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

Abstract

Assisted reproductive techniques 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 assisted reproductive technique 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 postmortem. In total, 68 cumulus–oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in 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 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro–produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus–oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture.

Introduction

Assisted reproductive technologies (ARTs) are now strongly recommended to be applied toward 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], [4], [5], [6]. Some ARTs have already been successfully applied in wild felids, such as the leopard cat (F bengalensis) [7], tiger (P tigris) [8], cheetah (Acinonyx jubatus) [9], and the black-footed cat (F nigripes) [10]. In the lion, embryo retrieval succeeded after the induction of ovulation and nonsurgical 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 [13]. 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 [14]. In another study, metaphase II oocytes were retrieved laparoscopically from three lionesses, followed by IVF of 19 oocytes [15], 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 [16]. 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 [4]. However, to our knowledge, there are no reports available on the successful production of blastocysts after combined IVM and intracytoplasmic sperm injection (ICSI).

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

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 ICSI.