
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

71804
10.1038/s41598-024-71804-6
Article
Improved surrogate eggshell incubation system for assisted reproductive technologies such as cloning, genome modification, and de-extinction efforts
Yang Hyeon 12
Lee Bo Ram 1
Lee Seunghoon 1
Ock Sun A. 1
Oh Keon Bong 1
Jo Yong Jin 1
Lee Poongyeon 1
Lee Haesun 1
Han Jae Yong 2
Byun Sung June pcs1778@korea.kr

1
1 grid.420186.9 0000 0004 0636 2782 Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, 55365 Republic of Korea
2 https://ror.org/04h9pn542 grid.31501.36 0000 0004 0470 5905 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826 Republic of Korea
5 9 2024
5 9 2024
2024
14 207962 6 2023
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
A germinal disc located on the egg yolk surface drives genetic modification. Windowed and surrogate eggshell incubation methods have been developed, but these exhibit limited abilities to generate transgenic chickens. In the present study, we investigated the frequency of observing the germinal disc according to the preincubation positioning direction and time and found that it depended on those conditions, but only a few chicks (2.8–5.6%) hatched using the windowed method. Then, we attempted to improve surrogate method via one- or two-step procedures. All eggs, including surrogates that were 10 g heavier than the donor eggs, were obtained from a poultry flock of the same age. With the one-step surrogate method, where the donor egg was transferred only once through a 3.5 cm hole on the point end, at the first day of preincubation, into the surrogate egg, the survival rate at day 4 was 30.8%, and the hatching rate was 11.8%. With the two-step surrogate method (transfer was on the 1st and 4th day of incubation), the survival rate at day 4 was improved to 90.7%, and a hatching rate of 70.0% was achieved. Therefore, this method can be effective for in ovo artificial incubation.

Subject terms

Biological techniques
Biotechnology
Developmental biology
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Germinal disc at Eyal-giladi and Kochav (EGK) stage X appears on the surface of the egg yolk, and eggshells make it physically impossible to directly observe and approach the disc. Thus, many attempts based on in ovo embryonic incubation have been conducted to reach the disc and modify the genome in chicken embryos1.

A windowed eggshell incubation system, which involves artificially making a 1-cm-diameter hole on top of eggshells and then covering it with sealing material, is a simple method2. On the other hand, a surrogate eggshell incubation system is a relatively complex method that involves transferring the egg yolk into surrogate eggshells once or twice. In the surrogate method, two types of surrogate eggs (the first chicken and second turkey eggs are approximately 4 g and 40 g heavier than the donor eggs, respectively) are used in System II and III3,4. Based on the windowed and surrogate eggshell incubation systems, assisted reproductive technologies can have potential in facilitating and enhancing via visualizing and enucleating the avian ovum, a crucial step in developing cloning birds, genome-modified chickens, and even in de-extinction efforts5,6.

The hatching rates by the windowed method have been improved by the development of various strategies7,8. However, few studies regarding location of the disc based on imaging methods are available9–12. Moreover, it is still difficult to determine the location of the disc in freshly laid fertile eggs within the 1-cm-diameter hole, which results from uncontrolled movement of egg yolk. Additionally, the surrogate method, which shows impressive hatchability4, is limited by the requirement of a second surrogate egg. In case of using the second surrogate egg obtained from chickens, the surrogate egg which is at least 25–30 g heavier than donor egg was required in System III13,14. These very large eggs must be obtained from other poultry flocks. However, the eggs externally obtained from other flocks are not perfectly free of avian transmittable and transovarian transmitted diseases such as Salmonella15 and mycoplasma16.

In this study, we investigated the frequency of observing the disc in fertile chicken eggs at EGK stage X based on positioning direction and time, and hatching performances according to preincubation positioning direction and time in the windowed method were evaluated. We then attempted to develop an improved surrogate eggshell method using surrogate eggs that 10 g heavier than donor eggs, obtained from a poultry flock of the same age.

Results

Germinal disc and chalaza position investigation

Shortly after the eggs were positioned blunt- (Group 1) and point-end up (Group 2), germinal discs and chalazas were observed through the blunt- (Group 1) and point-end holes (Group 2) created at the top of the eggshells (Fig. 1a). The frequency of observing germinal discs in the Group 1 eggs (41.2%) was significantly higher (p < 0.05) than that in the Group 2 eggs (3.8%). In contrast, the frequency of observing chalazas was significantly higher (p < 0.05) in Group 2 (51.7%) than in Group 1 (21.7%) (Fig. 1b). Additionally, the frequencies of observing germinal discs and chalazas in the eggs positioned blunt- (Group 1), point- (Group 2), and side-end up (Groups 3 and 4) for 24, 48, 72, and 96 h through blunt- (Groups 1 and 3) and point-end holes (Groups 2 and 4) were observed. As a result, the frequencies of observing discs and chalazas were dependent on the positioning direction and time (Fig. 1c). The frequency of observing discs in the eggs positioned for 48, 72, and 96 h were significantly higher (p < 0.05) in Group 1 (71.2, 78.1, and 82.3%) than in Group 2 (35.7, 44.4, and 70.6%), respectively. In contrast, germinal discs were almost not observed in any of the eggs in Group 3 and 4 after 24 h, and chalazas were mainly observed in the blunt- (Group 3) and point-end holes (Group 4). Thus, the positioning of the fertile eggs can affect the movement of the germinal disc together with the egg yolk, and the disc was rarely observed in the blunt- and point-end holes created in the eggs that were positioned side-end up.Fig. 1 Frequency of observing the germinal disc and chalaza. (a) Freshly laid fertile eggs positioned blunt- and point-end up allowed observation of the germinal disc (GD) or chalaza (CHL) at the blunt and point end. Scale bars: 1 cm. (b) The frequency of observing the GD and CHL through blunt- (Group 1) and point-end (Group 2) holes in freshly laid fertile eggs shortly after positioning blunt- (Group 1) and point-end up (Group 2) was investigated. The error bars represented the means ± standard errors of the mean (n = 3). **p < 0.01 and *p < 0.05 for the comparison between Groups 1 and 2. (c) The GD and CHL were observed through blunt- (Groups 1 and 3) and point-end holes (Groups 2 and 4) in the eggs positioned blunt- (Group 1), point- (Group 2), and side-end up (Groups 3 and 4) for 24, 48, 72, and 96 h. The error bars represented the means ± standard errors of the means (n = 3). a,bMeans with different superscripts are significantly different (p < 0.05).

Windowed incubation

Because the germinal disc was not observed through the blunt- and point-end holes in the eggs positioned side-end up, the discs of the eggs positioned blunt- (Group 1), point- (Group 2), and side-end up (Group 3) for 24 h and 96 h were additionally observed through artificially generated blunt- (Group 1), point- (Group 2), and side-end holes (Group 3). As a result, the discs were observed through blunt-, point-, and side-end holes in eggs positioned in the same direction. The frequencies of observing the germinal disc in the Group 1, 2, and 3 eggs positioned for 24 h were 45.5, 16.1, and 66.7%, respectively, and the frequencies for 96 h were 73.7, 52.6, and 84.7%, respectively (Fig. 2a). If the germinal disc could be observed through the hole, the eggs were sealed with parafilm and incubated with the control group. As a result, only a few chicks in Group 1 hatched, and poor hatching rates in Group 1 for 24 h (5.6%) and 96 h (2.8%) were obtained, respectively (Fig. 2b). Thus, the windowed method specifically used in this study was not effective for incubating the embryos of fertile eggs at EGK stage X.Fig. 2 Performance of the windowed eggshell incubation method based on the egg position. (a) The GD was observed through blunt- (Group 1), point- (Group 2), and side-end (Group 3) holes in the eggs positioned blunt- (Group 1), point- (Group 2), and side-end up (Group 3) for 24 and 96 h. The error bars represented the means ± standard errors of the means (n = 3). a,bMeans with different superscripts are significantly different (p < 0.05). (b) If the disc was observed through the hole, the egg was sealed and incubated, and the hatching performance was evaluated. The error bars represented the means ± standard errors of the means (n = 3). ***p < 0.001 for the comparison among all groups.

One-step surrogate eggshell incubation

Due to low hatching performance obtained with the windowed method specifically used in this study, we attempted to obtain an improved surrogate eggshell incubation method without using externally obtained eggs. Therefore, we established a one-step surrogate eggshell incubation method. In the one-step surrogate method used in this study, the egg yolk with blastoderm and thick and thin albumen was transferred into a surrogate eggshell only once on day 1 of preincubation, and the surrogate egg was approximately 10 g heavier than the donor egg. With the surrogate method, embryonic survival can be monitored daily (Fig. 3a), and as a result, the survival rates of the donor eggs at day 4 were relatively low (30.8%) compared with those obtained with the previously established surrogate eggshell incubation method14. However, approximately half (15.6%) of the live embryos that developed to Hamburger and Hamilton Stage 17-to-18 (HH stage 17-to-1817) were still alive at day 21 (Fig. 3b), and the hatching rate at day 22 was 11.8% (Fig. 3c). Thus, we confirmed that the embryos alive at day 4 may survive sufficiently and hatch in the unusual surrogate eggs, 10 g heavier than the donor eggs, used in this method.Fig. 3 Performance of the one-step surrogate eggshell incubation method. (a) The donors were transferred into surrogate eggshells only once on day 1 of preincubation in the one-step surrogate method, and the embryonic survival was monitored daily. Live (upper) and dead embryos (lower) at days 4, 12, 17, and 19 are presented. Scale bars: 1 cm. (b) The embryonic viability was evaluated at each time point (days 2–4, 5–12, 13–18, and 19–21). The error bars represent the means ± standard errors of the means (n = 5). (c) The hatching performances of the one-step surrogate method were evaluated. The error bars represent the means ± standard errors of the means (n = 5). ***p < 0.001 for the comparison between the control and surrogate groups.

Improved surrogate eggshell incubation

Because the embryos that were alive at day 4 could develop and hatch in the unusual surrogate eggs, additional strategies were investigated to improve the survival rates of the donors from EGK stage X to HH stage 17-to-18. We ultimately developed an improved two-step surrogate eggshell incubation method that consisted of System II (similar to the previously established protocol14) and System III (using second surrogate eggs that were 10 g heavier than the donor eggs). The transfer of donor eggs at day 4 into second surrogate eggshells was reportedly difficult, and yolk burst was confirmed immediately after transfer (Fig. 4a). In this study, the transfer was conducted by tilting the donors in the first surrogates into the empty second surrogate eggshells, and the transfer success rates (80.1%) were significantly higher (p < 0.05) than the failure rates (19.9%) (Fig. 4b). The survival rates of the donor eggs at day 4 were improved to 90.7% (Fig. 4c), and the hatching rates were improved to 70.0% (Fig. 4d). Additionally, we injected lentivirus particles driving expression of enhanced green fluorescence protein (EGFP) into germinal disc of freshly laid fertile eggs, and eggs were incubated with the improved two-step surrogate method. Then, the EGFP signal was confirmed in embryonic body at day 7 (Fig. 4e). Therefore, the improved surrogate method showed the possibility of obtaining adequate survival and hatching performances without using externally obtained large surrogate eggs.Fig. 4 Performance of the improved two-step surrogate eggshell incubation method. (a) Egg yolk burst causing death at the early phase can be confirmed immediately after transferring the donors into the second surrogate eggshells at day 4. Successful (left) and failed transfers (right) are presented. Scale bars: 1 cm. (b) The transfer performances are presented. The error bars represent the means ± standard errors of the means (n = 5). ***p < 0.001 for the comparison between the control and surrogate groups. (c) The embryonic viability was evaluated at each time point (days 2–4, 5–12, 13–18, and 19–21). The error bars represent the means ± standard errors of the means (n = 5). (d) The hatching performances of the improved two-step surrogate method was evaluated. The error bars represent the means ± standard errors of the means (n = 5). ***p < 0.001 for the comparison between the control and surrogate groups. (e) The expression of EGFP in the embryonic body cultured with the improved surrogate method. Arrows: EGFP. Scale bars: 0.5 cm.

Discussion

Many studies have attempted to develop transgenic chickens using windowed eggshell18–20 or surrogate eggshell incubation systems3,21,22. The method of chicken whole embryo culture using the windowed and surrogate eggshell can be important approaches in assisted reproductive technologies such cloning, genome modification, and even in de-extinction efforts5,6. We have utilized surrogate eggshell incubation to develop transgenic chickens for a long time23–25.

With this method, the second chicken surrogate eggs used in System III, which are usually at least 25 to 30 g heavier than the donor eggs, are externally obtained13,14. With a veterinary perspective, experimental eggs from specific pathogen free (SPF) facility can be recommended for donor and surrogate eggs. However, the introduction of external surrogate eggs, including the eggs from SPF, into the institutional poultry farm may be limited by regulations that detail that eggs must be inspected against avian transmittable diseases before being introduced. Additionally, consistently obtaining very large eggs is difficult; for example, second chicken surrogate eggs weighing 80 to 90 g are needed if the weight of the donor eggs is 55 to 60 g. Therefore, an in ovo embryonic incubation method for the development of transgenic chickens has to be established.

First, we attempted to establish a windowed eggshell incubation method due to its accessibility (donors can be incubated in intact eggshells). However, the frequency (41.2%) of observing germinal discs through a blunt-end hole is low and thus deficient when the freshly laid fertile eggs are briefly positioned blunt-end up (Fig. 1b). Because this low frequency results from high-viscosity albumen, it is difficult to localize the germinal disc at the point at which the disc is usually observed12. Similar to albumen, the chalaza appears to restrict the free movement of the yolk, which forces the disc into an oblique position10. Thus, we directly investigated the frequency of observing the disc and chalaza according to the preincubation positioning direction and time because if the chalaza was observed through the hole, the disc was rarely observed.

Burkhardt et al.12 mentioned that a positioning time of 96 h is necessary to find the disc at the top of the yolk and showed that the deviation of the position of the disc in the z-direction in magnetic resonance images is minimized after 96 h with horizontal positioning. In the current study, the disc of the eggs positioned for 96 h was observed through the blunt-, point-, and side-end holes of the eggs positioned in the same direction (Figs. 1c and 2a), which corresponded to the observation of the results obtained regarding horizontal positioning reported by Burkhardt et al.12. Moreover, the egg positioned of side-end up was identified as an ideal direction because the resulting frequency and depth from hole to the disc was higher and closer, respectively, than those obtained with other directions. However, very low hatching rates (2.8 to 5.6%) were obtained using the windowed method for all the groups positioned for 24 and 96 h, and no hatched chicks were obtained with from eggs positioned point- and side-end up (Fig. 2b).

Initial studies showing the hatching performance of the windowed method have obtained unsatisfying results (6.3 to 11.3% of the eggs incubated)2,26. The hatching rates were then improved by 32.0 to 45.3% through some modifications7,8. In our other study, hatching rates of approximately 60.0% were obtained using the same eggs that developed to HH stage 17–18 and then used for the transfer of chicken primordial germ cells (data not shown). Although the hole was made on the point-end of the eggshells of the eggs at HH stage 17–18, the eggs at EGK stage X in this study yield very poor hatching rates using the same method. Thus, the windowed method used here was not appropriate for the eggs at EGK stage X, and the hatching rates may depend on the survival condition of embryos at the early phase between EGK stage X and HH stages 17–18.

Accordingly, we attempted to improve the surrogate eggshell incubation method with respect to the transfer of the germinal disc of fertile eggs at EGK stage X. During the one-step surrogate method established in the current study, 2–6 mL of thin albumen needs to be discarded immediately after the transfer of the egg yolk and albumen of the donor eggs into the surrogate eggshells. Otherwise, the development of the blood circulation system is disturbed by sealing the materials during the rocking process in the incubator14. Although the hatching performance of the one-step surrogate method was low (11.8%), few embryos that developed to HH stages 17-to-18 (30.8% alive) showed subsequent development and hatching. Thus, additional methods for enhancing the survival rates at day 4 are needed, and we decided to use the method of System II previously established between EGK stage X and HH stage 17–1814.

The eggs, which weighed at least 50 to 65 g, were obtained from 80 hens of the same age in the flock. The procedure for System II of the improved surrogate eggshell method was similar to that established in a previous study4,14. However, the diameter of the hole was 4.2 cm, and the hole was prepared on the blunt end in System III14 which was different from the 3.5-cm hole made on the point end in the current study. The aforementioned difference made it possible to incubate the donor embryos using the unusual surrogate eggs used in this study, which were only 10 g heavier than the donor eggs. Additionally, there was no need to discard 2 to 6 mL of thin albumen, in contrast to the one-step surrogate method. Importantly, the surrogate eggs obtained from a specific flock of the same age have a significance that researchers can maintain only a flock for donor and surrogate eggs, leading to advantages in controlling flock sizes and operating poultry farm. In addition, this improved two-step condition such as preparation of the unusual second surrogate eggs showed that embryos were well developed and hatched (70.0%).

Differences of survival and hatching rates between one-step and improved two-step surrogate methods may be related to oxygen stress to embryos at the early phase. Kamihara et al.27 mentioned that most of the dead embryos in a previous surrogate method accumulated large amounts of lactate in amniotic and allantoic fluids, suggesting the deficiency of oxygen in the embryos. However, oxygen was incrementally needed according to embryonic development, and the oxygen supply during the initial stage of culture may reduce viability due to the toxic effect of oxygen27,28. In the current study, surrogate eggs in one-step method were sealed only with saran wrap and albumen. While, surrogate eggs in improved two-step method were well fixed using a pair of plastic rings and rubber bands in addition to sealing, which can make effect of differential oxygen penetration on embryos in one-step and improved two-step methods.

In conclusion, the improved surrogate eggshell incubation system established in this study has the potential to allow us to reach the germinal disc. With this method, the requirement for very large second surrogate eggs may be eliminated, and some hazardous factors, such as avian transmittable and transovarian transmitted disease caused by externally obtained eggs, can be minimized. Based on the results of the present study, these techniques may be useful for assisted reproductive technologies for various purposes in the field of avian biotechnology such as the development of cloned birds, genome-modified chickens with a higher success rate, and even in de-extinction efforts.

Methods

Animal care and use

The experimental eggs were produced from a poultry flock of wild-type (WT) White Leghorn (WL) hens of the same age at the institutional poultry farm. The protocols for the care and experimental use of WT WL chickens were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Animal Science (NIAS-2023-593) in the Republic of Korea.

Egg collection

The eggs used in this study were obtained from the WT WL poultry flock over the course of 23 weeks (33–56 weeks). To investigate the frequency of observing the germinal disc and chalaza and to analyze the hatching rate based on windowed eggshell incubation, fertile eggs were produced using artificial insemination with semen from WT WL roosters and WT WL hens. Additionally, fertile eggs as donors and unfertile eggs as surrogate eggs were obtained from the flock and used to improve the surrogate eggshell incubation method. The weights of the eggs were labelled on eggshells surface with a pencil, and fertile eggs within one day after oviposition were normally used.

Germinal disc and chalaza investigation

Freshly laid fertile eggs were used to investigate the frequency of observing the germinal disc and chalaza. The number and mean weight of the eggs are presented in Supplementary Table S1. The eggs, which were divided into two different groups, were briefly placed blunt-end up (Group 1) or point-end up (Group 2) immediately after collection (Supplementary Fig. 1a). A 1.5-to-2.0-diameter hole was artificially created on top of the blunt (Group 1) and point (Group 2) ends of the eggshells, and the frequency of observing the disc and chalaza in the holes was then investigated according to the preincubation positioning direction and time. The number and mean weight of the eggs are presented in Supplementary Tables S2–S5. In a separate experiment, the eggs were divided into four different groups and positioned blunt- (Group 1), point- (Group 2), and side-end up (Groups 3 and 4) for 24, 48, 72, and 96 h (Supplementary Fig. 1b). Immediately after positioning, the frequency of detecting the disc and chalaza through the generated blunt- (Subgroup 1) and point-end holes (Group 2) was determined. At the same time, the eggs in Groups 3 and 4 were vertically placed blunt- (Group 3) and point-end up (Group 4) to investigate the frequency of observing the disc and chalaza through the generated blunt- (Group 3) and point-end holes (Group 4). If the disc and chalaza were not observed, the egg was broken, and egg fertility was assessed to exclude unfertile eggs from the records.

Windowed eggshell incubation

The eggs were divided into three different groups and positioned blunt- (Group 1), point- (Group 2), and side-end up (Group 3) for 24 and 96 h (Supplementary Fig. 1c). The frequencies of detecting the disc through 1.0-to-1.5-diameter blunt- (Group 1), point- (Group 2), and side-end holes (Group 3) that were artificially generated on the eggshells were determined. The number and mean weight of the eggs are presented in Supplementary Tables S6 and S7. If the disc was observed through the hole, the eggs were sealed with parafilm. The sealed eggs were incubated while positioned blunt- (Groups 1 and 3) and point-end up (Group 2). The incubation conditions were 37.8 °C and 60–70% relative humidity, and the eggs were rocked at an angle of 30° at 30-min intervals for 18 d. The eggs were then incubated at 37.3 °C and 70–80% relative humidity without rocking to induce hatching.

One-step surrogate eggshell incubation

The donor and surrogate eggs obtained from the flock were used to develop a one-step surrogate eggshell incubation method. The number and mean weight of the eggs are presented in Supplementary Table S8. The surrogate method procedure was based on previous studies4,14 with some modifications, and illustration of one-step surrogate method are presented (Supplementary Fig. 2a). The donor and surrogate eggs which were 10.0 ± 0.7 g heavier than the donor eggs, were sterilized in a water bath supplemented with a sodium hypochlorite solution (1/4,000 dilution, Sigma, Saint Louis, MO, USA) for 3 min at 37.5 °C. The pointed end of the surrogate eggs was ground with 3.5-cm-diameter plastic rings. The entire contents of the surrogate eggs were discarded and washed with distilled water. The donor eggs were carefully broken, and the egg yolk with blastoderm and thick and thin albumen were transferred into the surrogate eggshells. Then, 2–6 mL of thin albumen was removed, and the reconstituted eggs were sealed with saran wrap and albumen. The eggs, which were positioned point-end up, were incubated at 37.8 °C and 60–70% relative humidity and rocked to an angle of 30° at 30-min intervals for 18 d. The eggs were incubated at 37.3 °C and 70–80% relative humidity without rocking to induce hatching. During incubation, the embryonic viability was visually monitored daily as described previously14.

Two-step surrogate eggshell incubation

The donor and surrogate eggs obtained from the flock were used to obtain an improved two-step surrogate eggshell incubation method. The number and mean weight of the eggs are presented in Supplementary Table S9. Illustration of the improved two-step surrogate method are presented in (Supplementary Fig. 2b). In System II, the donor eggs and the first surrogate eggs, which were 4.1 ± 0.2 g heavier than the donor eggs, were sterilized as described above. The pointed end of the first surrogate eggs was ground with 3.5-cm rings. The entire contents of the surrogate eggs were discarded and washed with distilled water. The donor eggs were carefully broken, and the yolk with blastoderm and thick albumen were transferred into the first surrogate eggshells. Then, only thin albumen from other eggs of the flock were added to fill the eggshells. The reconstituted eggs were sealed with saran wrap and albumen, and the eggs were well fixed using a pair of plastic rings and rubber bands. The eggs, which were positioned point-end up, were incubated at 37.8 °C and 60–70% relative humidity and rocked to an angle of 90° at 30-min intervals for 3 d. The second surrogate eggs, which were 10.0 ± 0.1 g heavier than the donor eggs, were sterilized, and the point end of the second surrogate eggs was ground with 3.5-cm rings. The entire contents of the surrogate eggs were discarded and washed with distilled water. Only live donor eggs that had developed to HH stage 17-to-18 at day 4 were transferred into the second surrogate eggshells, which started System III. The reconstituted eggs were sealed with saran wrap and albumen. Next, the eggs were positioned and incubated point-end up with embryonic viability monitored as describe above.

EGFP vector construction and injection

Lentivirus particles driving expression of EGFP were produced using lentiviral plasmid DNA purchased from Vector Builder (VectorBuilder, Guangzhou, China). The ultra-purified viral stocks contained approximately 1.1 × 109 transduction units per mL. The virus particles supplemented with polybrene (10 μg/mL) were microinjected into the subgerminal cavity of freshly laid fertile eggs produced by WT WL hens. Thereafter, the eggs were incubated using the improved two-step surrogate method.

Statistical analysis

Statistical analyses were performed using GraphPad Prism statistical software (GraphPad Prism 5.03 software, San Diego, CA, USA). A t test was used to compare the frequency of observing the germinal disc and chalaza in the freshly laid fertile eggs, and one-way ANOVA was used to compare the frequency of observing these in the eggs positioned for 24, 48, 72, and 96 h. One-way ANOVA was used to compare the hatching performances with the windowed and surrogate eggshell methods, and a p value lower than 0.05 was considered to indicate statistical significance.

Supplementary Information

Supplementary Tables.

Supplementary Figures.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71804-6.

Acknowledgements

This work was supported by the “Animal Science & Technology Development (Project No. PJ01720201)” from the Rural Development Administration of the Republic of Korea. The authors would like to thank Jayeon Yoo for providing illustration used in this study.

Author contributions

H.Y., J.Y.H. and S.J.B. conceived and designed the experiments. H.Y., B.R.L. and Y.J.J. performed the experiments. H.Y., S.L., S.A.O., K.B.O., P.L. and H.L. analyzed the data. H.Y., B.R.L. and S.J.B. wrote the main manuscript text and prepared the figures. S.L., S.A.O., K.B.O., P.L., H.L. and J.Y.H. reviewed and corrected the manuscript. All authors reviewed the manuscript.

Data availability

The data presented in this study are available on reasonable request from the corresponding author.

Competing interests

The authors declare no competing interests.

Ethical statement

The use of all experimental animals has been thought through according to the 3Rs rule (replacement, reduction, refinement), and the animals ultimately were euthanized using common methods supported by the American Veterinary Medical Association (AVMA) guidelines. The ARRIVE guidelines (https://arriveguidelines.org/) were followed while conducting the experiments on animals in this study. The points of the guide have been respected.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Farzaneh M Attari F Khoshnam SE Mozdziak PE The method of chicken whole embryo culture using the eggshell windowing, surrogate eggshell and ex ovo culture system Br. Poult. Sci. 2018 59 240 244 10.1080/00071668.2017.1413234 29206486
Farzaneh, M., Attari, F., Khoshnam, S. E. & Mozdziak, P. E. The method of chicken whole embryo culture using the eggshell windowing, surrogate eggshell and ex ovo culture system. Br. Poult. Sci. 59, 240–244 (2018).29206486 10.1080/00071668.2017.1413234
2. Petitte JN Clark ME Liu G Gibbins AMV Etches RJ Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells Development 1990 108 185 189 10.1242/dev.108.1.185 2351062
Petitte, J. N., Clark, M. E., Liu, G., Gibbins, A. M. V. & Etches, R. J. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108, 185–189 (1990).2351062 10.1242/dev.108.1.185
3. Mozdziak PE Borwornpinyo S McCoy DW Petitte JN Development of transgenic chickens expressing bacterial beta-galactosidase Dev. Dyn. 2003 226 439 445 10.1002/dvdy.10234 12619130
Mozdziak, P. E., Borwornpinyo, S., McCoy, D. W. & Petitte, J. N. Development of transgenic chickens expressing bacterial beta-galactosidase. Dev. Dyn. 226, 439–445 (2003).12619130 10.1002/dvdy.10234
4. Borwornpinyo S Brake J Mozdziak PE Petitte JN Culture of chicken embryos in surrogate eggshells Poult. Sci. 2005 84 1477 1482 10.1093/ps/84.9.1477 16206571
Borwornpinyo, S., Brake, J., Mozdziak, P. E. & Petitte, J. N. Culture of chicken embryos in surrogate eggshells. Poult. Sci. 84, 1477–1482 (2005).16206571 10.1093/ps/84.9.1477
5. Kjelland ME Romo S Kraeme DC Avian cloning: Adaptation of a technique for enucleation of the avian ovum Avian Biol. Res. 2014 7 131 138 10.3184/175815514X14065426847114
Kjelland, M. E., Romo, S. & Kraeme, D. C. Avian cloning: Adaptation of a technique for enucleation of the avian ovum. Avian Biol. Res. 7, 131–138 (2014).10.3184/175815514X14065426847114
6. Kjelland ME Novak B Blue-McLendon A Romo S Kraeme DC Manipulating the avian egg: applications for embryo transfer, transgenics, and cloning Avian Biol. Res. 2017 10 146 155 10.3184/175815617X14951979279268
Kjelland, M. E., Novak, B., Blue-McLendon, A., Romo, S. & Kraeme, D. C. Manipulating the avian egg: applications for embryo transfer, transgenics, and cloning. Avian Biol. Res. 10, 146–155 (2017).10.3184/175815617X14951979279268
7. Speksnijder G Ivarie R A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs Poult. Sci. 2000 79 1430 1433 10.1093/ps/79.10.1430 11055849
Speksnijder, G. & Ivarie, R. A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs. Poult. Sci. 79, 1430–1433 (2000).11055849 10.1093/ps/79.10.1430
8. Andacht T Hu W Ivarie R Rapid and improved method for windowing eggs accessing the stage X chicken embryo Mol. Reprod. Dev. 2004 69 31 34 10.1002/mrd.20155 15278901
Andacht, T., Hu, W. & Ivarie, R. Rapid and improved method for windowing eggs accessing the stage X chicken embryo. Mol. Reprod. Dev. 69, 31–34 (2004).15278901 10.1002/mrd.20155
9. Hutchison MJ Lirette A Etches RJ Towner RA Janzen EG An assessment of egg yolk structure using magnetic resonance imaging Poult. Sci. 1992 71 2117 2121 10.3382/ps.0712117 1470595
Hutchison, M. J., Lirette, A., Etches, R. J., Towner, R. A. & Janzen, E. G. An assessment of egg yolk structure using magnetic resonance imaging. Poult. Sci. 71, 2117–2121 (1992).1470595 10.3382/ps.0712117
10. Klein S Localization of the fertilized germinal disc in the chicken egg before incubation Poult. Sci. 2002 81 529 536 10.1093/ps/81.4.529 11989753
Klein, S. et al. Localization of the fertilized germinal disc in the chicken egg before incubation. Poult. Sci. 81, 529–536 (2002).11989753 10.1093/ps/81.4.529
11. Bain M Fagan AJ Mullin JM McNaught I McLean J Condon B Non-invasive monitoring of chick development in ovo using a 7T MRI system from day 12 of incubation through to hatching J. Magn. Reson. Imaging. 2007 26 198 201 10.1002/jmri.20963 17659540
Bain, M. et al. Non-invasive monitoring of chick development in ovo using a 7T MRI system from day 12 of incubation through to hatching. J. Magn. Reson. Imaging. 26, 198–201 (2007).17659540 10.1002/jmri.20963
12. Burkhardt A Meister S Bergmann R Koch E Influence of storage on the position of the germinal disc in the fertilized unincubated chicken egg Poult. Sci. 2011 90 2169 2173 10.3382/ps.2010-01179 21933997
Burkhardt, A., Meister, S., Bergmann, R. & Koch, E. Influence of storage on the position of the germinal disc in the fertilized unincubated chicken egg. Poult. Sci. 90, 2169–2173 (2011).21933997 10.3382/ps.2010-01179
13. Perry MM A complete culture system for the chick embryo Nature 1988 331 70 72 10.1038/331070a0 3340149
Perry, M. M. A complete culture system for the chick embryo. Nature 331, 70–72 (1988).3340149 10.1038/331070a0
14. Yang H Research note: embryonic viability by weight difference between donor and surrogate eggs in a surrogate eggshell incubation system Poult. Sci. 2022 101 101733 10.1016/j.psj.2022.101733 35303688
Yang, H. et al. Research note: embryonic viability by weight difference between donor and surrogate eggs in a surrogate eggshell incubation system. Poult. Sci. 101, 101733 (2022).35303688 10.1016/j.psj.2022.101733
15. Liljebjelke KA Vertical and horizontal transmission of salmonella within integrated broiler production system Foodborne pathog. Dis. 2005 2 90 102 10.1089/fpd.2005.2.90 15992303
Liljebjelke, K. A. et al. Vertical and horizontal transmission of salmonella within integrated broiler production system. Foodborne pathog. Dis. 2, 90–102 (2005).15992303 10.1089/fpd.2005.2.90
16. Feberwee A An experimental model to quantify horizontal transmission of mycoplasma gallisepticum Avian Pathol. 2005 34 355 361 10.1080/03079450500180770 16147573
Feberwee, A. et al. An experimental model to quantify horizontal transmission of mycoplasma gallisepticum. Avian Pathol. 34, 355–361 (2005).16147573 10.1080/03079450500180770
17. Hamburger V Hamilton HL A series of normal stages in the development of the chick embryo J. Morphol. 1951 88 49 92 10.1002/jmor.1050880104 24539719
Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92 (1951).24539719 10.1002/jmor.1050880104
18. Maeda T Yamakawa Y Masuda K Terada T Distribution of blastodermal cells transferred to chick embryos for chimera production using windowed eggs Br. Poult. Sci. 1997 38 241 244 10.1080/00071669708417979 9280347
Maeda, T., Yamakawa, Y., Masuda, K. & Terada, T. Distribution of blastodermal cells transferred to chick embryos for chimera production using windowed eggs. Br. Poult. Sci. 38, 241–244 (1997).9280347 10.1080/00071669708417979
19. Lee SH Development of transgenic chickens expressing human parathormone under the control of a ubiquitous promoter by using a retrovirus vector system Poult. Sci. 2007 86 2221 2227 10.1093/ps/86.10.2221 17878453
Lee, S. H. et al. Development of transgenic chickens expressing human parathormone under the control of a ubiquitous promoter by using a retrovirus vector system. Poult. Sci. 86, 2221–2227 (2007).17878453 10.1093/ps/86.10.2221
20. Lee SH Gupta MK Ho YT Kim T Lee HT Transgenic chickens expressing human urokinase-type plasminogen activator Poult. Sci. 2013 92 2396 2403 10.3382/ps.2013-03223 23960123
Lee, S. H., Gupta, M. K., Ho, Y. T., Kim, T. & Lee, H. T. Transgenic chickens expressing human urokinase-type plasminogen activator. Poult. Sci. 92, 2396–2403 (2013).23960123 10.3382/ps.2013-03223
21. Cao D Expression of recombinant human lysozyme in egg whites of transgenic hens PLoS One 2015 19 e0118626 10.1371/journal.pone.0118626
Cao, D. et al. Expression of recombinant human lysozyme in egg whites of transgenic hens. PLoS One 19, e0118626 (2015).10.1371/journal.pone.0118626
22. Liu T Oviduct-specific expression of human neutrophil defensin 4 in lentivirally generated transgenic chickens PLoS One 2015 10 e0127922 10.1371/journal.pone.0127922 26020529
Liu, T. et al. Oviduct-specific expression of human neutrophil defensin 4 in lentivirally generated transgenic chickens. PLoS One 10, e0127922 (2015).26020529 10.1371/journal.pone.0127922
23. Byun SJ Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens Biosci. Biotechnol. Biochem. 2011 75 646 649 10.1271/bbb.100721 21512248
Byun, S. J. et al. Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens. Biosci. Biotechnol. Biochem. 75, 646–649 (2011).21512248 10.1271/bbb.100721
24. Byun SJ Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken BMB Rep. 2013 46 404 409 10.5483/BMBRep.2013.46.8.251 23977988
Byun, S. J. et al. Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken. BMB Rep. 46, 404–409 (2013).23977988 10.5483/BMBRep.2013.46.8.251
25. Byun SJ Transgenic chickens expressing the 3D8 single chain variable fragment protein suppress avian influenza transmission Sci. Rep. 2017 7 5938 10.1038/s41598-017-05270-8 28724948
Byun, S. J. et al. Transgenic chickens expressing the 3D8 single chain variable fragment protein suppress avian influenza transmission. Sci. Rep. 7, 5938 (2017).28724948 10.1038/s41598-017-05270-8
26. Thoraval P Germline transmission of exogenous genes in chickens using helper-free ectopic avian leucosis virus-based vectors Transgenic Res. 1995 4 369 377 10.1007/BF01973755 7581517
Thoraval, P. et al. Germline transmission of exogenous genes in chickens using helper-free ectopic avian leucosis virus-based vectors. Transgenic Res. 4, 369–377 (1995).7581517 10.1007/BF01973755
27. Kamihara M Oguchi S Tachibana A Kitagawa Y Iijima S Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel Dev. Growth Differ. 1998 40 449 455 10.1046/j.1440-169X.1998.t01-2-00010.x 9727359
Kamihara, M., Oguchi, S., Tachibana, A., Kitagawa, Y. & Iijima, S. Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel. Dev. Growth Differ. 40, 449–455 (1998).9727359 10.1046/j.1440-169X.1998.t01-2-00010.x
28. Tahara Y Obara K A novel shell-less culture system for chick embryos using a plastic film as culture vessels J. Poult. Sci. 2014 51 307 312 10.2141/jpsa.0130043
Tahara, Y. & Obara, K. A novel shell-less culture system for chick embryos using a plastic film as culture vessels. J. Poult. Sci. 51, 307–312 (2014).10.2141/jpsa.0130043
