Exp. Anim. 54(1), 7Ð11, 2005

Culture System for Embryos of Blue-Breasted from the Blastoderm Stage to Hatching

Tamao ONO1), Yoshifumi NAKANE2), Takahiro WADAYAMA1), Masaoki TSUDZUKI3), Kenjiro ARISAWA1,4), Shoko NINOMIYA1), Toshihiko SUZUKI5), Makoto MIZUTANI6), and Hiroshi KAGAMI1)

1)Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, 2)Graduate School of Medicine, Kyoto University, Kyoto 606-8501, 3)Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, 4)United Graduate School of Agricultural Sciences, Gifu University, Gifu 5501-1193, 5)Junior College, Toyama Prefectural University, Kosugi 939-0311, and 6)Nippon Institute for Biological Science, Kobuchizawa 408-0041, Japan

Abstract: The blue-breasted quail ( chinensis), the smallest in the order Galliforms, is a candidate model for avian developmental engineering because it is precocious and prolific. This species requires 17 days to hatch and 8 to 9 weeks to mature to an adult body weight of about 50 g, whereas the Japanese quail (Coturnix japonica) requires 16 days to hatch and 6 to 8 weeks to mature to an adult body weight of 100 to 150 g. The early embryo is the most challenging embryonic stage in terms of culture and manipulation for avian biotechnology. We have evaluated various conditions for the culture of blue-breasted quail embryos from the blastoderm stage to hatching. A hatchability rate of 26% (10/39) is among the best of the various culture conditions examined in the present study and the embryo culture system should facilitate advances in avian biotechnology. Key words: avian biotechnology, blue-breasted quail, Coturnix chinensis, embryo culture, Japanese quail.

Introduction names for this species, Coturnix chinensis and chinensis, which is a member of the fam- The blue-breasted quail, the smallest species of quail, ily [6, 13Ð15]. We will refer to this species is a potential model animal for avian developmental as the blue-breasted quail (Coturnix chinensis) based engineering because it is precocious and prolific. This on the complete nucleotide sequence of its mitochon- species is also known as the Button quail in the United drial genome and phylogenetic analysis [6]. The States, the Chinese painted quail in Europe, and the blue-breasted quail requires 17 days to hatch and 8 to 9 in Australia. Other vernacular names in- weeks to mature to an adult body weight of ~50 g (Fig. clude Indian , Asian blue-breasted quail, and 1a, b), whereas the Japanese quail (Coturnix japonica) blue quail [3, 6, 14]. There are also two scientific requires 16 days to hatch and 6 to 8 weeks to achieve

(Received 26 April 2004 / Accepted 30 July 2004) Address corresponding: T. Ono, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa Kamiina, Nagano 399-4598, Japan 8 T. ONO, ET AL.

Fig. 1. and culture systems. a, Male blue-breasted quail. b, Female blue-breasted quail. c, Female Japanese quail. d, Male Japanese quail. e, Blue-breasted quail egg. f, Japanese quail egg. g, First step of culture system A for blue-breasted quail. h, Second step of culture system A for blue-breasted quail. Bars, 1 cm.

an adult body weight of 100 to 150 g (Fig. 1c, d). Materials and Methods One of the advantages of avian embryos for experi- mental analysis of developmental events is the relative Animals ease with which they can be cultured, manipulated, and Fertile eggs of blue-breasted quail (Fig. 1e) and Japa- observed. The early embryo is the most challenging nese quail (Fig. 1f) maintained in our laboratory were stage of avian development with regard to its culture used for the present study. The eggs were collected and manipulation. It has proved possible, however, to daily and maintained at 12°C to 14°C for not more than culture embryos of certain avian species ex ovo (out- 1 week. Development of embryos was staged accord- side of their own shell and shell membrane) from the ing to Hamburger and Hamilton’s standard [1]. single-cell stage, which normally exists in the oviduct, through to hatching [5, 8, 11]. The problem of how to Culture of embryos gain access to the avian embryo while allowing it to The culture of blue-breasted quail embryos consisted grow normally has been the subject of many studies of two steps and was based on the protocol developed [12]. Chicken and Japanese quail embryos have served for Japanese quail embryos [7, 8]. Eggshells were as model systems for studying the development both of wiped with 70% ethanol prior to manipulations. Seven avian species and of higher vertebrates in general. The series of culture experiments for blue-breasted quail blue-breasted quail, however, may provide an alterna- embryos (systems A through G) and one control series tive animal model because of its smaller body size and for Japanese quail embryos (system H) were performed efficient reproductive performance. Thus, in the present until the embryos hatched or stopped developing. study we examined the culture protocol of blue-breasted System A: For the first step, a surrogate eggshell of quail embryos from the blastoderm stage leading to the Japanese quail was prepared by cutting the narrow end hatching. of the egg where it was 24 mm in diameter and remov- ing the contents. A blue-breasted quail embryo together with the egg yolk and albumen was removed from the eggshell of an unincubated egg. The thick albumen capsule surrounding the embryo was removed and the CULTURE OF BLUE-BREASTED QUAIL EMBRYOS 9

Table 1. Systems for the culture of embryos from the blastoderm stage to hatching

First culture step Second culture step Type of System embryo cultured Diameter of shell Surrogate Thick albumen Type of thin Surrogate opening (mm) eggshell capsule albumen eggshell

ABq24JqRemoved C C BBq19JqRemoved C C CBq14BqRemoved C C DBq24JqRemoved Jq C EBq24JqRemoved Bq C FBq24JqAttached C C GBq24JqRemoved C Jq HJq19JqRemoved C C

Abbreviationns: Bq, blue-breasted quail; Jq, Japanese quail; C, chicken.

naked embryo and egg yolk were transferred to the System C: In the first step, the surrogate shell was surrogate shell. The surrogate shell was then filled prepared from blue-breasted quail; the opening of the with chicken thin albumen and sealed tightly with cling eggshell was 14 mm in diameter, and the inner diam- film (polyethylene wrap) and a pair of plastic rings eter of the rings was 15 mm. (inner diameter, 25 mm), which were secured by plac- System D: The surrogate shell was filled with thin ing elastic bands on their four screw bolt projections albumen of Japanese quail. (Fig. 1g). The egg was then incubated for 50 to 52 h at System E: The surrogate shell was filled with thin 37.5°C and 70% relative humidity, with rocking around albumen of blue-breasted quail. the long axis at a 90-degree angle at 30-min intervals, System F: In the first step, the blue-breasted quail until the embryo had developed to stages 14 to 18 of embryo as well as its egg yolk and surrounding thick Hamburger and Hamilton’s standard. For the second albumen capsule were transferred to the surrogate shell. step, a surrogate chicken eggshell was prepared by cut- System G: In the second step, the surrogate shell was ting the narrow end of the egg where it was 35 mm in prepared from Japanese quail with an opening 19 mm diameter and removing the contents. The embryo, to- in diameter. gether with yolk and albumen, from the first culture System H: Japanese quail embryos were cultured. In step was then transferred to the new surrogate shell the first step, the opening of the surrogate eggshell was (Fig. 1h). The shell was sealed with cling film and the 19 mm in diameter and the inner diameter of the rings embryo was cultured under conditions similar to those was 20 mm. used in the first step, with the exception that the cling film surface was directed upward and rocking was per- Results formed round the short axis of the shell at a 30-degree angle. One or two days before the expected hatching The viability and hatchability of embryos cultured time, the film was perforated to facilitate embryonic according to systems A through H are shown in Table respiration. Rocking of the embryos was stopped half 2. The embryos surviving after incubation for 50 to 52 or one day before hatching. Chicks were considered to h were found to be at stages 14 to 18 of development. have hatched when they were completely free from the The effect of size of the first surrogate eggshell was shell. Changes in the culture protocol for systems B evaluated by comparing the results obtained with Japa- through H are specified below; other aspects of these nese quail shells opened at a diameter of 24 mm (system systems were the same as those for system A (Table 1). A) or 19 mm (system B) or with blue-breasted quail System B: In the first step, the surrogate eggshell shells opened at a diameter of 14 mm (system C). The was opened where the egg was 19 mm in diameter, and viability obtained with systems A, B, and C at the end the inner diameter of the rings was 20 mm. of the first step of culture (50 to 52 h of culture) was 10 T. ONO, ET AL.

Table 2. Viability and hatchability of cultured embryos

No. of embryos No. of embryos surviving (viability) No. of hatchlings System cultured Day 2 Day 6 Day 10 Day 16 (hatchability)

A3935 (90%) 33 (85%) 24 (62%) 19 (49%) 10 (26%) B3730 (81%) 20 (54%) 11 (30%) 3 (8%) 3 (8%) C2315 (65%) 7 (30%) 2 (9%) 0 (0%) 0 (0%) D5741 (72%) 31 (54%) 10 (18%) 0 (0%) 0 (0%) E5142 (82%) 23 (45%) 10 (20%) 0 (0%) 0 (0%) F7970 (89%) 57 (72%) 35 (44%) 2 (3%) 0 (0%) G5753 (93%) 28 (49%) 14 (25%) 6 (11%) 3 (5%) H3636 (100%) 23 (64%) 16 (44%) 13 (36%) 12 (33%)

The values for day 2 were actually determined after culture for 50 to 52 h, when the surviving embryos were trans- ferred to the second step of culture.

90% (35/39), 81% (30/37), and 65% (15/23), respec- tively high viability during the second step of culture, tively. Hatchlings were obtained only for systems A resulting in a hatchability rate of 26%. Systems B and B, with a hatchability rate of 26% (10/39) and 8% (8%) and G (5%) yielded low percentages of hatched (3/37), respectively. The effect of the type of thin al- embryos, whereas the remaining systems did not yield bumen used for culture was evaluated by comparing any hatchlings. In systems A, B, and C, the amount of the results obtained with systems A (chicken), D (Japa- culture medium (thin albumen) was ~4.5, 6.5, and 3.0 nese quail), and E (blue-breasted quail). The viability ml, respectively. Given that the volume of albumen obtained with systems D and E at the end of the first present in normal blue-breasted quail eggs is ~3.5 ml, step of culture was 72% (41/57) and 82% (42/51), re- the amount of albumen in systems A and B may be in spectively, but no hatchlings were generated. The effect excess of the usual amount, although it did not prevent of the absence or presence of the albumen capsule was hatching in these systems. The eggshell of blue-breasted evaluated by comparing systems A and F. Although quail was not large enough for use as a surrogate shell viability was 89% (70/79) at the end of the first culture during the first culture step because more than half of step, no hatchlings were obtained for embryos with an the yolks were ruptured by contact with the edge of the attached albumen capsule. Finally, the effect of use of shell or by adherence to the shell membrane or wrap. a surrogate shell of Japanese quail in the second culture The basic components of albumen are similar among step was examined in system G; a hatchability rate of chicken, Japanese quail, and blue-breasted quail. How- 5% (3/57) was obtained. Culture of Japanese quail ever, no hatchlings were obtained with culture systems embryos as a control (system H) yielded 100% (36/36) that used albumen from Japanese quail or blue-breasted viability at the end of the first culture step, and 33% quail, probably because of contamination by eggshell (12/36) of the embryos hatched. fragments or other factors, given that it was difficult to collect albumen from these eggs. In the previous cul- Discussion ture of Japanese quail embryos with and without albumen capsule the hatchabilities were 38.1 and 48.4%, We have compared various culture conditions for respectively, indicating that removal of the surrounding embryos of blue-breasted quail. The highest viability albumen capsule seems to be subcritical for survival (≥90%) at the end of the first culture step (incubation (P=0.20; Fischer’s exact probability test) [8, 9]. In the for 50 to 52 h) was obtained with systems A and G. present study, however, the embryo culture with albu- Systems B, E, and F also yielded a high viability (>80%) men capsule failed to produce hatchlings even though a at the end of the first step of culture, whereas the corre- small number of embryos survived until the final stage sponding values for systems C and D were only 65 and of development. The movement of embryos cultured 72%, respectively. System A also maintained a rela- with an albumen capsule was not smooth in the surro- CULTURE OF BLUE-BREASTED QUAIL EMBRYOS 11 gate shells in cases of both Japanese quail and blue- References breasted quail cultures. But, none the less, it seems unlikely that the albumen capsule has a fatal impact on 1. Hamburger, V. and Hamilton, H.L. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. hatching of the blue-breasted quail embryos. In fact, 88: 49Ð92. carefull handling was required for the small-sized eggs 2. Kamihira, M., Oguchi, S., Tachibana, A., Kitagawa, Y., of blue-breasted quail. It remains a matter of improv- and Iijima, S. 1998. Improved hatching for in vitro quail ing our technique to obtain viable hatchlings from the embryo culture using surrogate eggshell and artificial vessel. Dev. Growth Differ. 40: 449Ð455. embryo culture with intact albumen capsule. Avian 3. Monroe, B.L.Jr. and Sibley, C.G. 1993. A World Check chimeras are obtained by transfer of blastoderm cells List of . Yale University Press, New Haven, CT. [4]. When trying to introduce blastoderm cells to the 4. Naito, M. 2003. Development of avian embryo manipulation recipient embryo, we found, however, that it was diffi- techniques and their application to germ cell manipulation. cult to insert a micropipette into the subgerminal cavity Anim. Sci. J. 74: 157Ð168. 5. Naito, M., Nirasawa, K., and Oishi, T. 1990. Development of the blastoderm through the capsular thick albumen. in culture of the chick embryo from fertilized ovum to About 80% of the calcium of a newly hatched chick or hatching. J. Exp. Zool. 254: 322Ð326. Japanese quail is derived from the eggshell and is trans- 6. Nishibori, M., Tsudzuki, M., Hayashi, T., Yamamoto, Y., ported through the chorioallantoic membrane [10, 16]. and Yasue, H. 2002. Complete nucleotide sequence of the Coturnix chinensis (Blue-breasted quail) mitochondorial An essential feature of the second half of the culture genome and a phylogenetic analysis with related species. concerns calcium absorption from the shell. In the cul- J. Hered. 93: 439Ð444. tured embryos the area of the chorioallantoic membrane 7. Ono, T. 2000. Ex ovo culture of avian embryos. pp. 39Ð46. extending along the inside of the eggshell during em- In: Developmental Biology Protocols, I. (Tuan, R.S., and bryonic development is less than that of normal Lo, C.W eds.), Humana Press, Totowa. 8. Ono, T., Murakami, T., Mochii, M., Agata, K., Kino, K., embryos. In a previous study, the supply of calcium Otsuka, K., Ohta, M., Mizutani, M., Yoshida, M., and lactate and oxygen aeration to cultured Japanese quail Eguchi, G. 1994. A complete culture system for avian embryos enhanced the hatchability rate; furthermore, transgenesis, supporting quail embryos from the single- the addition of calcium to the calcium-free artificial cell stage to hatching. Dev. Biol. 161: 126Ð130. 9. Ono, T., Muto, S., Matsumoto, T., and Yoshida, M. 1995. culture system made it possible to hatch Japanese quail Production of quail chimeras by transfer of early blastoderm embryos [2]. This system may also be applicable to cells: plumage chimeras and a germline chimera without the culture of blue-breasted quail embryos. plumage mixture. Jpn. Poult. Sci. 32: 252Ð256. In conclusion, of the various culture conditions ex- 10. Ono, T. and Wakasugi, N. 1984. Mineral content of quail embryos cultured in mineral-rich and mineral-free amined in the present study, system A was the best for conditions. Poult. Sci. 63: 159Ð166. the culture of blue-breasted quail embryos from the 11. Perry, M.M. 1988. A complete culture system for the chick blastoderm stage leading to hatching. This system is embryo. Nature 331: 70Ð72. similar to the practical system for the culture of Japa- 12. Selleck, M.A. 1996. Culture and microsurgical manipulation nese quail embryos, however, enrichment of calcium of the early avian embryo. pp. 1Ð21. Methods in Avian Embryology (Bronner-Fraser, M. ed.), Academic Press, San and oxygen to the culture system should be considered Diego. for improving the hatching rate of the blue-breasted 13. Shirai, S. 1992. Lexicon of the Names of the World. quail. Scientific Names. Hara Shobo, Tokyo (in Japanese). 14. Sibley, C.G. and Monroe, B.L.Jr. 1990. Distribution and of Birds of the World. Yale University Press, Acknowledgments New Haven. 15. Tsudzuki, M. 1994. Excalfactoria quail as a new laboratory This study was supported in part by a Grant-in-Aid research animal. Poult. Sci. 73: 763Ð768. (no. 13460132) from the Japan Society for the Promo- 16. Tuan, R.S. and Zrike, J. 1978. Functional involvement of tion of Science to T.O. carbonic anhydrase in calcium transport of the chick chorioallantoic membrane. Biochem. J. 176: 67Ð74.