Research Progress in the Establishment of Sterile Hosts and Their Usage in Conservation of Poultry Genetic Resources

Abstract

Many local chicken breeds are rapidly declining and even facing extinction due to a variety of factors such as indiscriminate crossbreeding, climate fluctuations, epidemic outbreak, and environmental changes. Developing effective preservation strategies is important to address this situation. The special reproductive characteristics and gamete morphology of chickens pose challenges for preserving genetic heritage through the cryopreservation of genetic materials. Currently, gonad and primordial germ-cell cryopreservation and transplantation are the most promising approaches, especially for the genetic information in the W chromosome of female birds. The study of establishing sterile recipients is crucial for increasing the efficiency of the colonization of transplanted donor tissues and cells. Several classical methods, including ovariectomy and testectomy, busulfan, and irradiation, have been employed to deplete endogenous germ cells in recipient males before transplantation. These methods rely on the toxicity of chemical reagents and physical stimulation to kill germ cells. Recent advances in gene-editing technology have introduced sterile hosts via the knocking out of genes relevant to germ cells’ development. This review explores state-of-the-art technologies for preparing infertile avian recipients (mainly chickens) and aims to provide guidance for the conservation of poultry genetic material and breed restoration.

1. Introduction

Regionally adapted chickens (considered as indigenous breeds or local ecotypes) are found in every country and are genetically diverse. Due to their being well adapted to scavenging feeding, environmental challenges, and climatic conditions, regionally adapted chickens have become important variety resources for breeding and ecological diversity [1]. As rural farming practices are being replaced by centralized commercial poultry breeding, the number of local chicken varieties and their genetic diversity are rapidly decreasing [2]. The development of cryopreservation technology provides a platform for the long-term, safe, and efficient preservation of germplasm resources. Although it is not possible to preserve chicken oocytes or zygotes, preservation techniques for semen, testis, and ovary cells; primordial germ cells (PGCs); spermatogonial stem cells (SSCs); and somatic cells have been developed in succession [3].

The most valuable usage for the cryopreservation of genetic materials is in the reconstruction of a population in case of variety extinction. Semen can be used in artificial insemination; several consecutive generations of backcrossing programs are required to recover or reconstitute a germline. However, other frozen materials need to be transplanted to a new host to complete the recovery of strains. For example, the cryopreserved testis or ovary is heterotopically or orthotopically transplanted to day-old recipients. The PGCs are injected to the host chicken embryos [3,4]. The germline chimera composed of donor germ cells and surrogate hosts can basically complete the reproduction of donor strains under ideal conditions [5,6]. A major constraint of this system is that the colonization and differentiation ability of exogenous germ cells in recipient chickens vary among different individuals and breeds, which directly affects the strain-transmission rate. The niche of germ cells is limited in gonads. Most foreign germ cells cannot colonize and differentiate normally as endogenous ones do; therefore, a large portion of these surrogate hosts produce more recipient-derived progeny. The low germline propagation rate has become another key issue in restricting the genetic material preservation and restoration efficiency, besides the technical problem of the cryopreservation of this germplasm.

Chemical and physical methods have played an important role in killing the native gonadal germ cells of the recipients and enhancing the chimerism rate of germ cells of genetic materials within the recipients [7,8]. The method for removing key genes in the reproductive cells of mammals, poultry, and fish through gene knockout has been developed and utilized in the past few years [9]. The improvement in transplant efficiency and strain-transmission rate is of a higher level of importance, although there are clear differences in limitations in terms of practicality, feasibility, efficiency, and cost. In the production of transgenic animals, the colonization process of gene-edited cells in the recipients will also be affected by endogenous germ cells. Therefore, the sterile recipients play an important role in improving the efficiency of transgenesis [10,11,12]. This review examines these available technologies, which will be utilized more and serve as the foundation for new technology development in this area.

2. Preparation Strategy for Sterile Host

2.1. Ovariectomy and Testectomy

Over the past decade, there has been a complete experimental process for gonad transplantation in mammals and birds [13,14]. Because the primary gonad will directly prevent the attachment and colonization of exogenous gonads, the removal of recipients’ testicles or ovaries surgically before the gonadal transplantation is recommended. In other words, after anesthetization, the entire left ovary or paired testicles of the chicks are carefully surgically removed.

Although the cost of gonad preservation and transplantation is low, several key difficulties need to be overcome. Reasonable surgical methods and superb surgical skills are needed. First, it should be ensured that the recipients do not die during the surgery and later development. Because the ovary and testis are close to the aorta and vena cava, rough manipulation may cause massive bleeding or even mortality during gonadectomy [15]. Electrocautery is encouraged to reduce local bleeding in the middle or tail of the gonad [16].

Secondly, the attachment rate of donor gonads in the recipients should be improved. It has been suggested that part of the ovarian tissue should be kept during the ovariectomy to facilitate better attachment of the donor ovary [16]. Contrary to the above, studies have shown that complete castration can reduce the generation of gonadal chimeras without decreasing the attachment rate, which results in gametes mostly from donor sources [17]. The abdominal air sacs have often been used to cover the transplanted ovarian tissue to ensure the attachment [16]. However, in subsequent experiments, it has been found that the presence or absence of abdominal air sacs has no significant effect on the attachment rate. The presence of abdominal air sacs may block the surgical field, leading to the unintentional destruction of important blood vessels and the death of the hosts [18]. Therefore, it not recommended here that the abdominal air sacs be used.

The third important issue is the immune state of the recipients. Immune rejection is controversial in the study of gonadal transplantation. According to the theory of acquired tolerance, chicks will not experience immune rejection if they have contact with foreign gonads before the set-up of the complete immune system [15]. However, other articles have described how different degrees of lymphocyte infiltration have occurred after the surgery, which proves that immune rejection and inflammatory reaction also occurred even though the transplantation was performed on two-days-old chicks [18]. Therefore, the use of immunosuppressants should be emphasized in specific experiments. Hall et al. compared the ability of three immunosuppressants (mycophenolate mofetil, cyclophosphamide, and cyclosporin A) to prevent ovarian transplantation rejection in turkeys and found that cyclosporin A reduced lymphocyte infiltration effectively without impairing the development and maturation of transplanted gonads [18].

Furthermore, in some cases of ovarian transplantation, the ovaries of non-laying chickens were surrounded by a membranous sac with ovulatory yolk inside [16]. Whatever the mechanism of the membranous sac, it does seriously prevent the eggs from reaching the oviduct. Why did such an organ come into being, and how can the generation of such a capsule be prevented? This may be a problem that needs to be considered in future ovariectomy and transplantation procedures.

2.2. Busulfan

Busulfan (1,4-butanediol dimethanesulfonate) is an alkylating agent with high cytotoxicity, specifically to germ cells. After entering the body, the ring structure of the sulfonate ester group is opened to destroy the structure and function of DNA by alkylating with guanine in cellular DNA [19]. It has been used to deplete endogenous germ cells for generating transgenic individuals and for conserving genetic resources in birds [20].

Busulfan was firstly applied via direct embryo injection [21]. The optional concentration and processing technology of busulfan solution for embryo injection is ongoing. Song et al. compared the effect of the endogenous PGC depletion of two delivery formulations of busulfan (75 mg/embryo), a busulfan/sesame oil suspension (BS) and a solubilized busulfan DMF/sesame oil emulsion (SBE) [22]. Both treatments induced a significant reduction in PGCs, and the frequency of germ-line chimerism and the number of donor-derived offspring in SBE-treated recipients increased 5-fold and 8-fold, respectively, as compared to the control recipients. Using the chicks from eggs treated with 250 ug busulfan after 24 h of incubation as recipients for ovarian transplants, the ratio of donor-derived to host-derived offspring was significantly higher (0.57 for the control group vs. 2.36 for the treated group). Nakamura et al. later prepared a sustained-release emulsion that emulsified equal amounts of Ca²⁺- and Mg²⁺-free phosphate-buffered saline containing 10% busulfan solubilized in N,N-dimethylformamide and sesame oil by using a filtration method [23]. Then, 75 µg busulfan was injected into the yolk at 24 h incubation in a vehicle. The results showed that the sustained-release emulsion of busulfan could completely remove the germ cells from embryos. It had a stronger ability to remove PGCs than the busulfan short-release emulsion and suspension. They later injected 100 µg busulfan solubilized in a sustained-release emulsion into the yolk before incubation and obtained a high proportion of donor PGCs (98.6%) and a high germline-transmission rate (99.5%) [20].

The intraperitoneal injection of busulfan was also used in chickens to prepare SSC transplant recipients [24]. It was suggested that several doses be used over a period of time to reduce the physiological stress caused by a single dose application. A single high dose of busulfan (60 mg/kg of bodyweight) resulted in the death of all treated pubertal-age roosters, whereas the same amount of busulfan applied in two doses resulted in a considerable suppression of spermatogenesis [25]. In the study by Ghadimi et al., two different levels of busulfan, 60 mg (40 + 20) and 50 mg (30 + 20), with 10-day intervals were injected intraperitoneally to adult roosters, and none of the treatments resulted in death [24]. Although busulfan can severely damage the Sertoli cells of the recipient testes, transplanted quail SSCs still undergo spermatogenesis in the treated recipient roosters [8].

Given the concern that its residues may target donor PGCs, a safer means of administering busulfan should be sought. With the development of gene-editing, the manipulation of donor PGCs also provides the chance to increase the success of using busulfan. Glutathione-S-transferase (GST) provides a major route for the detoxification of a variety of xenobiotics including busulfan. A previous study in humans suggested that the overexpression of GST and MGSTII confers resistance to busulfan in HEK293 cells [26]. Recently, an in vivo selection model, which used microsomal MGSTII-overexpressing PGCs that were resistant to busulfan, was also developed in chickens. These PGCs may provide significant resistance to busulfan compared with wild-type PGCs and increase the rate of germline transmission (95%) and the number of offspring [27]. Jung et al. devised a similar method to enrich donor cells by transplanting busulfan-resistant PGCs into the adult zebra finch testis, eliminating endogenous germ cells by means of busulfan treatment of the recipients, and donor cell-derived spermatogenesis was accomplished [28].

SSCs and PGCs transplanted into the testes of infertile chickens were able to repopulate the seminiferous tubules and differentiate into functional SSCs [10,24]. After entering the blood circulation, busulfan can specifically kill more sensitive germ cells and increase the proportion of exogenous germ cells in the gonad. Therefore, the transmission rate can be improved by intraperitoneal injection or embryo injection, and the latter is suitable for a wider range. The appropriate dose of busulfan solution intraperitoneally injected into adult cocks had no significant effect on the health and survival of chickens [25]. Embryo injection may cause high mortality and deformity rates, but the use of a slow-release agent greatly improves the hatching rate of embryos [20] (Figure 1). The introduction of an in vivo selection model system highly increased the chimera production or breed-reconstruction efficiency [20].

2.3. Irradiation

X-rays and gamma irradiation are the most used irradiation sources for eliminating germ cells. Exposure time, duration, and dose are the main factors in optimizing irradiation in different poultry species in order to avoid the negative side effects of improper exposure, such as malformation or lethality [29].

Lim et al. confirmed that a 40 s exposure of stage X, stage 9, or stage 14 chicken embryos to a low dose of soft X-rays (1.5–1.8 Gy, i.e., 15–18 kV) was effective in depleting the endogenous PGC population without affecting embryo hatchability or somatic cells viability [30]. Nakamura et al. showed that the exposure of recipient chicken embryos after 52 h of incubation to 3 Gy of soft X-rays significantly reduced the endogenous PGCs and increased the germline transmission of donor PGCs, with no negative effect on hatchability [7]. The irradiated PGCs still retained the ability to migrate toward the germinal ridges [29]. The usage of gamma irradiation on stage X embryos was also effective in decreasing PGCs [31]. Extensive colonization of donor cells was promoted by exposing the recipient fertilized eggs of chickens to 500–700 rads from a 60Co source within one hour after oviposition [32,33]. A similar effect was achieved in Macdonald’s study, where fertile eggs were irradiated by a MDS Nordion Gammacell 1000 Elite, which has a Cs137 source, before incubation [34]. Using these techniques, more donor-derived PGCs colonized the recipient gonads, but the development of the treated embryos fell behind the non-treated control embryos.

Gamma irradiation was also used to suppress spermatogenesis in adult male recipients before SSC transplantation. Repeated moderate doses of gamma irradiation treatment over a period of 2 weeks (based on 60Co isotope, 5 × 8 Gy) applied in the adult male recipients successfully eradicated the endogenous spermatogenesis 45 days after the last treatment [35]. The spermatogenesis had not re-established even 12 months after the treatment. The follow-up study did not detect any negative influence on the cockerels’ condition and health or on the offspring from the donor-originated sperm ejaculated by the sterile recipients [35]. Ghadimi et al. compared the effects of 40 Gy gamma radiation in repeated doses of 8 Gy 5 times over 13 days and 30 Gy of gamma radiation in repeated doses of 10 Gy 3 times over 6 days on the 38-week ROSS roosters, and the results showed that both treatments significantly reduced the diameter of seminiferous tubules and the thickness of epithelial cells, and depleted endogenous spermatogenesis in one week [24].

The efficiency of radiation is dependent on the dose and the age of the animals at the time of exposure. The need for specialized facilities and equipment is another obstacle [36].

2.4. Genetically Engineered (GE) Sterile Chickens

Recently, precise gene-editing techniques have also been proven to be useful in this field. The reproductive ability of animals is regulated by many key genes, and at the level of reproductive stem cells or fertilized eggs, the production and development of individual reproductive cells are prevented by means of knocking out genes or introducing antagonistic genes. The development of gene editing in the preparation of mammalian infertile hosts is more widespread [9]. Avian PGCs, the precursors for ova and sperm, have unique migration characteristics. They are initially localized in the central region of the area pellucida at the oviposition stage. With the development of the embryo, they first migrate to the germinal crescent, then circulate through the blood vessels, and finally reach the genital ridge [37]. These migratory properties enable the manipulation of PGCs to produce transgenic chickens [38]. Furthermore, and exhilaratingly, genes controlling germ-cell formation, development, and migration in poultry have been identified, including the DEAD box helix 4 gene (DDX4, also known as chicken vasa homologue, CVH) and deleted in azoospermia-like (DAZL). With the advances in genome-editing technologies, it is possible to generate genome-edited sterile chicken recipients by knocking out these genes [39].

Vasa, a DEAD box RNA helicase, is essential for proper germ-cell formation in multiple species. DDX4 marks the germ-cell lineage at the early stages of embryonic development and is hypothesized to be a determinant in oogenesis in chickens [40]. Taylor et al. and Aduma et al. used TALE nuclease (TALEN) technology to knock out DDX4 [41,42]. In the DDX4 Z^-W embryonic gonads, the number of PGCs decreased with increasing embryonic age, and the ovaries of chicks did not contain germ cells [41]. Artificial insemination of the DDX4 Z^-W layer hosts with cryopreserved semen produced offspring completely derived from donor-heritage PGCs [43]. The high efficiency of line restoration will greatly reduce the workload and improve the safety of germplasm conservation. However, the DDX4 sterile recipients must be produced by heterozygote Z⁺Z⁻ males and wild-type females. The probability of producing suitable hosts (Z^-W) is only one fourth. Subsequently, the Roslin Institute utilized caspase/cas9 gene-editing technology and produced a more promising iCaspase 9 sterile-recipient system based on DDX4 receptor chickens [44]. Surprisingly, the iCaspase9 line produced a larger proportion of donor/host chimeras than the DDX4 line, which was able to stably pass on endogenous transgenes to its offspring.

DAZL protein is a germ-line-specific RNA-binding protein essential for gametogenesis [45,46]. Ballantyne et al. produced a surrogate host-chicken line in which the germ-cell lineage can be conditionally ablated by indirectly knocking down this gene. Mediated by CRISPR/Cas9, an inducible caspase-9 gene and GFP are recombined into the coding exon of the DAZL locus, which may lead to the death of apoptotic cells. Donor PGCs are introduced into the sterile male and female host embryos to produce adults chickens that only carry exogenous germ cells. Their direct mating recreates the donor breed in a single generation [44]. The strategy vastly enhances the efficiency of gene editing and breed reconstruction using cryopreserved germplasm. Given favorable circumstances, simple experiments could profitably be conducted to verify whether these gene-knockout methods can interfere with the differentiation of endogenous germ cells, and whether the established model can support stem-cell transplantation.

Although the costs are relatively high and technical support is needed, gene editing produces better sterilization of individuals and does not affect the genotype transmission of donor offspring. Gene editing of chickens has the most potential and the best comprehensive effects on strain-transmission rates and survival rates. Strict policies on genetically modified organisms (GMO) and even the complete prohibition of GMO animals may be an obstacle to the application of this technique [47].

3. Concluding Remarks

As described above, there have been multiple options for creating infertile host chickens, depending on transplantation materials, variations in cost, and technique requirements (

Table 1. Comparison of preparation methods for sterile recipients.

Method		Materials	Operation Difficulty	Cost	Application
Ovariectomy and testectomy		Day-old chicks	↑	↓	Gonad translation in day-old chicks
Busulfan		Stage HH 1–4 embryos	↓	↓	PGC translation in stage HH 14–16 embryos SSC translation in day-old chicks and mature roosters Gonad translation in day-old chicks
		Mature roosters	↓	↓	PGC translation in mature roosters SSC translation in mature roosters
Irradiation		Stage X and stage HH 14 embryos	↓	↓	PGC translation in stage HH 14–16 embryos SSC translation in day-old chicks and mature roosters Gonad translation in day-old chicks
		Mature roosters	↓	↓	SSC translation in mature roosters
Gene editing	DDX4	Cultured PGCs	↑	↑	PGC translation in stage HH 14–16 embryos Gonad translation in day-old chicks
	iCaspase9	Cultured PGCs	↑	↑	PGC translation in stage HH 14–16 embryos Gonad translation in day-old chicks SSC translation in mature roosters

* The description of embryo stages follows the method proposed by Hamburger and Hamilton [49].

). Compared with chicks, adult chickens experience severe immune rejection in the process of tissue transplantation. Therefore, the recipients required for gonadal transplantation can be obtained almost all preparation methods except for the sterilization of adult chickens; however, the gonadectomy method is only valid for gonad transplantation. There are still some uncontrollable factors in the preparation of sterile recipients. For example, strong chemical toxicity or physical stimulation may result in a lower fertilization rate, hatching rate, and survival rate. In the gene-knockout method, the outcome of the knockout of target genes on the growth and development of the individual is not clear enough. Many studies have indicated that there are no off-target mutations [48]. In all evaluations of preparation methods, the germline-transmission rate should be the main consideration, as it represents the transmission of donor genotypes. Factors such as survival rate, fertilization rate, or hatching rate can be used as the criteria for evaluating the methods. The preparation technology for sterile recipients for poultry is becoming more mature and enhancing the efficiency and safety of the conservation of cherished poultry species.