Animal husbandry – Avian egg treatment or production
Reexamination Certificate
2002-02-12
2004-02-17
Abbott, Yvonne (Department: 3644)
Animal husbandry
Avian egg treatment or production
C424S582000
Reexamination Certificate
active
06691638
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns methods of transferring primordial germ cells to birds for the production of gametes therein. Such methods are useful in the conservation of endangered avian species, in reducing the time required to produce spermatozoa from slowly maturing species such as turkeys, decreasing the costs of maintaining breeder flocks, and altering the sex ratio of offspring flocks (e.g., to enhance the efficiency of production).
BACKGROUND OF THE INVENTION
The ability to more easily produce gametes of particular avian species would be extremely useful to the avian veterinary and poultry production fields. For endangered species such as the whooping crane, it would be extremely useful to have a ready supply of male spermatozoa. For commercial birds such as turkeys, it would be desirable to more quickly and economically produce male spermatozoa. For meat-producing flocks, it is desirable to have ways to increase the ratio of male birds in the flock. Accordingly, there is a need for new ways to obtain avian spermatozoa.
Chimeras are composite organisms consisting of cells derived from more than one zygote. Experimental chimeras have been used to study cell to cell interaction and cell lineage analysis during development (A. McLaren,
Mammalian Chimeras
. Cambridge University Press, Cambridge (1976)). When chimeras are produced using material derived from very early embryos, organisms develop containing a full mixture of somatic tissues. If the starting material includes early germ cells or their precursors, the resulting individuals will produce gametes of both the donor and recipient genotypes. In addition, chimeras can be intraspecific, i.e. between two zygotes of the same species, or interspecific, i.e. between two different species.
Avian primordial germ cells (PGCs) like other vertebrate germ cells are extragonadal in origin and must undergo a complex journey to reach the gonad. The transfer of blastodermal cells and primordial germ cells has produced avian germline chimeras. Reynaud (
J. Embryol. Exp. Morphol
. 21:485-507 (1969)), a pioneer in the production of avian germline chimeras, reported the production of turkey-chicken germline chimeras by the intravascular transfer of dissociated turkey germinal crescent cells into previously sterilized chick embryos (accomplished by exposure of the recipient germinal crescent to ultra-violet light). PGCs obtained by mechanical dissociation of the endoderm of the germinal crescent (stage 5) were injected into the blood vessels of chicken embryos (3-5 days of incubation). Prior to injection the recipient embryos were sterilized at stage 8-10 (H&H) with ultraviolet light; however, the sterilization was not complete and caused problems with development and mortality. The turkey PGCs in the chick embryo were identified solely on the basis of their nucleo-cytoplasmic ratio. This method of identification was difficult and tenuous and could not be used for actively dividing turkey PGCs since the dividing germ cells gave an aberrant nucleo-cytoplasmic ratio.
In a succeeding study, the transferred PGCs were allowed to undergo maturation in the host gonads and apparently could give rise to gametes but they were not suitable for fertilization (Wilhelm,
Roux's Arch. Dev. Bio
. 179:85-110 (1976)). The spermatozoa were incapable of fertilizing turkey eggs. They fertilized chick eggs but there was no normal development. Chicken spermatozoa were capable of activating the eggs obtained from female interspecific chimeras but they did not give rise to embryos. When the eggs were fertilized by turkey spermatozoa they developed into abnormal embryos that did not survive beyond stage 38 (H&H). Reynaud (
J. Embryol. Exp. Morphol
. 21:485-507 (1969)) used morphology as the only distinguishing characteristic in an attempt to identify turkey germ cells from chicken germ cells. Morphology alone is not sufficient for identifying chimeras and must be substantiated with other markers.
By reducing endogenous PGCs, the efficiency of generating germline chimeras, by repopulating the gonads with the desired donor PGCs, may be enhanced. A number of approaches to reduce PGCs have been utilized with varying degrees of success. Continuous exposure (20 days) to gamma irradiation (0.3-3.4 R/hr,
60
Co) resulted in the complete destruction of oocytes at a dosage level of 3.4 and 1.8 R/hr (Mraz and Woody,
Radiation Research
54:63-68 (1973)). However, hatchability was reduced at levels of 0.9 R/hr or higher. The application of continuous low-level gamma irradiation to reduce endogenous PGC is limited due to the relatively small numbers of eggs that can be exposed at any one time and the long period of exposure required.
Short-term exposure to a gamma source has also been attempted (Carsience et al.,
Development
117:669-75 (1993); Thoraval et al.,
Poultry Sci
. 73:1897-1905 (1994); Maeda et al.,
Poultry Science
77:905-07 (1998)). In these studies, unincubated eggs were exposed to 500-700 rads just prior to the injection of stage X blastodermal or area pellucida cells. The incidence of germline chimerism following short-term gamma irradiation was highly variable. The basis for the inconsistent results were ascribed to “donor cells being injected into an inappropriate location . . . ” (Carsience et al.,
Development
117:669-75 (1993)).
Attempts to sterilize recipient embryos using ultraviolet light have been described (Reynaud,
J. Embryol. Exp.Morphol
. 21:485-507 (1969); Reynaud, J.,
Roux's Archives of Developmental Biology
179:85-110 (1976); Aige-Gil and Simkiss (
Brit. Poul. Sci
. 32:427-438 (1991)). Aige-Gil and Simkiss concluded “it is not possible to irradiate the germinal crescent, particularly at stage 4 of incubation, without inducing major abnormalities”. The level of sterility appeared to be positively correlated with developmental abnormalities, thus limiting the practical use of UV-light as a means to reduce endogenous PGC.
The compound busulfan (1,4-butanediol dimethane sulfonate, BU) has been used as a chemotherapeutic agent in the treatment of leukemia (Bhagwatwar et al.,
Cancer, Chemotherapy & Pharmacology
37:401-08 (1996)). In 1963, Hemsworth and Jackson demonstrated that the administration of BU in rats could markedly impair the development of PGCs (Hemsworth and Jackson,
J. Reproduction
&
Development
6:229-33 (1963)). Injection of BU into the yolk sac of chick embryos resulted in multiple malformations (Swartz,
Teratology
21:1-8 (1980)). Hallett and Wentworth (
Poultry Science
70:1619-23 (1991)) also report significant declines in hatchability following injection of an albumen suspension of BU into quail eggs. In some BU treated quail, there appeared to be an absence of germ cells in the gonads, while other similarly treated birds appeared normal. The authors suggested that “inconsistencies in the delivery of BU to the embryo” might explain the observed variation. They concluded that discovering a non-toxic solvent system would be necessary to eliminate the inconsistent results associated with use of a suspension. Aige-Gil and Simkiss (
Brit. Poul. Sci
. 32:427-438 (1991)) used saline or sesame oil suspensions of BU, or solublized BU in dimethyl sulphoxide (DMSO) in chick embryos. Administration of DMSO alone produced embryonic mortality, developmental delays, and malformations that exceeded those observed with saline. The teratogenic effects were greatly minimized when BU was suspended in sesame oil and injected into yolk. Injection of 100 &mgr;g BU in sesame oil resulted in a sterility index of 95+%. In a subsequent experiment, Vick and co-workers (
J. Reproduction
&
Fertility
98:637-41 (1993)) reported that the injection of 25, 50 and 250 &mgr;g BU significantly reduced gonadal germ cells in chick embryos. They estimated that BU treatment increased the rate of germline chimerism 3.5-fold when compared to non-BU treated embryos. Bresler et al. (
British Poultry Science
35:241-47 (1994)) demonstrated that treatment with BU and subsequent injection of PGCs could result in a signific
D'Costa Susan
Pardue Samuel
Petitte James
Song Yonghong
Abbott Yvonne
North Carolina State University
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