Embryo Manipulation Techniques in the Rabbit

4.1. Embryo coats

The extracellular coverings of rabbit embryos are structurally more complex than in most mammalian embryos. The peculiarity of rabbit embryos is that they are surrounded not only by the zona pellucida but also by a mucin coat (Figure 1).

Zona pellucida is an extracellular embryonic coat that surrounds the early embryo (Figure 1). Zona pellucida proteins are synthesised by the oocyte and the granulosa cells to form concentric layers consisting of cross-linked zona proteins [38]. The steadiness of zona pellucida allows the intrazonal diameter of the embryo to remain constant (around 120 μm) up to 72 hpc [33, 39]. The zona pellucida thickness itself ranges between 15.9 and 19.2 μm [32, 40]. The zona pellucida selects morphologically normal spermatozoa [41], indices the acrosomal reaction [42] preventing polyspermy [43] and protects early embryo integrity during the transport through the oviduct [44]. It affects fertilisation; a thicker zona pellucida was found in oocytes with failed fertilisation collected from the oviducts compared with those of embryos [40]. Moreover, recent studies have shown that zona pellucida thickness is 8% lower in females under heat stress than in thermal comfort conditions, decreasing the number of normal embryos [45].

The mucin coat is a layer of acid mucopolysaccharides, which is deposited on the embryos during the passage through the oviduct [46]. The mucin coat thickness depends on the time spent in the oviduct [46], and it is half the thickness for embryos at 48 hpc (around 50 μm) than at 72 hpc (around 100 μm) [33]. The thickness of the mucin coat is essential for rabbit embryos to develop at term because it physically prevents the embryos from direct exposure to a deleterious uterine environment and allows them to expand until the appropriate time for implantation [46]. When in vitro embryos are transferred into recipients, the lack of a mucin coat predisposes to subsequent failure of pregnancy [47].

The embryo is covered by the zona pellucida and a mucin coat until 72–96 hpc. The zona pellucida disappears by then, and it is substituted by neozona (4.5 days) when the blastocysts enter into the uterus, while the mucin coat is covered by a new layer, named gloiolemma, around 6 days of gestation [38].

7.1. Embryo recovery and transfer

The ability to recover and transfer preimplantation embryos has numerous associated applications which are inevitably linked to translational molecular genetics, cell biology and assisted reproductive technology [79]. In 1890, the first successful mammalian embryo transfer was performed in rabbits [80]. Nowadays, this technique has become a routine practice in human medicine [81].

Embryo transfer includes the generation of preimplantation embryos along with the development of those embryos until term in different recipient females. Production of preimplantation embryos can be achieved in vivo or in vitro. In both cases, superovulation protocols can be applied to ensure the maximum number of embryos recovered per donor. The classical protocols have been performed with equine chorionic gonadotropin (eCG) or FSH (Table 2) [21, 82, 83, 84, 86, 87, 88]. But, both the number and quality of the oocytes obtained are highly variable when eCG is administrated [21, 83, 86]. The use of LH on super stimulation has been studied using porcine FSH (pFSH). The results are also variable due to the different LH concentration present, contamination from other hormones, inconsistencies within and among batches, and the possibility of the spread of diseases [88]. Nowadays, because of break-through in recombinant technology, it is possible to easily dispose of LH and FSH in an isolated manner [81], and the treatment with recombinant human FSH (rhFSH) alone or supplemented with LH is effective in stimulating superovulation without affecting the embryo quality [85].

Fertilisation of the oocytes can be achieved by natural mating or artificial insemination, if embryos are produced in vivo. Artificial insemination is usually performed with fresh or cooled semen stored for short periods of time (under 36 h) [89, 90], and obtains a high fertility rate and prolificacy. However, when fresh semen is used the sperm concentration per ml should be 4 million [91], while it should be increased to 15 million if the semen is refrigerated [89]. Fresh or cooled semen is used in the rabbit industry to improve breeding management [22]. However, frozen semen presents poor fertility after thawing [92, 93] and it is used mainly for conservation of banking resources, international exports and research [94].

A good understanding of the chronology of events that follows the ovulation is crucial to the recovery of preimplantation embryos [78]. At 24 hpc, all the embryos can be found in the isthmus [95]. At 78 hpc about one-third of the embryos are already found beyond the uterotubal junction, whereas at 84 hpc more than 90% of the embryos have reached the uterus [95]. Embryo implantation occurs between 120 and 144 hpc [96]. Considering the chronology of embryo development some techniques have been developed to recovery embryos at any stage and location.

Embryos can be recovered by non-surgical, post-mortem or laparoscopy. In the non-surgical method, PGF_2α is administered 50–55 h after mating. It produces the expelling of embryos, located in the oviduct or the uterus, toward the vagina, where embryos can be recovered without surgery [97, 98]. This procedure has an efficacy of 40% [97, 98]. When embryos are obtained post-mortem, the entire reproductive tract is removed after the female is euthanised. The embryos are recovered by perfusion of each oviduct and the first third of the uterus with 5 ml of phosphate-buffered saline containing 0.2% of BSA [33]. This method allows a retrieval rate of 64% [99]. Laparoscopy is a method that allows the recovery of both preimplantation embryos and also of oocytes [100]. If multiple cycles of embryo collection are required, this method is chosen because it guarantees minimal invasion through the use of a small entrance in the peritoneal cavity. However, this procedure has a lower efficacy in primiparous or multiparous females (around 50%), compared with nulliparous females (73%) [99].

Recovered embryos are subjected to morphological grading (Table 3, [101, 102, 103]); they are usually classified as having ‘good quality’ when they present homogenous cellular mass and intact zona pellucida and mucin coat [104]. Normal embryos can be maintained under in vitro conditions or cryopreserved until transferred. Zygotes have been successfully developed into blastocyst in vitro [10, 31, 104, 105, 106, 107, 108]. One example of culture media is TCM199 supplemented with 0.1% of BSA and culture is performed in 500 μl of medium layered under paraffin oil at 38.5°C, 5% CO₂ and saturated humidity [109]. However, these culture media do not mimic the uterine environment, and as a consequence, embryos developed in vitro have fewer cells and lower intrazonal diameter and mucin coat thickness than embryos developed in vivo, which reduces pregnancy rates after embryo transfer [40, 106].

Embryo transfer is performed by surgery (laparotomy) [110, 111] or by laparoscopy [112], into the oviducts or each one of the uterine horns. The use of laparoscopy aims at minimising the invasion site of the reproductive organs and in situ manipulation [79]. To guarantee the success of the transfer, a minimum number of embryos have to be transferred, and asynchrony between the embryo development and the recipient female has to occur [65, 70, 113]. On one hand, at least two foeto-placental units are required to maintain the gestation [114, 115], and on the other hand, when the survival rate is high, competition between foetuses can lead to a lower foetal survival [115]. So, the number of transferred embryos recommended per recipients ranges between 10 and 13 for both fresh and cryopreserved embryos [71, 116]. If the number of transferred embryos per donor female is lower than this range, it is possible to transfer embryos from two donor females to each of the uterine horns of the recipient female, to ensure the survival of the embryos [71]. In case it is necessary to know the origin of the transferred embryos, it is recommended to perform a caesarean to the recipient female [71]. In general, asynchrony is obtained by artificially inducing ovulation in recipient female between 0 and 6 h before the donor females, for fresh embryos, or between 6 and 12 h before, for in vitro cultured or cryopreserved embryos [60, 65, 70, 113]. Besides, an association of the recipient genotype with implantation rate, foetal losses and offspring rate at birth has been documented [70, 71]. The recipient female usually belongs to a maternal line because recipient genotype is crucial in providing an adequate uterine environment to support adhesion, embryo-uterine cross-talk and placental and foetal development to term after vitrified and fresh embryo transfer [70, 111].

Embryo recovery and transfer influences mRNA expression of late blastocyst before implantation and may result in faulty embryonic implantation [117]. Transferred embryos have a lower transcript abundance of the transcription factor octamer binding 4 (OCT4) and higher epithelial membrane protein 1 (EMP1) [117]. OCT4 is a key regulator of the pluripotency maintenance system [118], and the main function of this transcriptional factor is to repress or activate several target genes involved in cell differentiation and early embryonic development [119]. The altered expression of OCT4 in the preimplantation embryo is associated with lower embryo quality [120]. EMP1 is involved in the regulation of cell cycle or cell-cell recognition, and high levels of EMP1 expression have been related to cell differentiation and arrest [121]. Signals involved in cell proliferation and differentiation during gastrulation and implantation events could be disturbed with embryo manipulation.

7.2. In vitro fertilisation and intracytoplasmic sperm injection

Oocyte collection and extracorporeal fertilisation represent an important embryo production in rabbits [73]. These embryos are of scientific interest for cloning and transgenesis [73].

Both in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) have been successfully developed [122, 123]. For IVF, oocytes are incubated with sperm for 5 h, and then moved to the embryo culture medium containing TCM199 medium suspended with 1.25 mM pyruvate, 0.1 mM EDTA, 10% FBS, and cultured at 37°C and 5% CO₂ [122, 123]. For ICSI, the micromanipulation is performed in the fertilisation medium on a warm microscope stage at 37°C. Cumulus cells of mature oocytes are removed by hyaluronidase treatment and gentle pipetting. The injection needle used for rabbit sperm is of 5.0 μm inner diameter. After injection, the oocytes were transferred to embryo culture medium [123].

7.3. Cloning and transgenesis

Cloning involves the transfer of a nucleus from a multicellular embryo, foetal or adult cell into an enucleated metaphase II (MII) oocyte [124]. This oocyte has the ability to incorporate the transferred nucleus and support development of a new embryo. Cloning of embryos by nuclear transfer technology (NT) has been developed in several species [125, 126, 127]; the rabbit was a pioneering species in NT by introducing embryonic cells into enucleated oocytes, at the end of the 1980s [128, 129]. However, the use of highly differentiated somatic cells as nuclear donors remained a challenge. It was not until 2002 that live clones were generated from NT with freshly prepared adult rabbit cumulus cell [130]. And later, clones were produced from foetal [131] and adult fibroblast [132].

Overall, rabbit NT efficiency depends on the enucleation of the recipient oocyte, fusion of the transplanted nucleus to the enucleated oocyte, activation of the oocyte and reprogramming of the transferred nucleus. On enucleation, the visualisation of the MII is difficult, because of the presence of dark cytoplasmic granules. To overcome this problem, MII can be detected with low ultraviolet light, which allows the removal of MII and polar body under visible light using an enucleation pipette with a minimal volume of oocyte cytoplasm [133]. Enucleation rates vary from 60 to 90% [134, 135], and the age of the recipient oocyte plays an important role in successful NT [136, 137]. Briefly, for nuclear transplantation, single donor cell is introduced beneath the zona pellucida of the enucleated oocytes by micromanipulators. Electro-cell fusion (3.2 kV/cm, 20 μs and three pulses) is applied to fuse the donor cell with the cytoplasm of the reconstructed embryos [138].

Classically, gene transfer has been carried out by microinjection of DNA constructs into fertilised oocytes or by using viral factors. Notwithstanding, with the progress in molecular technology, embryos have been genetically modified using most recently developed tools including TALEN [14] and CRISPR-Cas9 [139, 140] (Figure 2).

Both techniques, cloning and transgenesis, have low applications in livestock production due to problems derived from detection of genetically superior animals and evaluation of the clones and the transgenic animals [141]. Some implications for the use of transgenic rabbits nowadays include to act as bioreactors [142] or model for detailed analysis of spermatogenesis [143], and more recently, to establish embryonic stem cell lines from blastocyst stage rabbit embryos cloned by somatic cell NT [138, 144].