Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.


INTRODUCTION
Indonesia has around 43 breeds of local chickens (Sartika et al., 2016). Some of the local chicken populations are very limited in number and even many of them are in the status of endangered species. Some of them have potentially high benefit that have to be preserved in-situ or ex-situ. Kampung Unggul Badan Litbang Pertanian (KUB) chicken is one of the genetic resources of Indonesian Kampung chicken breeds, selected for improved egg production conducted at the Indonesian Research Institute for Animal Production. KUB chickens are able to produce eggs about 50% hen-day production, reaching peak productions of 65%-70% (Sartika et al., 2016). As a new breed, KUB chicken may be categorized as a new genetic resource.
Preservation can be divided into in-situ preservation and ex-situ preservation. The primary objective of preservation is to ensure the survival of threatened species and the maintenance of associated genetic diversity. In-situ or ex-situ preservation is a form of the living individual or population that needs high costs and risks associated with the disease. To solve the problem, it is necessary to develop a preservation strategy through the cryopreservation of genetic material, such as the storage of sperm, ova, and embryos in the frozen form (cryopreserved). However, the method of genetic resource preservation cannot be applied to poultry species primarily because of the technical difficulties on the cryopreservation of poultry eggs. The approach was developed to overcome the current limitations of in-situ and ex-situ preservations of genetic resources by collecting and transferring avian primordial germ cells (PGCs), collected from developing embryos.
Primordial germ cells are the progenitor cells for gametes that colonize the developing gonad and subsequently differentiate into spermatogonia in the testis or into oogonia in the ovary (Kohara et al., 2008;Qian et al., 2010;Glover & McGrew, 2012). Chicken PGCs are located at the center of the pellucida area . At the primitive streak stage, PGCs locates at the extra embryonic region that called germinal crescent region (Nakamura et al., 2007). As soon as the blood vessel is formed at stage 10 of Hamburger & Hamilton (HH) (1951), The PGCs from the germinal crescent start to circulate temporarily through the embryonic bloodstream on day 2 of incubation. The cells leave the capillary system and migrate to the form of genital ridge, where they colonize the developing gonad (Minematsu et al., 2004;Glover & McGrew, 2012). After migrating to the gonadal anlage, the PGCs are called gonadal germ cells (GGCs) (Nakajima et al., 2011). Gonadal germ cells or gonadal primordial germ cells (gonadal PGCs) in the gonad, ultimately differentiate into spermatogonia, which takes place in the testes or into oogonia, which takes place in the ovaries. Primordial germ cells are cells with pluripotent characteristic making the PGCs as a good model to study embryonic development in-vitro (Wang et al., 2010).
Numerous studies had reported the isolation and collection of PGCs from gonadal embryos (Nakamura et al., 2011;Nakajima et al., 2011;Nakajima et al., 2016). Moreover, germline chimeras had been produced in chicken by transferring PGCs or GGCs into the bloodstream at 2-day-old recipient embryos (Furuta et al., 2007(Furuta et al., , 2008 so that PGCs could be used as a genetic resource to produce germ line chimera (Furuta, 2012). Nakajima et al. (2012) reported that gonadal PGCs collected from 7-day-old chick embryos and incubated in PBS [-] can be used to produce germline chimeras. It has been reported that gonadal PGCs are used to generate interspecies germline chimeras by transferring pheasant gonadal PGCs into chicken embryos (Kang et al., 2008) and by transferring chicken gonadal PGCs into duck embryos (Liu et al., 2012).
One of the problems with the PGCs collection of embryonic blood at the age of 2.5 days is the low number of cells found in Indonesian local chicken (Kostaman et al., 2013). Therefore, to obtain more harvested PGCs, collection of PGCs from embryonic gonad can be used as an alternative method. There are several methods used to isolate avian gonadal PGCs, one is by using proteinases, such as trypsin solution Kohara et al., 2008) and the other is by using a phosphate-buffered saline without Ca 2+ and Mg 2+ solution (PBS [-]) (Nakajima et al., 2011). The present study was conducted to apply this method to isolate gonadal PGCs from KUB chicken embryos by using phosphatebuffered saline without Ca 2+ and Mg 2+ solution (PBS [-]) (Nakajima et al., 2011). The main objective of this study was to determine the number of gonadal PGCs of KUB chicken at different developmental stages of embryos.
The results of the research are expected to be basic information for determining the appropriate stage for the isolation of gonadal PGCs.

Preparation of Fertilized Eggs
Sixty fertilized eggs of KUB chickens, produced at the chicken research laboratory of Indonesian Research Institute for Animal Production, Bogor-Indonesia, were used in this research for gonads collection. Those eggs were divided into 4 treatment groups based on embryonic development on days 6, 7, 8, and 9 of incubation to obtain embryos at stage 29-35 of HH. The collected eggs were incubated at 38 o C with humidity of 60% and rotated 90 o every 30 min in a portable incubator (P-008B Biotype; Furanki Showa, Saitama, Japan). The harvesting of embryos for each group was based on the age of embryonic development on days 6 to 9.

Isolation and Collection of Gonadal PGCs
Gonadal PGCs were isolated on days 5.5-6 or more than 6 days of embryonic gonads (stage 26-28 HH) (Chojbacka-Puchta et al., 2012). In this study, gonadal PGCs were collected from the right and left gonads of KUB chicken embryos at the age of 6-9 d. Each embryo was separated from yolk and placed in a petri dish of 90 x 15 mm of LBS60001PT, BIOLAB. Thereafter, the embryos were rinsed with PBS [-]. The abdomen of the embryos and their contents were carefully dissected under microscope and the gonads were collected using sharp tweezers (Dumont No. 5L4 ¼ in., Inox alloy, Sigma Aldrich/F6521-1EA). The collected gonads were placed in an Eppendorf tube of 1.5 mL containing 500 µL PBS [-]. The primordial germ cells were then collected using PBS [-] solution. The gonads were incubated using PBS [-] for one hour (Nakajima et al., 2011). The morphological characteristics and the total number of gonadal PGCs of KUB chicken were observed microscopically.

Statistical Analysis
The total number of gonadal PGCs of each embryonic development at the ages of 6, 7, 8, and 9 d were analyzed statistically using Completely Randomized Design with 15 replications.
Note: Y ij = any observation for which X 1 = i µ = general location parameter t i = effect of having treatment level i e ij = random error Data were analyzed by ANOVA after their normal distribution was analyzed by Kolmogorof-Smirnov Z. The differences among groups were examined by Duncan's multiple-range test.

KUB Chicken
Embryonic gonad is generally attached to the mesonephros (Figure 1). For the PGCs isolation, embryonic gonads should be separated from mesonephros attachment. Results under microscopic observations showed that gonadal PGCs that were released from the gonads reached a high number in 1 h after incubation of the gonads (Figure 2). This results show that gonadal  The results were in line with the results reported by Nakajima et al. (2011;2013)

in White Leghorn (WL) and
Rhode Island Red (RIR) chickens. The result also confirmed that PGCs isolation from gonad was easier and better with larger number of collected PGCs compared to the isolation of circulated PGCs.
Gonadal PGCs of KUB chicken had morphological characteristics: (1) large cell size, (2) bright lipid droplet distributed in the cytoplasm (3) spherical and large nucleus and located asymmetrically, (4) peripheral bright ring surrounding the cytoplasmic membrane. More details about KUB chicken PGCs characteristics are summarized in Table 1.

Number of Gonadal PGCs
The number of gonadal PGCs per embryo isolated from embryonic development on days 6, 7, 8, and 9 of incubation period (stage 29-35 of HH) are presented in Table 2. Chojbacka-Puchta et al. (2012) reported that PGCs could be collected from embryonic gonad on days 5.5 to 6. Further result of Minematsu et al. (2004) showed that PGCs began to migrate to the embryonic gonad starting on day 5 after incubation and subsequent PGCs began to play a part in the gonad between days 6 and 7 (at the onset of sexual differentiation) and sexual differentiation of the gonads were occurred after the arrival of germ cells. Meanwhile, germ cells multiplications are occurred at a later stage. Germ cells differentiation phase in female embryos took place between days 8 and 11 of incubation, whereas in male embryos, germ cells differentiation took place between days 13 and 15 of the incubation period.
In this study, gonads sampling of embryos was conducted on day 6 of incubation period, since most of PGCs had left the blood vessels and had begun to arrive in the gonadal anlage (Han, 2009). Collection of embryo gonads was terminated on day 9 of incubation because the PGCs remained stayed in the gonads. After that period, PGCs gradually differentiated into sex cells.

KUB Chicken
The results of morphological characteristics of PGCs were consistent with the results reported by Kostaman et al. (2013) in Gaok chicken, Minematsu et al. (2004) in RIR chicken, Nakajima et al. (2016) in Oriental stork and Northern Bald ibis, and Sopiyana et al. (2016) in KUB chicken using the lysis buffer ACK method.

Number of Gonadal PGCs
Based on the results of previous studies, one of the problems in the PGCs collection of embryonic blood on day 2.5 was the low numbers of cells in local chickens that could be collected (Kostaman et al., 2013). On the contrary to those studies, our results revealed that the PGCs collection from days 6-9 yielded more PGCs compared to the collection method conducted on day 2.5. The highest number of gonadal PGCs (P<0.05) was obtained in the embryonic development at the stage of day 7 among the number of gonadal PGCs collected on days 6, 8, and 9. This result showed that the number of gonadal PGCs was apparently influenced by the age of embryonic development.
Starting on day 6 of embryonic development, the number of gonadal PGCs significantly increased (P<0.05) and reached the peak number of gonadal PGCs on the embryonic development on day 7, which was probably that all PGCs might already reach the embryonic gonad as reported by Hong et al. (1995). Furthermore, this opinion was supported by Nakamura et al. (1988) that the PGCs had mostly settled down in the gonadal ridge on the day 7 th of the incubation period.
The number of PGCs slowly decreased (P<0.05) on days 8 th to 9 th incubation period. The reason of the decreased number of PGCs on the day 8th of incubation might be due to the start of PGCs to differentiate sexually to be a female embryo (Hong et al., 1995). Furthermore, PGCs began the meiotic division actively forming oogonia on days 8-9 of the incubation period (Van Krey, 1990).
Collection of PGCs from avian embryonic gonad, especially in Indonesian chickens, had not been done so far, so this preliminary study can be applied for genetic material preservation technology of embryonic gonad PGCs on Indonesian local chicken, especially for KUB chicken. The advantage of the collection of PGCs using PBS [-] has also been reported by Nakajima (2011)   embryo at embryonic age of 7 d. Nakamura et al. (2011) harvested PGCs from gonads of WL chicken at the age of 7 d and obtained 237 cells per embryo. This is the first report about gonadal PGCs in Indonesian local chicken, particularly in KUB chicken, although has lower number of PGCs compared to the number of PGCs resulted from exotic chicken as reported previously by Atsumi et al. (2008), Nakajima et al. (2011), andNakamura et al. (2011). However results of this study were still comparable with the results reported by Nakajima et al. (2016) on Oriental stork and Northern Bald ibis with the averages number PGCs as much as 130 and 160 cells. The lower number of gonadal PGCs was influenced by the type of chicken (Zhao et al., 2003). The results of this present study indicated generally that KUB chicken as one type of Indonesian local chicken had lower number of PGCs compared to exotic chicken. This lower number of PGCs seems likely to be correlated with the lower productivity of local chicken, as was also stated by Zhao et al. (2003) that the number of chicken PGCs correlated with the number of eggs.

CONCLUSION
The highest number of gonadal PGCs of KUB chicken was obtained on day 7 of incubation period and it can be recommended that the best period time to isolate gonadal PGCs is on day 7.