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Offspring Produced from Orthotopic Transplantation of Chicken Ovaries¹

Agassiz Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada, V0M 1A0

² Corresponding author: Silversidesf{at}agr.gc.ca

	ABSTRACT

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The loss of avian genetic variation and the threat of disease lend urgency to the cryopreservation of remaining poultry stocks. However, techniques for freezing ova and embryos are not available for birds, and the recovery of genetic material has been a major obstacle to cryopreservation. To overcome this problem, we transplanted chicken ovarian tissue just after hatch with or without subsequent treatment of the recipient with an immunosuppressant. Nine of 12 hens in the nonimmunosuppressed group and 6 of 9 birds in the immunosuppressed group produced eggs, whereas 3 hens in each group produced donor-derived offspring. These results suggest that transplantation of ovarian tissue of chickens is possible if performed just after hatch. This finding should allow efficient cryopreservation of female germ cells in chickens with regeneration in live birds. In addition, ovarian transplantation could be useful for studies in genetics or developmental biology or could provide convenient access to the female germline for genetic manipulation.

Key Words: ovarian transplantation • offspring • chicken

	INTRODUCTION

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Avian genetic resources are invaluable for fundamental research in medicine and agriculture and for continued efficient production of meat and eggs. However, poultry stocks kept by industry and public institutions are being eliminated at an alarming rate (Fulton and Delany, 2003; Miller, 2004), and infectious disease poses a constant threat to live birds, making it imperative that remaining lines be stored cryogenically. Techniques for freezing ova and embryos, although used widely for some mammalian species, are not available for birds because the avian ovum is attached to the large structure of the yolk. Avian embryonic (blastodermal, primordial germ) cells can be frozen and used to generate germline chimeras, but their use for genetic conservation is limited by low efficiency and complex procedures (Petitte, 2006). Semen can be used to cryogenically store avian genetic material, but without the counterpart of the ovum is only efficient for the recovery of single genes. Transplantation of ovarian tissue could allow cryopreservation of ova in birds.

Attempts at transplanting ovaries were pioneered in the chicken nearly 100 yr ago but were first successful in the mouse. The first report of ovarian transplantation was in an inbred strain of mice maintained by forced heterozygosis (Robertson, 1940). The techniques for orthotopic ovarian transplantation were further developed using mice of inbred strains and various hybrids among them (Russell and Hurt, 1945; Stevens, 1957; Jones and Krohn, 1960). Currently, normal fertility in rodents that differ by a single genetic character (Sztein et al., 1998; Dorsch et al., 2004) or in immunodeficient mice (Gunasena et al., 1997) is routinely restored by ovarian transplantation.

In 1908, Guthrie exchanged ovaries between white-feathered and black-feathered chickens to study the effect of a foreign soma on the germplasm and suggested that all the transplanted ovaries had normal function (Guthrie, 1908). However, the genetic background of the chickens that Guthrie used was questioned by other researchers (Castle, 1911; Davenport, 1911). Using the same transplantation procedure, Davenport (1911) demonstrated that the offspring of the transplanted hens were from regenerated host ovarian tissue. A later attempt at ovarian transplantation using chicks from 24 to 30 d of age did not succeed (Grossman and Siegel, 1966) because of difficulties with surgery and immunological incompatibilities between the host and the donor. We recently developed a surgical technique for orthotopic transplantation of ovarian tissue in newly hatched chickens and demonstrated that the donor ovaries could attach and undergo development in the host (Song and Silversides, 2006). The present study aimed to determine whether the hens with transplanted ovaries could lay normal eggs and produce offspring derived from the transplanted ovarian tissue.

	MATERIALS AND METHODS

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Birds
Barred Plymouth Rock (BPR, Line 60) and White Leghorn (WL, Blue Line) chicks from pure lines maintained at the Agassiz Research Center (Silversides et al., 2006) were used as donors and recipients, respectively. All methods used were approved by the Animal Care Committee of the Agassiz Research Center and followed principles described by the Canadian Council of Animal Care (1993).

Ovarian Transplantation
Ovarian transplantation was carried out as described previously (Song and Silversides, 2006). Briefly, ovaries were isolated from BPR chicks that had been freshly euthanized by cervical dislocation, cleaned of connective tissue, and cut into 2 or 3 pieces. Ovarian tissue was kept in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St. Louis, MO) on ice until transplantation within 4 h. The WL chicks to be used as recipients were anesthetized by i.m. injection of 0.5 mg of ketamine (Ketaset, Ayerst Veterinarian Laboratories, Guelph, Ontario, Canada) and 0.1 mg of xylazine (Rompun, Bayer Inc., Toronto, Ontario, Canada). With the chick on a heated operating surface, an incision was made distal to the last rib to expose the left side of the abdominal cavity. The yolk sac was removed and abdominal organs displaced to expose the ovary, which was removed using fine forceps. In chickens and most other birds, only the left ovary is functional (Johnson, 1986). Two pieces of BPR ovarian tissue were placed into each WL recipient in the original position of the ovary and were covered with the greater abdominal air sac. Immediately after surgery, chicks were given an i.m. injection of 5 mg of an antibiotic (EXCENEL, Pharmacia Animal Health, Orangeville, Ontario, Canada). The procedure was performed when both donors and recipients were <24 h old.

Surgery was successful for 21 chicks, and 9 of these were given an oral dose of the immunosuppressant mycophenolate mofetil (CellCept, Hoffmann-LaRoche Ltd., Mississauga, Ontario, Canada) at 100 mg/kg per day for 2 wk, followed by once a week until they were 2 mo of age. The remaining 12 chicks received no immunosuppressant treatment.

Progeny Test
Surgically manipulated birds were kept in a brooder for 2 wk with an initial temperature of 33°C and subsequently raised in pens with a temperature of 25°C and a day length of 10 h. At approximately 15 wk of age, the pullets were individually caged with the same conditions of day length and temperature.

White Leghorn chickens (white-feathered) are homozygous for the dominant white gene (II; Smyth, 1990), and BPR chickens (black-feathered) are homozygous for the wild-type allele at this locus (ii). A cross of WL and BPR chickens produces offspring that are white with a few randomly distributed black spots (Ii). At the start of egg production, recipient hens were inseminated with pooled semen from 4 to 6 BPR roosters to determine the genetic origin of their offspring. Eggs were incubated and candled at 5 to 7 d of incubation to determine fertility. Some of the eggs were opened at 15 to 17 d of incubation, by which time feather color is evident, and some were allowed to continue development until hatch. Any black chicks (ii) produced from this cross were from transplanted ovarian tissue, and any white chicks (Ii) were from regenerated host ovarian tissue. The progeny test was terminated when the hens had laid eggs for at least 2 mo.

Statistical Analysis
The PROC GLM procedure of SAS (Littell et al., 1991) was used to compare the number of donor-derived offspring from hens in the immunosuppressed and nonimmunosuppressed groups, and contingency ² (Zar, 1999) was used to compare the fertility of eggs from the 2 groups. Statistical significance was set at P < 0.05.

	RESULTS

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All manipulated birds grew to sexual maturity. Nine of the 12 hens in the nonimmunosuppressed group and 6 of the 9 birds in the immunosuppressed group produced eggs (Table 1). Of these, 3 hens in each group produced donor-derived (black) offspring (Figure 1, panel A). Among these hens, 2 in each group produced only donor-derived offspring, and 1 hen in each group produced both donor-derived and host-derived chicks (Table 2). The remaining 9 hens produced only white chicks from regenerated host ovarian tissue until they were killed after 57 to 89 d of lay. Necropsies were performed on hens that did not lay eggs and showed that the ovary was enclosed by a membranous sac containing internally ovulated yolks.

View larger version (118K): [in this window] [in a new window]   Figure 1. Offspring from chicken ovarian transplants. White eggs opened at 15 to 17 d of incubation were laid by White Leghorn hens transplanted with Barred Plymouth Rock ovaries. Black embryos were from transplanted ovaries, and white embryos were from regenerated ovaries (panel A). Live black chicks were hatched from each successfully transplanted hen. The White Leghorn hen 62553 produced 42 black chicks in 67 d of collecting period and is shown here with 5 black chicks produced from eggs laid on 5 consecutive days (panel B).

	DISCUSSION

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This study demonstrated that ovarian transplantation between 2 lines of chickens performed immediately after hatch with or without subsequent immunosuppression can result in the production of normal eggs giving rise to donor-derived offspring. Avian yolk is synthesized in the liver, transported in the blood, and transferred to the oocytes by receptor-mediated endocytosis (Bujo et al., 1997). Transplantation of ovarian tissue well before follicular maturation allowed development of the required blood supply, and freshly ovulated yolks from transplanted tissue were accepted by the infundibulum for incorporation into normal eggs. Others (Gilbert and Wood-Gush, 1970) have shown that yolks from an ovary that had been dislocated within the abdomen can enter the infundibulum, showing that exact repositioning of the ovary is not required.

This report is the first of successful ovarian transplantation in avian species. Although ovarian transplantation can be performed in adult mice differing in a single genetic character (Robertson, 1940; Stevens, 1957), previous efforts to transplant ovaries in chickens of at least 3 wk of age have been unsuccessful (Guthrie, 1908; Davenport, 1911; Grossman and Siegel, 1966). In the present study, chicken ovaries were transplanted within 24 h of hatching. This timing was based on a report (Silversides and Smyth, 1986) that skin transplantation among chicks at day-of-age is possible and on a pilot experiment showing that skin grafts in day-old chicks were usually accepted and those in 2-wk-old chicks were usually rejected (Song and Silversides, 2006). The donor and recipient lines of chickens used in this study are genetically diverse and have been maintained as pure lines for more than 50 and 35 generations, respectively (Silversides et al., 2006). Our successful production of offspring from ovarian transplants suggests that there is a window just after hatch that allows grafting in chickens. This finding is significant for transplantation experiments in avian species, because there is no immunodeficient animal model available for birds as there is in mice.

In the present study, donor-derived offspring were produced from both immunosuppressed and nonimmunosuppressed recipients. Although immunosuppression was not necessary for interline ovarian transplantation, fertility of eggs was higher for the immunosuppressed group, and the number of donor offspring with immunosuppression appeared to be higher, suggesting that selectively inhibiting T-cell and B-cell proliferation with mycophenolate mofetil (Morris et al., 1990) could be useful for ovarian transplantation among outbred lines of poultry or even among different species of birds.

The immediate application of the present research is to design cryopreservation protocols for the conservation of female gametes in poultry. The large, yolky egg of birds prevents cryogenic storage of female gametes. Without the counterpart of ovum, cryopreservation of semen cannot be used to store and recover highly inbred or specially selected lines of poultry. Recent advances in the manipulation of avian embryonic cells (Van de Lavoir et al., 2006) suggest that these cells might offer a means to preserve and reconstitute poultry. However, the low efficiency and complexity of the procedures (Petitte, 2006) make them too costly for germplasm conservation. Cryopreservation and transplantation of ovarian tissue have been used for genomic banking of genetically important laboratory mice and rats (Sztein et al., 1998; Dorsch et al., 2004). The texture and structure of ovarian tissue in day-old chicks is very similar to that of adult mice and is well-suited for cryostorage, because the primary oocytes are peripherally located and developmentally dormant (Hughes, 1963). The transplantation of chicken ovaries should allow efficient cryopreservation of female germ cells with subsequent regeneration in live birds.

Transgenic chickens are considered to be an ideal bioreactor for the production of therapeutic proteins in eggs (Ivarie, 2003) and are a useful tool for developmental biologists (Mozdziak and Petitte, 2004). However, production of transgenic chickens has been technically challenging, partially owing to the nature of the reproductive system of the hen and the difficulty in genetic modification of avian embryonic germ/stem cells (Sang, 2004; Van de Lavoir et al., 2006). Transplantation of ovarian tissue may allow access to the female germline in chickens for the transfer of genes. In addition, the techniques used in the present study might be applied to other poultry or to rare and endangered birds with regeneration in closely related species.

	ACKNOWLEDGMENTS

 
We thank Beth McCannel, Lee Struthers, Harold Hanson, and Kathy Ingram for the care of the birds. This research was funded by Poultry Industry Council (Guelph, Ontario, Canada) and Agriculture and Agri-Food Canada.

	FOOTNOTES

 
¹ Agriculture and Agri-Food Canada contribution number 744.

Received for publication July 31, 2006. Accepted for publication August 28, 2006.

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