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Poult Sci 2007. 86:937-943
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Fertilization and Blastoderm Development of Quail Oocytes After Intracytoplasmic Injection of Chicken Sperm Bearing the W Chromosome

S. Takagi*, T. Ono{dagger}, A. Tsukada*, Y. Atsumi{dagger},{ddagger}, S. Mizushima*, N. Saito* and K. Shimada*,1

* Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Aichi 464-8601, Japan; and {dagger} Faculty of Agriculture, Department of Food Production Science, and {ddagger} Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Minamiminowa, Nagano 399-4598, Japan

1 Corresponding author: kshimada{at}agr.nagoya-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous study demonstrated that elongated spermatids and sperm carrying the female-specific W-chromosome of the sex-reversed domestic fowl can activate the mouse oocyte, but whether they can fertilize the avian oocyte and lead to a developing zygote remains undetermined. A single sperm isolated from the semen and testis of normal rooster and from a testis of sex-reversed hen was microinjected into a quail oocyte and cultured for 20 to 24 h. Blastoderms were fixed, cleaved, nuclei stained by 4',6'-diamidino-2-phenylin-dole, and developmental stages were assessed. In the normal rooster group, ejaculated and testicular sperm induced blastodermal development in 22.6 and 20% of the quail oocytes, respectively. The developmental stages ranged from IV to VII. In the sex-reversal group, 20% of injected testicular sperm induced blastodermal development. The blastodermal stages varied from stage III to VI. Blastoderms after 4',6'-diamidino-2-phenylindole staining were assayed by PCR to identify the W chromosome of either chicken sperm or quail oocyte. The PCR assay results showed that 2 out of 9 developed blastoderms microinjected with sperm of sex-reversed hen were identified containing the female-specific W chromosome derived from sex-reversed hen. From these results, it is concluded that chicken sperm bearing the W chromosome possess fertilizing ability and can function to stimulate blastoderm development similar to that of normal chicken sperm carrying the Z chromosome.

Key Words: sex reversal • W chromosome • sperm • fertilization • blastodermal development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, reduction of avian genetic resources, in particular chicken lines, has been reported, and future conservation of avian genetic resources has been proposed (Qureshi, 2006). Among the various conservation methods, keeping live birds is preferred but expensive (Fulton, 2006). Several other methods of germplasm preservation are available such as cryopreservation of blastodermal cells, embryonic stem cells, and primordial germ cells; however, despite the relatively low cost, recovery of live birds can be difficult due to limitations in terms of practicality, efficiency, and success rate (Fulton and Delany, 2003; Petitte, 2006). Artificial insemination is relatively practical, but semen cryopreservation is only applicable for the male genome. This eliminates or severely limits the usefulness of this conservation technique with inbred or selected stocks. If, however, the female genome is available as sperm, the disadvantage may be overcome (Shimada et al., 2007).

Reports of experimental sex reversal in chickens have been accumulated, and they demonstrated that sex-reversed chicken can produce sperm carrying the female-specific W chromosome (Abinawanto et al., 1998). Although, sex-reversed chickens have developed testes containing essentially the same cellular components as those of normal male testes and capable of complete spermatogenesis (Elbrecht and Smith, 1992), no offspring has ever been produced due to low sperm counts (Abinawanto et al., 1998) and blind-ended vas deferens (Rashedi et al., 1983). One exception, however, is the report of Frankenhuis and Kappert (1980), who inseminated epididymal sperm of sex-reversed hens to normal hens and obtained offspring including male and female chicks. However, they did not identify the sperm bearing the female-specific W chromosome (W sperm) in the offspring. Therefore, before employing the W-containing sperm as a means of conservation, it is necessary to evaluate whether these sperm are fertile. Our previous study has shown that the sperm certainly possess an oocyte-activating ability (Takagi et al., 2007). In this study, the sperm was injected into the mouse oocyte, and the resultant 2-pronuclei oocyte that carried W DNA derived from a sex-reversed hen was identified by PCR assay. Although oocyte activation is an essential part of the fertilization process, it is necessary to demonstrate that the W chromosome-bearing sperm fertilizes the oocyte and further develops to an embryo. To this end, we established intracytoplasmic sperm injection (ICSI) in the quail (Hrabia et al., 2003a), and this technique was used for the present study. Namely, chicken sperm from a normal rooster or sperm from a sex-reversed hen was injected into a quail oocyte and cultured to allow development and screening for the presence of a chicken W chromosome. Because the chicken-quail hybrids have been produced by artificial insemination to quail hen (Wilcox and Clark, 1961; Ogasawara and Huang, 1963; McFarquhar and Lake, 1964; Takashima and Mizuma, 1981, 1982; Wentworth et al., 1989; Okamoto et al., 1991; Khosravinia et al., 2005), the ICSI approach should have been reasonable in this study. Herein, we report that W sperm of the sex-reversed chicken can fertilize the quail oocyte, and the chicken-quail hybrid developed into blastoderm similar to that using normal chicken sperm carrying the Z chromosome.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Quail

Male and female Japanese quail (Coturnix coturnix japonica) were raised up to 6 wk of age at Shinshu University, transferred to Nagoya University, and kept in an environmentally controlled room (room temperature at 24 ± 1°C with 50 to 60% humidity). Quail were caged under a photoperiod of 14L:10D (lights on at 0500 h and off at 1900 h) with free access to food and water. To obtain the fertilized eggs, female quail were mated with male quail, and female quail were caged individually for collection of unfertilized eggs. All female quail were from the ages of 8 to 20 wk. Time of egg laying was recorded for each quail by video camera recorder system (camera: ExwaveHAD SSC-DC430, Sony, Tokyo, Japan; camera adapter: YS-W170, Sony; VHS time lapse: SVT-S 960 ES, Sony). The birds laid eggs consecutively for more than 7 d in a sequence from 1400 to 1900 h. Time of ovulation was estimated by the time of oviposition. Ovulation was considered to occur within 30 min after oviposition of the previous egg in the series.

Collection of Quail Oocytes

One quail oocyte was collected from 1 female quail. Attempts at multiple ovulations in quail were inefficient for egg collection because of variable responses of quail to pregnant mare’s serum gonadotropin and ovine luteinizing hormone treatment (Hrabia et al., 2003b). The quail oocytes were collected in relation to a midsequence oviposition, followed by ovulation of the next egg.

Quail Oocytes Unfertilized and Fertilized with Quail Sperm. For morphological standardization of early embryo development and examination of the effect of culture medium, various positive and negative control ova were prepared. Positive controls were as follows: 1) in vivo fertilized ova collected from the oviduct about 1 to 2 h after oviposition of the previous egg and cultured in the same conditions as those after ICSI, 2) in vivo fertilized ova collected from the oviduct at 20 h after ovulation, and 3) in vivo fertilized ova 30 to 40 min after spontaneous oviposition. The negative controls were as follows: 1) unfertilized ova collected within 50 min after ovulation and 2) unfertilized eggs collected from virgin quail after oviposition.

Quail Oocytes for Microinjection with Chicken Sperm. Unfertilized quail oocytes were collected from the infundibulum or upper magnum 30 to 40 min after oviposition of the previous egg in the sequence, and these eggs were used for ICSI.

Induction of Sex Reversal in Chickens

Sex-reversed chickens were made as previously described (Takagi et al., 2007). Briefly, fertile White Leghorn eggs were incubated under humid conditions at 37.8°C in a commercial incubator. On d 4 of incubation, eggs were treated with a nonsteroidal aromatase inhibitor (Fadrozole, Novartis, Basel, Switzerland). The genotypic sex of all hatchlings after Fadrozole treatment was determined by PCR as previously described (Takagi et al., 2007). Treated female and untreated male chicks were reared to 18 mo of age under conventional management and used for collection of ejaculated sperm and testicular sperm for ICSI.

Preparation of Ejaculated Sperm and Testicular Sperm

Semen was collected from normal adult males after ejaculation induced by lumbar massage. Ejaculated sperm were washed with Dulbecco’s modified Eagle medium (DMEM) 3 times by centrifugation. Testicular sperm from normal roosters and sex-reversed hens were isolated and prepared for ICSI as previously described (Takagi et al., 2007).

Culture Media and Reagents

The medium used for micromanipulation and for culturing oocytes after microinjection was DMEM containing 4 mg/mL of BSA (fraction V; Sigma-Aldrich, St Louis, MO). The medium used for collection of sperm was DMEM containing 12% (wt/vol) polyvinylpyrrolidone (360 kDa, Sigma-Aldrich). All inorganic and organic reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise stated.

Microinjection and Culture

Intracytoplasmic sperm injection was carried out according to Hrabia et al. (2003a). Under a Hoffman modulation contrast microscope (IX70, Olympus, Tokyo, Japan), a single ejaculated or testicular sperm was picked up tail first into an injection pipette, and sucking in and blowing out of the pipette was repeated until the sperm was immobilized. The oocyte was placed in DMEM in a 6-well dish, and the isolated sperm was injected immediately into the central area of the germinal disc using a micromanipulator connected to the injector (IM-9B, Narishige Instruments, Tokyo, Japan) under a stereomicroscope (SZ11, Olympus). This manipulation was performed with the aid of an image processor system (Image S-III, Nippon Avionics, Tokyo, Japan). The injected oocytes were incubated at 41.5°C under 5% CO2 in air for 20 to 24 h.

4',6'-Diamidino-2-Phenylindole Staining of the Blastoderm

Development of the embryo after culture was staged as described by Eyal-Giladi and Kochav (1976). The blastoderms were dissected from the yolk and fixed in ethanol-glacial acetic acid (3:1) and used for 4',6'-diamidino-2-phenylindole (DAPI) staining either in paraffin section or in whole mount as described by Hrabia et al. (2003a) and Waddington et al. (1998), respectively. Photographs of the DAPI-stained blastoderms were taken under a fluorescent microscope (Axioskop 2, ZEISS, Oberkochen, Germany) using a digital camera (AxioCam, ZEISS). Following the photography, all blastoderms were transferred to a 0.5-mL tube individually and stored at –30°C until assayed using the PCR for W chromosome identification of sperm.

W Chromosome Identification in Chicken-Quail Hybrid Blastoderms

The Identification of W Chromosome Derived from Testicular Sperm of Sex-Reversed Chicken. The blastodermal samples were mixed with 30 µL of sterile H2O, heated to 99°C for 15 min to expose the DNA, and were then placed on ice. One microliter of each DNA sample was amplified with initial denaturation at 97°C for 1 min followed by 5 cycles of 97°C: 5 s, 72°C: 30 s; 5 cycles of 97°C: 5 s, 65°C: 10 s, 72°C: 30 s; 5 cycles of 97°C: 5 s, 60°C: 10 s, 72°C: 30 s; and 35 cycles of 97°C: 5 s, 55°C: 10 s, 72°C: 30 s with a final extension at 72°C for 7 min. Primer sets used were W chromosome-specific XhoI primers, XhoI-For.: 5'-CCCAAATATAACACGCTTCACT-3' and XhoI-Rev.: 5'-GAAATGAATTATTTTCTGGCGAC-3' (Clinton et al., 2001). The PCR product in 9 µL of the reaction was separated through a 2% agarose gel by electrophoresis in Tris-borate-EDTA buffer and stained with ethidium bromide before visualization under ultraviolet light. The electrophoretic results were photographed (AE-6905 H Image Saver HR, ATTO, Tokyo, Japan).

The Identification of W Chromosome Derived from Quail Oocyte. One microliter of the DNA sample was amplified with initial denaturation at 97°C for 1 min followed by 40 cycles of 97°C: 5 s, 50°C: 10 s, 72°C: 30 s with a final extension at 72°C for 7 min. Primer sets used were quail W chromosome-specific Wpkci primers, quWpkci-For.: 5'-CTTCTTGGGCATTTGAA-3' and quWpkci-Rev.: 5'-CGTGAAATCCATTCGGTGGCCG-3'. The PCR products were separated by electrophoresis, and these results were photographed as above.

Animals

All procedures involving animals were conducted after approval by the Institutional Animal Care and Use Committee of Nagoya University.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Control

Figure 1Go shows examples of control blastoderm images under stereomicroscope prepared for morphological standardization of early development of quail embryos classified by Eyal-Giladi and Kochav (1976; Figure 1Go, panels A and C) as well as the presence or absence of nuclei in these blastoderms verified by DAPI staining (Figure 1Go, panels B and D). Figure 1Go, panels A and B, shows an in vivo fertilized egg of stage X after oviposition, and Figure 1Go, panels C and D, shows an unfertilized egg after oviposition. The in vivo fertilized blastoderm shows numerous nuclei stained by DAPI (Figure 1Go, panel B), whereas the blastodisc of unfertilized egg contains many vacuoles (Figure 1Go, panel C). Additionally, several granulosa cells were identified instead of blastodermal cells (Figure 1Go, panel D).


Figure 1
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  		Figure 1. Quail blastoderm and blastodisc of fertilized and unfertilized oocytes after oviposition. A) Blastoderm image under stereomicro-scope. Black arrow and arrowhead show area opaca and area pellucida, respectively. B) 4',6'-diamidino-2-phenylindole (DAPI) staining of blastoderm. White arrow shows nuclei. C) Blastodisc image under stereomicroscope. V = vacuoles. D) DAPI staining of blastodisc. White arrowhead shows granulosa cells. Bar = 50 µm.

 
In vivo fertilized oocytes collected from the infundibulum or upper magnum developed to stage IX in DMEM 20 to 24 h after culture (Figure 2Go, panels A and B). Oocytes collected from the oviduct 20 h after fertilization developed to stage X (Figure 2Go, panels C and D). Fertilized eggs after oviposition developed to stage XI (Figure 2Go, panels E and F). The DAPI-stained nuclei showing intensive blue fluorescence in fertilized oocytes were distinguished by their round shape, size, and location. The DAPI staining showed more distinguishable nuclei. Control embryos of in vivo fertilized oocytes developed in the oviduct or in culture for 20 to 24 h always showed numerous nuclei identified by DAPI staining (Figure 2Go, panels B, D, and F).


Figure 2
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  		Figure 2. Blastoderms of in vivo fertilized oocytes and unfertilized oocytes under stereomicroscope and after 4',6'-diamidino-2-phenylindole (DAPI) staining. A, C, E, G, I) Blastoderm images under stereomicroscope. B, D, F, H, J) DAPI staining of blastoderms. A, B) Blastoderms 20 to 24 h after culture of in vivo fertilized oocyte 1 to 2 h following oviposition of previous egg. C, D) Blastoderms of in vivo fertilized oocyte collected from oviduct 20 h after ovulation. E, F) Blastoderm of in vivo fertilized oocyte after spontaneous oviposition. G, H) Blastodisc of unfertilized oocyte just after ovulation. I, J) Blastodisc of unfertilized oocyte after oviposition. A, C, E) Black arrow and arrowhead show area opaca and area pellucida, respectively. I) V = vacuoles. B, D, F) Numerous nuclei in the blastoderms. The high magnification (insets) reveals nuclei stained by DAPI. H, J) Absence of nucleus in the blastoderm. Bar = 1 mm, inset bar = 20 µm.

 
Oviposited eggs from virgin quail always contained numerous vacuoles at the periphery of germinal disc, and often part of their cytoplasm was segmented into pseudo cells (Figure 2Go, panels I and J), unlike fertilized oocytes (Figure 2Go, panels A to F) and unfertilized oocytes just after ovulation (Figure 2Go, panels G and H). In unfertilized eggs, no nuclei were seen after staining, although a part of their cytoplasm showed cleavage. Some of the negative control oocytes after DAPI staining showed irregularly fragmented DNA bodies or granulosa cells.

Intracytoplasmic Injection of Ejaculated Sperm and Testicular Sperm from Normal Roosters and Testicular Sperm from Sex-Reversed Hens

Figure 3Go shows representative photos of blastoderms of chicken-quail hybrids after ICSI. The blastoderm developed to stage VII after microinjection of an ejaculated sperm from a normal rooster into a quail oocyte (Figure 3Go, panel A), whereas the blastoderm developed to stage VI after microinjection of a testicular sperm from a normal rooster into a quail oocyte (Figure 3Go, panel B). The developmental stages of oocytes injected with ejaculated and testicular sperm from a normal rooster varied from IV to VII. Figure 3Go, panels C, D, and E show blastoderms developed to stages VI, VI, and V, respectively, after microinjection of a testicular sperm from a sex-reversed hen into a quail oocyte. The DAPI-stained nuclei were evident in all blastoderms, as observed after fertilization of quail oocytes with ejaculated sperm and testicular sperm of normal rooster and sex-reversed hen, although developmental stages varied.


Figure 3
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  		Figure 3. Typical examples of blastoderms from chicken-quail hybrids after 4',6'-diamidino-2-phenylindole (DAPI) staining. A) Blastoderm of quail oocyte injected with ejaculated sperm from a normal rooster. B) Blastoderm of quail oocyte injected with testicular sperm from a normal rooster. C, D, E) Blastoderms of quail oocyte injected with testicular sperm from a sex-reversed hen. Bar = 1 mm, inset bar = 20 µm.

 
Table 1Go summarizes the results in which a single ejaculated sperm and testicular sperm from normal rooster and testicular sperm from sex-reversed hen were microinjected into quail oocytes. Seven of 31 oocytes (22.6%) injected with ejaculated sperm from a normal rooster were fertilized, whereas 6 out of 30 oocytes (20.0%) injected with testicular sperm from a normal rooster were fertilized. On the other hand, 9 out of 58 oocytes (15.5%) injected with a sperm from a sex-reversed hen were fertilized.


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  		Table 1. Development of quail oocytes injected with ejaculated sperm from normal roosters and testicular sperm from normal roosters and sex-reversed hens
 
W Chromosome Identification of Chicken-Quail Hybrid Blastoderms

Nine chicken-quail hybrid blastoderms developed after injection of a sperm from a sex-reversed chicken into a quail oocyte were analyzed by the PCR assay to identify the source of the W chromosome, viz. chicken sperm or quail oocyte. Figure 4Go, panel A, shows the PCR result of chicken Xho-I family DNA fragments obtained from red blood cells (RBC) of a normal male and female chicken and quail and sperm from a sex-reversed hen injected into quail oocyte. The predicted band of about 416 bp is characteristic for female DNA in the chicken and is absolutely absent from the male chicken and male and female quail RBC samples (Figure 4Go, panel A). The female-specific chicken DNA products were detected in only 2 out of 9 hybrid blastoderms. Figure 4Go, panel B, shows the PCR result of quail Wpkci DNA fragments obtained from the same samples as those used for the above analysis of chicken Xho-I family DNA fragments. The predicted band of about 221 bp is characteristic for the female-quail Wpkci DNA fragments and is absolutely absent from the male quail as well as from male and female chicken RBC samples. The female-specific quail DNA products were detected in 6 out of 9 hybrid blastoderms. Neither chicken Xho-I family DNA nor quail Wpkci DNA fragments were detected in 1 hybrid blastoderm of the number 5 sample (Figure 4Go, panel B, lane 5).


Figure 4
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  		Figure 4. Polymerase chain reaction identification of W chromosome derived from sperm of a sex-reversed hen and quail oocyte. A) PCR products were amplified by chicken W chromosome-specific XhoI primer. B) PCR products were amplified by quail W chromosome-specific quail Wpkci primer. Left lane shows molecular marker. N = no template; C = chicken; Q = quail; F = female red blood cell; M = male red blood cell; 1 to 9 = blastoderms of quail oocyte after injection with testicular sperm of sex-reversed hen.

 
Table 2Go shows the stages of blastoderm development after microinjection with ejaculated or testicular sperm from a normal rooster. The developmental stages of these blastoderms were as follows: 2 blastoderms developed to stage IV, 2 blastoderms developed to stage V, 2 blastoderms developed to stage VI, and 1 blastoderm developed to stage VII (Table 2Go). The developmental stages of blastoderms injected with testicular sperm were as follows: 2 blastoderms developed to stage IV, 1 blastoderm developed to stage V, 2 blastoderms developed to stage VI, and 1 blastoderm developed to stage VII (Table 2Go).


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  		Table 2. Developmental stages of quail oocytes injected with ejaculated or testicular sperm from normal roosters
 
Table 3Go summarizes the sex chromosomes and species constituents of the hybrids by PCR assay. For this summary, any sperm without chicken Xho-I family DNA are assumed to be those with chicken Z chromosomes. Likewise, any oocyte without quail Wpkci DNA are assumed to be those with quail Z chromosomes. Accordingly, there is 1 hybrid with chicken Z and quail Z chromosomes (cZqZ), 6 hybrids with chicken Z and quail W chromosomes (cZqW), and 2 hybrids with chicken W and quail Z chromosomes (cWqZ; Table 3Go). The developmental stages of those hybrid blastoderms varied as follows: 1 cZqZ hybrid developed to stage VI, 2 cZqW hybrids developed to stages III, 1 cZqW hybrid developed to stage IV, 2 cZqW hybrids developed to stage V, 1 cZqW hybrid developed to stage VI, 1 cWqZ hybrid developed to stage III, and 1 cWqZ hybrid developed to stage V (Table 3Go).


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  		Table 3. Developmental stages of chicken-quail hybrid blastoderms after injection with testicular sperm from sex-reversed hens
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present paper indirectly confirmed the report of Hrabia et al. (2003a) that a single sperm is capable of activating the oocyte for fertilization and subsequent embryonic development. The difference is that Hrabia et al. (2003a) injected a quail sperm into a quail oocyte, but I injected a chicken sperm. However, the rate of fertilization after ICSI of spontaneously ovulated oocytes (22.6%) is comparable to or higher than that of Hrabia et al. (2003a), who demonstrated 16.6% efficiency. The embryonic development is also comparable between the 2 studies (stage II to VII). The present study also demonstrated that testicular sperm from normal chickens can fertilize the quail oocyte, and the fertilized oocyte can develop into a blastoderm similar to ejaculated sperm from normal rooster. This is consistent with our previous finding when quail sperm were injected into quail oocytes (Hrabia et al., 2003a).

Furthermore, the testicular sperm from sex-reversed chickens also can fertilize the quail oocyte. More importantly, the sperm carrying W chromosome of sex-reversed chicken has a fertilizing ability similar to Z-carrying sperm, and the chicken-quail hybrid can develop to stages III to VI. Recently, we demonstrated that the sperm carrying the W chromosome from sex-reversed hens had oocyte-activating ability in the mouse oocyte (Takagi et al., 2007). In this study, 6 out of 32 mouse oocytes (18.8%) showed 2 pronuclei that were identified as those containing chicken W chromosome; however, this approach was not able to demonstrate fertilizing and developmental ability of the W sperm. In the present study, our principal aim was to reveal whether the W chromosome-containing sperm could become functional sperm and whether they could fertilize the oocyte. The present study clearly demonstrated that W chromosome-containing sperm are viable and functional after ICSI.

Kosin (1945) observed degenerated nuclei in the germinal disc of the unfertilized eggs of chicken. Some breeds of turkeys and strains of chicken exhibit limited parthenogenesis. In most cases, this spontaneous development without fertilization lead to very early degeneration of the embryo. In a selected line of turkeys, a low percentage of parthenogenetically derived embryos hatched. To our knowledge, there has been no report of parthenogenesis in quail.

In summary, the results obtained demonstrate for the first time that intracytoplasmic injection of a single sperm isolated from chicken semen or testicular sperm into quail oocytes can activate the oocyte and lead to fertilization, and testicular sperm of the sex reversed-hen carrying W chromosome can fertilize and further develop into a blastoderm. Accordingly, these results suggest that the production of W sperm could contribute to avian genetic resource preservation.


   ACKNOWLEDGMENTS
 
We thank H. Hasegawa (Japan-Ciba-Geigy, Takarazuka, Japan) for providing us with aromatase inhibitor (CGS16949A, Fadrozole).

Received for publication December 12, 2006. Accepted for publication January 10, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Eyal-Giladi, H., and S. Kochav. 1976. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev. Biol. 49:321–337.[Web of Science][Medline]

Frankenhuis, M. T., and H. J. Kappert. 1980. Experimental transformation of right gonads of female into fertile testes. Biol. Reprod. 23:526–529.[Abstract]

Fulton, J. E. 2006. Avian genetic stock preservation: An industry perspective. Poult. Sci. 85:227–231.[Abstract/Free Full Text]

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Hrabia, A., S. Takagi, T. Ono, and K. Shimada. 2003a. Fertilization and development of quail oocytes after intracytoplasmic sperm injection. Biol. Reprod. 69:1651–1657.[Abstract/Free Full Text]

Hrabia, A., S. Takagi, and K. Shimada. 2003b. Variable responses to luteinizing hormone on induction of ovulation in PMSG-primed laying quail. J. Poult. Sci. 40:231–238.

Khosravinia, H., H. N. Narasimha Murthy, and K. Prathap Kumar. 2005. Scope for interspecific hybridization of chicken and quail. J. Poult. Sci. 42:363–368.

Kosin, I. L. 1945. Abortive parthenogenesis in the domestic chicken. Anat. Rec. 91:245–251.

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