Peak Abundance and Developmental Dynamics of Circulating Primordial Germ Cells in Cemani Chicken (Gallus gallus domesticus) Embryo

Multipotent stem cells known as primordial germ cells (PGCs) can give rise to either oogonia or spermatogonia [1]. These PGCs play a crucial role as progenitor cells, transmitting essential genetic information to subsequent generations [2]. When it comes to chicken PGCs, they exhibit distinctive developmental characteristics, beginning in the epiblast and subsequently migrating toward the germinal crescent [3]. These cells circulate through the circulatory system, eventually settling in the germinal ridges and colonising the developing gonadal anlage [4, 5]. Although PGCs can be readily isolated from the embryonic bloodstream, it is important to note that their numbers tend to be limited during the circulation period [6]. Therefore, it is imperative to consider this limitation while conducting further investigations and experiments.

Previous investigations have demonstrated that the quantity and concentration of circulating PGCs (cPGCs) in chick embryos vary significantly, with notable differences observed at various stages of embryogenesis and among embryos [7, 8]. De Melo Bernardo et al. [4] conducted a different study that, in striking contrast, found a remarkable consistency in the number of cPGCs during stages 5 to 19. Stages 5 to 19 include the time from cPGCs presence at the germinal crescent to their arrival at the vaginal ridge.

Cemani chicken is an Indonesian breed of chicken that originated in Temanggung, Central Java (Figure 1) [9]. Cemani chicken, especially in the Javanese Islands, is not only used in ancient ceremonies but also as traditional medicine for cardiovascular and respiratory illnesses [10]. Furthermore, Cemani chickens play a significant role in Bali, as they are used in cockfighting, a long-standing custom ingrained in the island's culture [11]. In addition, the Cemani chicken is recognised as an attractive breed in Europe, lending a touch of grace and beauty to poultry fans [12]. Cemani chicken has secured its status as a highly sought-after and appreciated breed due to its numerous applications and widespread recognition. Due to its cultural and economic value, the Cemani chicken has potential as a biological model for the study of germ cell migration and reproductive biotechnology applications. However, information on the temporal dynamics of PGCs in Cemani embryos is still limited, making this research crucial to fill this knowledge gap and support conservation and utilisation efforts for local germplasm. Furthermore, studies on the peak abundance and developmental dynamics of PGCs in Cemani embryos are expected to provide a scientific basis for more efficient germ cell isolation.

Figure 1. Cemani chickens

Based on the above information, this study aims to identify the peak period of PGCs abundance in the circulating blood of Cemani chicken embryos during early development and to analyse the migration of germ cells to the recipient. Thus, this study provides a scientific basis for more efficient cPGCs isolation strategies and contributes not only to a fundamental understanding of developmental biology but also has practical relevance in reproductive biotechnology and genetic conservation.

3.1 Collection of cPGCs

The collection of cPGCs occurs at stages 14-18, encompassing an incubation period of 55-69 hours. Within the embryo's circulatory system, two distinct cell types exist. cPGCs are distinguished by a larger diameter (ranging from 12 to 16 mm) than red blood cells (approximately 8 mm) at the corresponding stage. This size difference is a key feature used to identify and isolate cPGCs during embryonic development [16]. In addition, collecting cPGCs from the bloodstream of adult chickens is impossible because cPGCs have differentiated into precursor cells that generate egg cells or sperm.

The morphology of cPGCs in Cemani chickens is readily identifiable, as they are round with a textured surface, rendering them larger than red blood cells when observed under a microscope at 20× magnification (Figure 5) [17]. The cPGCs of Cemani chickens closely resemble those reported in previous studies [14, 18] for Gaok and Chinese Meiling chickens, respectively. In addition to their size, cPGCs usually have numerous small cytoplasmic spikes projecting from the cell surface and an abundance of refractive granules in the cytoplasm.

Figure 5. Morphology of circulating primordial germ cells (cPGCs) from Cemani chicken embryos under light microscopy (20×)

Blue arrows are cPGCs, and red arrows are other cells.

Cytoplasmic spikes are believed to be involved in cell adhesion and migration, which are crucial for the movement of cPGCs to the developing gonads. Refractive granules indicate high metabolic activity and the presence of specific proteins and organelles that are essential for the development and differentiation of germ cells [19]. Cemani chickens may have more varied core characteristics, whereas commercial chickens may have more uniform cores due to selective breeding for certain traits.

3.2 Number of cPGCs in the embryo's blood circulation

Optimal collection of cPGCs was observed when PGC harvesting occurred at stage 15 of embryonic development (Figure 6). Notable differences were observed when cPGCs were harvested at different stages (p < 0.05). A disparity in embryonic development was demonstrated between stages 14, 16, 17, and 18 (p < 0.05). These findings suggest that the quantity of cPGCs can be influenced by embryonic development.

Figure 6. Average cPGCs yield per embryo in Cemani chickens

The average number of cPGCs varies across stages of embryonic development. At stage 15, cPGCs can actively migrate through the bloodstream, and their number subsequently declines beyond that stage. This investigation has substantiated that cPGCs in Cemani chickens decrease after embryos reach stages 16, 17, and 18. With this reduction at this stage, they are likely to have exited the bloodstream, as they exist only briefly. Subsequently, they aggregate into colonies within the germinal ridges. Consequently, when obtaining blood containing cPGCs, collection was suboptimal, likely due to challenges in isolating cPGCs and variations in their number across different developmental stages [20]. Comparable findings were observed in White Leghorn, Kureko Dori, Barred Plymouth Rock, and Silkie chickens [21-24], where it was ascertained that as the embryo ages, the number of cPGCs that can be isolated diminishes.

The average number of cPGCs in Cemani chicken embryos peaked around stage 15 (25.53 ± 11.13 cells/μL) because this stage provides an optimal window for isolating and collecting cPGCs, as they are abundant and accessible in the embryo's blood vessels. Stage 15 is considered safe for isolation and preservation [24]; a similar result was observed in Gaok embryos [14]. Conversely, in Green-legged Partridge-like chick embryos, the peak occurred during stage 14 (18.42 cells/μL) [17]. A prior study [4] reported that cPGCs continuously migrate from the germinal crescent to the embryo's bloodstream (stages 11–16), which explains the differences in results. Cemani chickens might have cPGCs that are more dispersed throughout embryonic development, while commercial chickens may exhibit a more concentrated distribution due to selective breeding for reproductive efficiency.

Embryonic development may influence differences in PGCs characteristics, as reported by Dunislawska et al. [25], who found significant differences in PGCs characteristics on day 8 of embryonic development, with faster rates in Green-legged Partridge-like chickens than in White Leghorns. Genetic factors and migration efficiency are additional factors that influence PGCs characteristics, as genetic differences can affect the efficiency and success of this migration, ultimately shaping germline formation [26]. This is reinforced by Tagami et al. [26], who found that genetic factors play a significant role in PGCs migration and PGCs number [27].

The findings reveal a contrast in chick embryos, specifically in the Green-Legged Partridge, which reaches its peak at stage 14 (18.42 cells/μL) [28]. This discrepancy could be attributed to the breed of chickens, as the cPGCs in native chickens are reported to be fewer than those in commercial chickens [28]. Numerous investigations conducted thus far have employed purebred chickens, for instance, the White Leghorn, among others [29]. Furthermore, cPGCs are influenced by egg production performance, as highlighted by the study [30] in relation to laying and broiler hens. The weight of the egg also influences the number of cPGCs; a larger egg weight results in more cPGCs. This correlation is supported by a regression analysis of egg weight, which reveals a strong association between duck egg weight and the number of cPGCs, as observed by the researcher [31]. It turns out that not only female factors, but also the male factor that provides sperm influences the total number of cPGCs in different individuals [27].

From all that has been mentioned before, the factors that influence the success of cPGCs isolation are: (a) stage of embryonic development, as the concentration of cPGCs in the blood varies with embryonic development [20, 28]; (b) breed variations, perhaps within the same breed of chicken there may be significant variations in cPGCs concentrations [28]; (c) isolation method, the cPGCs isolation method greatly influences the success of cPGCs isolation [20, 28]; (d) purification and quantity, the ability to obtain cPGCs with high purity from blood is influenced by the success of isolation [20]; and (e) genetic, the total number of cPGCs may be lower in local or endangered chickens compared to commercial breeds [28].

3.3 Injection and migration cPGCs

Gonad migration efficiency in Cemani chicken embryos varies across five developmental stages (stages 14–18). The highest success rates were observed at stages 15 and 16 (each at 85%), followed by stage 17 (80%), stage 14 (75%), with a marked decline at stage 18 (65%) (Table 2). The consistent outcomes at stages 15–16 indicate that this developmental window is an optimal time for tracking cPGCs migration in Cemani chickens.

Table 2. Efficiency of cell migration in the embryo gonad recipient, using PKH26 labelling at stages 14-18

PGCs undergo a complex migration to reach the developing gonad, as this migration is critical and governed by intricate interactions between somatic cells and the surrounding microenvironment [32], making early tracking essential for understanding PGCs migration. In avian species, PGCs utilize the bloodstream for migration and subsequently extravasate to the gonadal ridge, where further colonization occurs [33].

The reduction in efficiency observed at stage 18 is likely attributable to increased gonadal tissue complexity and potential microstructural changes, which may hinder label penetration or reduce target visibility. Consistent with this, Furuta et al. [34] reported that PGCs can successfully migrate to the gonads of recipient embryos around stages 13–16.

In addition, the study by Tagami et al. [26] emphasized that molecular intervention and germ cell tracking are most effective when performed before stage 18, as subsequent gonadal tissue reorganisation may affect label distribution. Therefore, these findings further support that stages 15–17 represent an ideal window for PGCs migration-tracking studies and genetic interventions in local chicken embryos, such as Cemani. Accordingly, the migration and proliferation capacities of cPGCs in the recipient gonads appear to be highly variable.

Figure 7. Detection of PKH26-labelled cPGCs in the gonads of recipient embryos, observed under (A) a light microscope and (B) a fluorescence microscope, at 20× magnification

To assess migration, donor cPGCs were labeled with PKH26 before transferring to recipient embryos. Figure 7 illustrates PKH26-labelled cPGCs in the gonads of recipient embryos at the post-injection stage. Figure 7(A) presents a 20× magnification light microscopy image, where the gonadal cellular structure is clearly visible but without fluorescence. In contrast, Figure 7(B) shows a 20× fluorescent microscopy image, featuring bright red dots throughout the gonad tissue. These fluorescent dots confirm the presence of cPGCs, successfully tagged with PKH26, at a scale of 20 µm.

Fluorescent detection of PKH26-labelled cPGCs in the recipient embryos' gonads confirmed that the isolated and labelled cells successfully migrated and colonized the target site. The presence of a bright red signal under fluorescent microscopy (Figure 7(B)) indicated that the PKH26 labelling method was effective for tracking cPGC distribution.

PKH26 is a lipophilic fluorochrome that intercalates into the cell membranes and has been widely used for cell tracking in various biological systems [35]. Its effectiveness in migration and cell colonization studies has been demonstrated across multiple models, making it a reliable tool for long-term cell labeling [36].

The absence of a detectable signal in the light microscopy image (Figure 7(A)) further confirms that fluorescent detection is required for the specific identification of labelled cells. This finding supports the efficiency of the injection and labelling technique employed in cPGCs migration and colonization studies and highlights their potential for further applications in germline engineering and avian genetic conservation.

The successful detection further indicates that the observation timing and sampling locations were appropriately aligned with the physiologically established migration pathway of cPGCs. Accordingly, this approach can be integrated into germ cell tracking protocols for advanced studies investigating the migration competence and colonization capacity of donor cells.

In avian reproductive biology, studying primordial germ cells is crucial. Examining the occurrence and concentration of PGCs in Cemani chickens is of utmost importance, as it helps understand their reproductive capabilities and supports conservation efforts.