful IVM of oocytes has been reported with success rates of 73.1% of

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26 cumulus-oocyte complexes (COCs) obtained from one lioness, in which the oocytes reached

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telophase I or metaphase II [14]. In another study, metaphase II oocytes were retrieved

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laparoscopically from three lionesses, followed by IVF of 19 oocytes [15], resulting in 30% of the

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cleaved oocytes developing to blastocysts. The combination of both IVM and IVF was performed in

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two studies. The first report resulted in a 22.9% oocyte maturation rate from 108 COCs recovered

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from two lionesses, and 11.4% successful fertilization [16]. The second study resulted in 77.2% of

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matured oocytes from 57 COCs retrieved from three lionesses and 31.6% embryos cleaved beyond

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the 8-cell stage [4]. However, to our knowledge, there are no reports available on the successful

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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

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medically-indicated euthanasia in captivity, or because of death either in captivity or in the field.

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Therefore, all methods of oocyte retrieval and gamete preservation developed in non-domestic

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felids are considered as basic steps towards gamete rescue programs [4].

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In our study, the aim was to test a full ART protocol combining oocyte retrieval, IVM and ICSI,

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previously developed in the domestic cat [17, 18], that could be established as a standardized

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embryo production approach in Panthera leo. Also, we compared two IVM culture media for their

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ability to support oocyte maturation as judged by embryo cleavage and blastocyst formation after

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ICSI.

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2. Materials and methods

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All chemicals were purchased from Sigma Aldrich (Taufkirchen, Germany) unless stated otherwise.

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Ovaries were obtained from four 2-year-old lionesses after euthanasia for population management

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in Ebeltoft Ree Park (Ebeltoft, Denmark). They were transported in Dulbecco’s PBS in a cooling

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box with ice packs, carefully avoiding direct contact of the ovaries with the frozen packs. After

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transportation lasting about eight hours, ovaries were processed immediately upon arrival at the

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laboratory. One half of each ovary was assigned for oocyte retrieval by slicing the outer surface in

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Washing Medium (WM), prepared from Medium 199 (M-199) with Earle's salts, supplemented

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with 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL HEPES, 0.25 mg/mL sodium pyruvate, 0.6

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mg/mL sodium lactate, 0.15 mg/mL L-glutamine and 0.055 mg/mL gentamicin. The remaining

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halves of the ovaries were used for ovarian cortex preservation for another experiment. This

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preparation was performed in Dulbecco’s PBS supplemented with 0.3% BSA [13]. For the present

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study, after cortex dissection COCs were directly collected from this solution and combined with

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the other oocytes in WM. Both types of oocyte retrieval were performed in 6 cm Nunc Petri Dishes

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(VWR International, Germany) and collection and quality assessment of COCs was performed

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under a stereomicroscope. Only oocytes of good quality, with dark, homogeneous cytoplasm and

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several granulosa cell layers were processed further. IVM was performed in WM supplemented

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with two different established hormone regimens: IVM_1: 0.02 IU/mL oFSH and 0.05 IU/mL oLH

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(ovine origin, Sioux Biochemical Inc., City, IA, USA) [6] and IVM_2: 1.64 IU/mL pFSH, 1.06

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IU/mL pLH (porcine origin, both from the National Hormone and Pituitary Program, Rockville,

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MD, USA) and 1 µg/mL 17ß-Estradiol [17, 19]. Our previous trials with lion oocytes (unpublished)

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indicated that maturation might take longer in this species than in the domestic cat. Therefore,

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maturation culture was performed for 32 to 34 hours at 38.5ºC in 5% CO2 in a humidified air

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atmosphere.

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The lion sperm used for fertilization was from a 12-year-old male, which was also euthanized in

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2012 for management reasons in the Givskud Zoo (Givskud, Denmark) that was already stored in

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our felid gametes bank. Prior to cryopreservation, the estimated fresh motility was 30%, and

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freezing was performed according to a modified protocol from Lengwinat and Blottner [20]. For

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thawing the sample, a cryo tube was rapidly plunged into a warm bath at 38°C, and the spermatozoa

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were washed in Sperm Medium, prepared from Tyrode's Salts Solution supplemented with

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6 mg/mL BSA, 1.2 mg/mL HEPES, 1.1 mg/mL sodium lactate, 0.15 mg /mL L-glutamine, and

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0.1 mg/mL sodium pyruvate. Finally, the spermatozoa were resuspended in the same medium.

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For ICSI, a 60 cm petri dish was prepared with two 10µL droplets of polyvinylpyrrolidone (PVP)

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diluted 1:2 (v:v) in WM, where sperm were placed, and ten 5 µL droplets of WM supplemented

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with 0.5 mg/mL HEPES, where the oocytes were transferred after being stripped of cumulus cells

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by gently pipetting with a micropipette (The Stripper®, BioTipp, Waterford, Ireland). All drops

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were covered with mineral oil. Each oocyte was assessed for morphology and extrusion of the first

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polar body as a sign of metaphase II, under 200 X magnification on an inverted microscope

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(Axiovert 100, Carl Zeiss, Jena, Germany). ICSI was performed as previously described for the

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domestic cat [6, 18]. In brief, under the inverted microscope, a sperm cell was immobilized and

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caught with an injection pipette, the oocyte was held in place with a holding pipette with the polar

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body between 11 to 1 or 5 to 7 o’clock, and the spermatozoon was pushed inside head first from the

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3 o’clock position.

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After ICSI, all oocytes were placed into 400 µL drops of embryo culture medium, consisting of

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Ham's F-10 supplemented with 5% fetal calf serum (FCS), 0.11 mg/mL sodium pyruvate, 0.075

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mg/mL L-glutamine, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Assessment of embryo

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development was performed every 24 hours. The medium was not changed during embryo culture.

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3. Results

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In total, 68 COCs were recovered from eight ovaries. The number of COCs obtained from the parts

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that were used for ovarian cortex preservation was lower than the COCs retrieved by surface cutting

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(12 vs. 56, respectively). Before maturation, all COCs were combined and then randomly divided

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into two maturation groups (IVM_1: n = 33; IVM_2: n = 35). After 32 to 34 hours of culture, 13

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oocytes from the IVM_1 group (39.4%) and 12 oocytes from IVM_2 group (34.3%), clearly

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showed a first polar body (Figure 1a). All these mature oocytes were microinjected with frozen-

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thawed lion sperm cells. After sperm thawing, only around 10% of the spermatozoa were motile, so

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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

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and 58.3% (seven out of 12) in the IVM_2 group (Figure 1b). Half of the cleaved embryos in the

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IVM_1 group reached advanced embryo stages (50%, two of four cleaved), and 28.6% (two of

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seven cleaved) in the IVM_2 group (Figure 1c). All the embryos that developed into morulae also

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reached the blastocyst stage (Figure 1d), independently of IVM group. Blastocysts were observed

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between Days 9 and 11 after ICSI. All results are presented in Table 1.

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4. Discussion

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Here we describe for the first time the successful production of blastocysts after IVM and ICSI

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procedures in the lion.

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As already shown for the domestic cat, strong morphological criteria to characterize good quality

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oocytes are also very important to achieve good rates of maturation and development in other felid

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species. Although working with scarce experimental material from a non-domestic species is

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difficult, attempts to increase oocyte numbers by collecting oocytes of suboptimal quality does not

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benefit the eventual outcome of the approach. In the domestic cat, high quality COCs were

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designated as those with uniformly dark cytoplasm and with several layers of cumulus cells

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surrounding the entire oocyte [5, 21]. When adopting these criteria, IVM rates in the domestic cat

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are between 50 to 80% [3, 16, 19, 21]. These criteria were also valid for lion oocytes [4, 14, 16].

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When the same guidelines were applied to the present experiment, from eight lion ovaries (four

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animals), 68 high quality COCs were obtained (~7 COCs per ovary). This rate is in accordance with

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or even slightly higher than the average of 3 to 4 good COCs per ovary recovered in the domestic

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cat [17, 19, 21] and other felids [4, 16]. However, fewer oocytes were retrieved from the solution

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where the ovary halves assigned for ovarian cortex preservation were prepared. One possible

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explanation for the higher retrieval of oocytes in the present study might be the young age of the

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lions. In other species, such as cattle, it was shown that the number of good quality COCs recovered

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per ovary is significantly higher in prepubertal females [22, 23], but at the expense of the oocyte

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maturation outcome. To overcome the deficient IVM competence of prepubertal COCs, higher

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gonadotropin levels in the maturation culture media were proposed [24, 25].

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Different maturation media have been tested in the domestic cat. The most pronounced differences

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were divergent levels of gonadotropins. We compared a medium with low FSH/ LH content [6, 26]

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with one having an 80-fold and 20-fold higher gonadotropin content for FSH and LH and 1 µg/mL

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17ß-Estradiol, respectively [19]. In the cat, the first protocol resulted in a blastocyst formation rate

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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.

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The maturation rate was 39% in the low gonadotropin medium (IVM_1) compared to 34% in the

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high gonadotropin medium (IVM_2). The overall lower rate (compared to the domestic cat) may be

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explained by the fact that the protocol used is not yet specific enough for the lion. Even in the

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domestic cat it has been suggested that different media compositions could influence the time

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needed for meiotic maturation and thus increase the proportions of matured oocytes [27].

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Despite previous studies indicating that IVM in lions can be attained in 24 hours and that

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maturation rates are comparable to those in the domestic cat [4, 14], recent trials in our laboratory

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(results not published) demonstrated that extrusion of the first polar body took longer than in the

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domestic cat. Since this stage of maturation is required for ICSI, we established the culture time for

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maturation at 32 to 34 hours, instead of the usual 24 hours indicated for Felis catus.

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Another point that could have a negative influence on the oocyte competence, was the time required

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for transporting ovaries from the zoo to the laboratory, which took about eight hours in the present

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study. Prolonged storage of ovaries after excision clearly affects the competence of the collected

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ovarian oocytes [28]. Furthermore, lionesses enter puberty around three years of age, and usually

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have their first litter at around four years [29, 30] but the lionesses in the present study were only

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two years old and therefore, probably were not yet fully mature. This may play a role in the

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maturation competence of the oocytes. In other species, it was shown that oocytes retrieved from

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prepuberal animals are less meiotically competent than oocytes from adult females [31, 32]. In the

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domestic cat, however, ovaries are usually obtained from local animal clinics, and the queens are

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often ovariectomized before their first heat, thus they are also prepuberal, as with the lions in our

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study. The impact of donor animals’ ages on oocyte developmental competence needs to be

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considered in future studies.

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Successful cryopreservation of feline spermatozoa has been reported many times, achieving rates

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higher than 50% of motility post-thawing [20, 33]. Nevertheless, poor sperm quality is a limiting

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factor in non-domestic felid ARTs, due to both lower quantity and quality of samples obtained from

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wild individuals [34, 35]. Because of this, ICSI has been suggested as a solution to increase embryo

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production by avoiding this male factor. Microinjection of fresh or frozen-thawed sperm cells into

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in vivo and in vitro matured oocytes has been successful in producing competent feline embryos

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[18, 27] and has led to the birth of live kittens [3, 36]. In this study, no sham injection was

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performed because of the limited number of COCs finally retrieved and the intrinsic value of the

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samples. We showed in another study that sham injection did not produce any parthenogenetic

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cleavage under our conditions [18]. 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].

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Also, it is known that the cell cycle is faster in parthenogenetic oocytes [38] which contrasts with

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the developmental speed of the embryos obtained here.

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The chronology of domestic cat embryo development has been established, comparing in vivo and

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in vitro fertilization and development [40]. At least until the morula stage, embryos derived from

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these two approaches have similar developmental timings. The morula stage is reached around Day

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4 post-fertilization. Also with both ICSI and IVF, embryos develop at similar speeds [36]. Thus, the

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blastocyst stage was reached on Day 7 after IVM and ICSI, with around 41% cleavage and 19%

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blastocyst formation [3]. In the lion, embryo development seems to be retarded in comparison to the

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pattern observed with domestic cats. At the first examination 30 hours post-fertilization, lion

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embryos reached the 2-cell stage, while on Day 4 we found 8- to 16-cell embryos. Morulae were

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observed only from Day 6 onwards, with all of them developing into blastocysts between Days 9

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and 11. Since no information about the timing of lion embryo development has been available until

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now, we cannot assess whether this “retarded” embryo development is an artifact of in vitro

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production (caused by prolonged transportation of ovaries at low temperature, suboptimal culture

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media or some other origin) or if it reflects a species-specific feature. However, the embryos in our

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study were competent to develop up to blastocyst stages (Figure 1d), overcoming the developmental

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block which is a typical problem of in vitro cultured feline embryos [26, 41]. In addition, the fact

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that all morulae were competent to develop to the blastocyst stage indicates the good quality and

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competence of these embryos.

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In summary, we showed that in the lion (Panthera leo), IVM and ICSI can be successfully

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performed using COCs, which were collected from ovaries of two year old females after 8 hours’

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transportation at cold temperatures. The in vitro produced embryos required at least nine days for

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development to the blastocyst stage, in contrast to our experiences in other felid species.

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Acknowledgments

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We thank Kim Rasmussen from the Givskud Zoo, in Denmark, for providing lion testes. Also we

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want to acknowledge Susanne Eder, Christiane Franz and Karin Müller for preparation and freezing

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of the sperm. For shipping the ovaries used in this experiment, we thank Susanne Pribbenow and

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Olga Amelkina.

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Karongwe Game Reserve, South Africa. Afr Zool 2008;43:230-6.

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Reproductive success in lions. In: Clutton-Brock TH, editor. Reproductive Success, Chicago:

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analysis of calf and cow oocytes during in vitro maturation. Mol Reprod Dev 1998;49:168-75.

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vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil

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[33] Toyonaga M, Sato Y, Morita M, Watanabe M, Oba H, Mizutani T, et al. The qualities of

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cryopreserved epididymal sperm collected from feline epididymides stored at low temperature. J

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comparative analysis of ejaculate and hormonal characteristics of the captive male cheetah, tiger,

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produced by intracytoplasmic sperm injection of cat oocytes. Anim Reprod Sci 1998;53:221-36.

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intracytoplasmic sperm injection (ICSI) with sperm derived from frozen-thawed cat testicular

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tissue. Thai J Vet Med 2011;41:144-5.

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[38] Comizzoli P, Wildt DE, Pukazhenthi BS. In vitro development of domestic cat embryos

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following intra-cytoplasmic sperm injection with testicular spermatozoa. Theriogenology

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2006;66:1659-63.

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Theriogenology 1995;43:302.

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fertilized in vivo versus in vitro. Biol Reprod 1994;51:441-51.

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[41] Merlo B, Iacono E, Regazzini M, Zambelli D. Cat blastocysts produced in vitro from oocytes

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vitrified using the cryoloop technique and cryopreserved electroejaculated semen. Theriogenology

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2008;70:126-30.

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Tables and figures

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Table 1: In vitro maturation, fertilization and embryo development of Panthera leo oocytes.

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Figure 1: Different stages of in vitro development of (a) a lion oocyte after in vitro maturation, and

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ICSI with homologous frozen-thawed sperm, (b) 2-cell embryo on Day 2, (c) morula on Day 6 and

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(d) blastocyst on Day 9.

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Number

Number

Number

Number

Group

oocytes

mature (%)

cleaved (%)

morulae (%)

blastocysts (%)

IVM_1

33

13 (39.4)

4 (30.8)

2 (50)

2 (50)

IVM_2

35

12 (34.3)

7 (58.3%)

2 (28.6)

2 (28.6)

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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

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still competent.

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.