Exp. Anim. 54(1), 7–11, 2005 Culture System for Embryos of Blue-Breasted Quail 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 (Coturnix chinensis), the smallest species in the order Galliforms, is a candidate model animal 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 Excalfactoria chinensis, which is a member of the fam- The blue-breasted quail, the smallest species of quail, ily Phasianidae [6, 13–15]. 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 King quail in Australia. Other vernacular names in- weeks to mature to an adult body weight of ~50 g (Fig. clude Indian blue quail, 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. Animals 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.