U.S. patent application number 12/357982 was filed with the patent office on 2009-05-28 for in ovo activation of an egg in the shell.
Invention is credited to Tim Cantrell, Andrew Wooten.
Application Number | 20090133633 12/357982 |
Document ID | / |
Family ID | 26878084 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090133633 |
Kind Code |
A1 |
Cantrell; Tim ; et
al. |
May 28, 2009 |
IN OVO ACTIVATION OF AN EGG IN THE SHELL
Abstract
The present invention relates to the field of avian
reproduction. In particular, the present invention provides a
method of activating an egg in a shell. The invention also provides
a method of activating an egg in a shell, whereby a live chick is
hatched.
Inventors: |
Cantrell; Tim; (Clermont,
GA) ; Wooten; Andrew; (Chandler, AZ) |
Correspondence
Address: |
Ballard Spahr Andrews & Ingersoll, LLP
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
26878084 |
Appl. No.: |
12/357982 |
Filed: |
January 22, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11824229 |
Jun 29, 2007 |
7481179 |
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12357982 |
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09784575 |
Feb 15, 2001 |
7237505 |
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11824229 |
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60182432 |
Feb 15, 2000 |
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60182969 |
Feb 16, 2000 |
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Current U.S.
Class: |
119/6.8 |
Current CPC
Class: |
A01K 67/02 20130101;
A01K 45/007 20130101; A61D 19/00 20130101 |
Class at
Publication: |
119/6.8 |
International
Class: |
A01K 67/00 20060101
A01K067/00 |
Claims
1-87. (canceled)
88. A method of producing a live chick from an oviposited
unfertilized avian egg in the shell, wherein the live chick
contains heterologous nucleic acid, wherein the egg comprises a
yolk enclosed by a membrane and an ovum, wherein the method
comprises: a) activating the ovum; b) introducing heterologous
nucleic acid into the egg; and c) incubating the egg until
hatching.
89. The method of claim 88, wherein the heterologous nucleic acid
encodes a pharmaceutical protein, an antigen, a hormone or an
antibody.
90. The method of claim 88, wherein the heterologous nucleic acid
comprises Avian Leukemia Virus-derived transducing particles.
91. The method of claim 88, wherein the heterologous nucleic acid
encodes a protein.
92. The method of claim 88, wherein the heterologous nucleic acid
is stably integrated into the avian genome.
93. A method of producing a live chick from an oviposited
unfertilized avian egg in the shell, wherein the live chick
contains heterologous nucleic acid, wherein the egg comprises a
yolk enclosed by a membrane and an ovum, wherein the method
comprises: a) activating the ovum by delivering a sperm sample
comprising avian sperm in a physiologically acceptable carrier into
the egg; b) introducing heterologous nucleic acid into the egg; and
c) incubating the egg until hatching.
94. The method of claim 93, wherein the heterologous nucleic acid
encodes a pharmaceutical protein, an antigen, a hormone or an
antibody.
95. The method of claim 93, wherein the heterologous nucleic acid
comprises Avian Leukemia Virus-derived transducing particles.
96. The method of claim 93, wherein the heterologous nucleic acid
encodes a protein.
97. The method of claim 93, wherein the heterologous nucleic acid
is stably integrated into the avian genome.
Description
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/824,229, filed on Jun. 29, 2007 (now
allowed), which is a continuation-in-part of U.S. patent
application Ser. No. 09/784,575, filed on Feb. 15, 2001 (now U.S.
Pat. No. 7,237,505), which claims priority to U.S. Provisional
Patent Application No. 60/182,432, filed Feb. 15, 2000, and U.S.
Provisional Patent Application No. 60/182,969, filed Feb. 16, 2000,
which applications are hereby incorporated herein by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of avian egg
activation. In particular, the present invention relates to methods
of activating an egg in a shell. The invention also relates to a
method of activating an egg in a shell, whereby a live chick is
hatched.
[0004] 2. Background Art
Traditional Breeding
[0005] Typically, breeding in the poultry industry is carried out
by either one of two systems:
[0006] Floor Breeding Program. The first system is called "floor
breeding" and it is utilized to produce the vast majority of all
commercial hatching eggs. In this system males are simply added
into the flocks of females at a typical ratio of between 10 and 15
percent. The floor breeding system, even with its inefficiencies,
is currently the low-cost system for producing hatching eggs
because it requires less labor than competing systems. Average
hatch rates range from approximately 83% for broiler breeders to
92% for layer breeders. Even though this system has been the
backbone of the poultry industry for many years, it has many
limitations.
[0007] Size Versus Reproductive Capacity: Floor breeding is no
longer practiced at all in turkeys due to the intense selection for
increased muscle yield that has rendered commercial turkey breeds
incapable of natural mating. The same trend is being seen in
broilers. Selection for increased size in broilers has compromised
fertility and mating ability and it is predicted that fertility
will continue to decline as body weights increase. This presents a
dilemma for poultry producers because decreases in fertility have a
direct negative impact on their bottom line.
[0008] Inefficient Waste Removal: Natural mating must be performed
on solid floors to avoid injury to the birds. This design
requirement precludes the use of automated waste removal systems
and necessitates manual cleaning between successive flocks of
birds. This adds to labor and overhead costs while decreasing the
productive use of facilities.
[0009] Egg Production & Quality: Since eggs remain in the
houses with the flock until collection time; eggs are frequently
contaminated with dirt and fecal material which can reduce hatch
rates. In addition, typically between 3 and 5% of the eggs produced
in floor houses are laid directly on the floor rather than in the
provided laying boxes and must be discarded.
[0010] Inefficient Space and Equipment Utilization: Maintaining
males and females together in a floor house requires the
installation of two independent feed and watering systems because
of different nutritional and production requirements for each sex.
It also requires the installation of laying boxes and automated egg
collection systems. All of this equipment occupies limited floor
space in the house. For these reasons floor rearing is not an
efficient use of housing space and equipment when compared to
stacked cage systems.
[0011] Mortality & Fertility: Aggressive males tend to fight,
leading to higher male mortality rates. Male mortality rates
average 13% in floor houses versus 2% in cage houses. Male
aggressiveness towards hens during mating gradually takes a toll in
the form of increased female mortality, decreased fertility, and a
decrease in the length of the egg production cycle. As the males in
one flock get older, fertility starts to decline. The standard
solution is to "spike" the flock with young males to improve
fertility. However, this sets off another round of aggression with
a short-term decrease in fertility and an increase in mortality.
Disease is more common in floor houses because of the constant
contact of the birds with bedding and waste material that harbor
pathogenic organisms.
[0012] Decreased Feed Conversion: Controlling feed costs is
critical to running a competitive poultry operation. Feed costs can
account for up to 60% of the cost of raising a broiler chick, for
instance. In one study, birds raised on the floor consumed 20% more
food for the same amount of production when compared to those
raised in cages. This difference is due to the increased level of
social interactions as well as the generally higher level of
physical activity seen in floor houses. Males consume more feed
than females, making the floor breeding system inefficient with
respect to feed consumption due to the large numbers of males that
must be maintained.
[0013] Limited Flexibility in Breeding Strategies: Due to the fact
that males and females are housed in one large group in the floor
breeding house arrangement, the breeder is very restricted in their
ability to perform advanced crosses and selections on the breeding
stock. For this reason floor houses are primarily utilized as a
tool for the multiplication of pre-selected genetic stocks to
produce final commercial crosses.
[0014] Artificial Insemination Breeding Program: Another system
utilized to generate hatching eggs is called artificial
insemination (AI). AI is widely practiced by "primary breeders" at
the top of the breeding pyramid but not generally used by
commercial producers at the bottom of the pyramid. Primary breeders
are companies that own and improve the elite pedigreed genetic
lines that are crossed to produce the final commercial
products-broilers, layers and turkeys. The quantities of birds
increase exponentially as you move down the breeding pyramid from
the pedigreed lines through the grandparent stock, parent stock,
and finally to the actual commercial birds. While birds of elite
genetic makeup at the top of the pyramid are very expensive, birds
at the bottom are inexpensive. For these reasons, different
operational models are utilized for reproduction at different
level.
[0015] In the AI system, males and females are housed in the same
houses but are caged separately. The female cages typically hold
between two and five hens, while the male cages hold a single
rooster. AI programs address many of the limitations of the floor
breeding houses listed above. For example, since cage houses are
utilized, waste removal can be performed automatically. Houses are
generally much cleaner, leading to fewer disease problems. Egg
production is improved because eggs roll out of the cages and is
not laid on dirty floors. Equipment and housing space are utilized
more efficiently. Mortality is minimized due to a decrease in
social aggression and disease. Fertility levels are maintained more
consistently because social and physical interaction are eliminated
from the process of reproduction. Feed conversion is increased. And
finally, the production system has increased flexibility for doing
advanced crosses and selections. This capability is absolutely
required by primary breeders in order to improve their genetic
stocks and to stay competitive in the marketplace. While most of
the advantages listed above are also important for commercial-level
multiplication breeders, they are offset by one crucial
shortcoming, the high labor costs associated with AI programs.
[0016] AI programs replace the innate sexual drive of poultry with
human labor. Workers must manually collect semen from males in
cages and inseminate females in cages on a 7-day rotation. The
level of sophistication required in these programs mandates a
skilled workforce. For this reason, the AI program, though
operationally superior, is economically impractical for
commercial-level breeding programs. Even the use of dwarf hens, an
innovation that allows similar egg production with about 30% less
feed consumption, can not justify the increased labor costs of the
AI program for commercial level multiplication breeders.
Reproductive Process
[0017] At the time of ovulation, the avian oocyte comprises a
blastodisc, or germinal disc, which contains the female pronucleus,
and a yellow yolk mass. The germinal disc and yolk mass are
surrounded by the oocyte cell membrane, called the oolemma.
Surrounding the oolemma is the perivitelline layer (PL), also
referred to as the inner perivitelline layer (IPL). The space
between the oolemma and the IPL is termed the perivitelline space,
which is traversed by granulosa cells. Once the oocyte is released
from its ovarian follicle, it is referred to as an ovum. The ovum
moves into the oviduct where it is engulfed by the infundibulum,
where fertilization occurs if sperm are present.
[0018] As the ovum passes into the posterior infundibulum, another
layer, the outer perivitelline layer (OPL), surrounds the ovum.
This membrane acts to prevent polyspermy, which is a lethal
condition that occurs when multiple sperm bind to and penetrate the
ovum at the region of the blastodisc. The egg is then surrounded
with additional layers of chalaza and thick and thin layers of
albumen. When the ovum moves into the isthmus, two shell membranes
are deposited, upon which small crystals of calcium carbonate are
deposited, thus beginning the formation of the shell.
[0019] The preceding events all occur within the first few hours
following fertilization. The ovum next moves into the uterus, where
over the next 18-20 hours, the calcium shell is completed. The egg
then moves into the vagina for several minutes, and then is
extruded from the vagina, or oviposited (i.e., "laid"). At this
point, if the egg has been fertilized, the embryo contained therein
will have 40,000 to 70,000 cells. (Johnston, "In Vitro Sperm
Binding, Penetration, and Fertilization of Recently Oviposited
Chicken Eggs," December 1988, Clemson University); Olsen, M. W., J.
Morph. 70: 413-533 (1942); Etches et al., in Methods in Molecular
Biology, vol. 62 Recombinant Gene Expression Protocols, Ed. R.
Tuan, Humana Press, Inc. Totowa, N.J., pp. 433-450 (1997); Petitte
et al., in Manipulation of the Avian Genome, Ed. Etches et al., CRC
Press, Boca Raton, Fla., pp. 81-101 (1993)).
Transgenesis
[0020] It has long been a goal of avian geneticists to supplement
traditional selection procedures by inserting desirable genes
directly into the avian germline. Substantial progress along these
lines has been made in mammalian species where early embryos are
accessible and where pronuclei can be visualized for the insertion
of exogenous DNA. The production of transgenic mice, cattle, sheep,
and goats has become a routine procedure in large commercial
operations. In avian species, by contrast, the early embryo has not
been easily accessed and the pronuclei are visually obscured,
making manipulation of the avian genome in early embryos in ovo an
almost insurmountable goal.
[0021] The development of genetically modified birds largely
requires access to early stage, pluripotent embryonic cells. As the
earliest stages of embryo development occur within the oviduct of
the female, the introduction of heterologous DNA into the early
embryo has only been possible by removing the embryo from the
female, requiring that she be sacrificed. Furthermore, while
culture of the early stage embryo to hatch has been accomplished,
the method is extremely laborious, and results in a post-hatch
survival rate of only about 10%. Sang et al., in Manipulation of
the Avian Genome, Ed. Etches et al., CRC Press, Boca Raton, Fla.,
pp. 121-133 (1993).
[0022] The present invention provides a ground-breaking improvement
in avian biology by making it possible to activate an oviposited
egg in its shell, and to hatch a live bird from the shell, herein
called in ovo activation. Such methods provide an alternative to
floor breeding and artificial insemination that can greatly
increase the efficiency of poultry production. Such methods also
provide access to early avian embryos such that transgenic avian
species can be more readily developed.
SUMMARY OF THE INVENTION
[0023] The present invention relates to the field of avian
reproduction. In particular, the present invention provides a
method of activating an egg in a shell. The invention also provides
a method of activating an egg in a shell, whereby a live chick is
hatched.
[0024] Also provided by the present invention is a developmentally
early stage oviposited avian egg.
DETAILED DESCRIPTION OF THE INVENTION
[0025] As used herein, "a," "an" or "the" may mean one or more. For
example, "an" egg may mean one egg or more than one egg. Moreover,
"the" egg may mean one egg or more than one egg.
[0026] As used herein, "activation" means the initiation of embryo
development in an unfertilized oviposited avian egg or oocyte.
Various forms of activation are set forth below. The process of
activating an oviposited egg in a shell is referred to herein as
"in ovo activation," (IOA).
[0027] The present invention provides a method of activating an
avian egg in a shell, wherein the egg comprises a yolk enclosed by
a membrane and an ovum, comprising activating the ovum. The present
invention relates to the unexpected and surprising discovery that
an unfertilized, oviposited avian egg can be activated in the shell
and produce a live chick. As used herein, reference to an avian egg
in a shell refers to an oviposited egg, that is, an egg with a
calcium carbonate shell that has been extruded from the vagina of
the bird. Extrusion of the egg is referred to as "oviposition."
Accordingly, all references herein to an "egg in a shell" or to an
"oviposited egg" should be understood to be equivalent in
meaning.
[0028] An avian egg comprises a hard, calcified shell at the time
the egg is oviposited. Within the shell is a yolk that contains
nutrients for supporting growth and development of an embryo. As
used herein, an "embryo" is a developing organism resulting from
the joining of a female pronucleus and a male pronucleus during the
process of egg fertilization. While a fertilized (single cell) ovum
may thus be called an embryo, the single cell embryo is also
specifically referred to herein as a zygote.
[0029] Although in ovo activation can be performed on eggs as old
as 2 weeks if the eggs are maintained at room temperature, ideally
newly oviposited eggs are used for the best results. In a preferred
embodiment, activation occurs between 0 and 96 hours following
oviposition. In a more preferred embodiment, the activation occurs
between 0 and 72 hours following oviposition. In an even more
preferred embodiment, activation occurs between 0 and 48 hours
following oviposition. In a highly preferred embodiment, the
activation occurs between 0 and 24 hours following oviposition.
Thus, it is preferred that the activating event occur as soon as
possible following oviposition. However, the precise timing can
depend on how the oviposited egg is maintained, e.g., temperature,
humidity, etc. For example, activation can improve if the
unfertilized oviposited egg is activated before it is allowed to
cool.
[0030] In the methods of the invention, activation of the avian egg
in the shell is accomplished by mechanically disrupting the ovum or
delivering a biological sample, e.g., a sample comprising one or
more of a sperm, cell or nucleus, into the oviposited egg.
Disruption or delivery of the biological sample may be accomplished
by any method which will allow the sample to be delivered inside
the shell, including, but not limited to, dissolving an area of the
shell with, e.g., an acid solution, using electroporation, and
creating an opening by penetrating or cracking an area of the
shell, for example using a tool such as a needle or a scalpel.
[0031] Preferably, the surface of the area of the shell to be
penetrated in order to deliver the sample or disrupt the ovum is
sanitized before the sample is delivered inside, to prevent
contamination of the egg. Any method which is compatible with the
delivery method may be used to sanitize the shell, including, but
not limited to, the disinfectant IOFEC-20.RTM., and 3% hydrogen
peroxide. The surface of the egg at the intended penetration site
may be wiped or sprayed with the disinfectant, or the egg may be
immersed in a vessel containing the disinfectant of choice.
[0032] As is described above, an opening in the shell can be made
with a tool such as a knife or a needle. Preferably, the tool will
be sterile. For example, in a two-step procedure, an opening in the
shell can first be made with a knife or other sharp instrument. In
a second step, a needle attached to a syringe containing a sample
can be passed through the opening to deliver the sample into the
egg. Introduction of the sample into the opening in the shell may
also be accomplished by other means, including, but not limited to,
the use of a pipette, such as a micropipettor. Alternatively, in
one step, a needle attached to a syringe containing the sample can
be used to penetrate and thus create the opening in the shell and
deliver by injection the sample into the egg. Thus, "opening" can
include a hole created by a needle. Of course, one of ordinary
skill in the art will be able to choose a needle whose gauge will
be large enough to allow the sample to be moved through the needle.
In one embodiment, the needle will be of the smallest gauge that
can deliver intact into the shell and also be sturdy enough to
penetrate the calcium eggshell. Alternatively, a separate needle or
other device could be used to make the opening in the eggshell.
Typically, needles varying from 30-gauge to 16-gauge can be used.
In one embodiment a 22-gauge needle is used.
[0033] The opening can be made anywhere in the shell that effects
viable activation, but is typically made in an area of the shell
that is near the germinal disc. While an egg may be manipulated so
as to place the germinal disc at different regions of the egg, the
germinal disc in a newly oviposited egg is typically located at the
large end of the shell, which overlies the air cell adjacent to the
yolk. Once an opening has been created in the shell, the sample is
preferably delivered by introducing the needle, pipette, etc.,
through the air cell and beneath a membrane lying below the air
cell (inner shell membrane).
[0034] Preferably, to prevent contamination of the egg and death of
an embryo, the opening in the shell is sealed. A non-toxic adhesive
can be applied directly to the opening in the shell to seal it.
Alternatively, a piece of eggshell can be used as a patch to close
the opening and may be attached to the shell with a non-toxic
adhesive. In one embodiment, the non-toxic adhesive is Elmer's.RTM.
glue. In another embodiment, the adhesive is a silicone sealant.
Moreover, any "tissue glue" can also be used to seal the shell. A
"tissue glue" is a sterile, non-toxic adhesive used during
surgical, operative procedures to bind tissues together.
[0035] The method of the present invention can be used to activate
oviposited eggs from avian species selected from the group
consisting of chicken, quail, duck, turkey, pheasant, ostrich, emu,
goose, peafowl, grouse, rhea, parrot, cockatiel, cockatoo,
parakeets, and other commercially valuable birds.
[0036] The present invention also provides a method of activating
an avian egg in a shell, wherein the egg comprises a yolk enclosed
by a membrane, and hatching a live chick.
[0037] After the activation according to the methods of the present
invention as described, the egg is incubated until the live chick
is hatched. One of ordinary skill will be aware of the amount of
time and the preferred conditions for incubating a fertilized egg
belonging to a particular species. The following are incubation
periods for various species of birds: Chicken--21 days, Quail--23
days, Corunix quail--17 to 18 days, Pheasant--23 days, Turkey--28
days, Duck--28 to 33 days, Goose--28 to 30 days, Parakeet--18 days,
Parrots--28 days, Dove--14 days, Mynah--14 days, Finch--14 days,
Button Quail--16 days, Valley Quail--21 to 22 days, Swan--30 to 37
days. Incubation of eggs fertilized by the methods of the present
invention as compared to naturally fertilized eggs may differ only
in that the length of incubation time may be lengthened to include
the amount of time that the fertilized egg would have spent within
the body of the female prior to oviposition. In a preferred
embodiment, the incubation period lasts from 21 to 23 days for
chicken eggs. While one of ordinary skill in the art will readily
be able to determine the optimal temperature for incubation of an
egg from a particular species of bird, typically the incubation
temperature is between 95.degree. F. and 100.degree. F. A chicken
egg will be incubated at about 99.5.degree. F. In a more preferred
embodiment, the temperature at which the chicken egg is incubated
will be lowered as the egg nears the point of hatching. Thus, in a
currently preferred embodiment, a chicken egg is incubated at
99.5.degree. F. from day 1 of incubation to about day 18 of
incubation, and at 98.5.degree. F. from day 19 of incubation to
hatching.
[0038] As is well known in the art, the humidity level at which an
egg is incubated can be important for bringing the egg to hatch.
Thus, typically the egg is incubated at between 75% and 90%
humidity. Preferably, the egg is incubated at about 80% humidity.
More preferably, the humidity level at which the egg is incubated
will be raised as the egg nears the point of hatching. Thus, in a
preferred embodiment, a chicken egg is incubated at 80% humidity
from day 1 of incubation to about day 18 of incubation, and at 85%
humidity from day 19 of incubation to hatching. In a specific
preferred embodiment, an egg is incubated at 99.5.degree. F. and
80% humidity from day 1 of incubation to about day 18 of
incubation, and at 98.5.degree. F. and 85% humidity from day 19 of
incubation to hatching. As is well known in the art, turning the
eggs during incubation is useful for promoting growth of the
embryo.
[0039] It is further preferred that the incubation of the eggs take
place in a commercial incubator. Commercial hatchers and setters
are produced by many companies including PAS Reform, Jamesway,
Chickmaster, Buckeye, Cumberland, Petersime, Humidaire Incubator
Co., etc. Preferably, the eggs are moved from a setter incubator to
a hatcher incubator at about 3 days prior to hatch. The hatcher
basket allows the egg to lie on its side where the chick can more
easily pip out. This basket also allows the chick to walk about
immediately after hatch, which is necessary for the chick's
development and viability.
[0040] In another embodiment, the present invention provides an
oviposited avian egg comprising a native embryo having fewer than
40,000 cells, wherein the embryo can develop into a live chick.
"Native" means growing, living or produced in its place of origin.
Thus, a native embryo is an embryo that develops and hatches in the
same shell in which the female pronucleus was formed. Thus, the
embryo is descended from the native ovum. By the time an ovum which
has been fertilized naturally has been oviposited, the developing
embryo typically has between 40,000 and 70,000 cells. However, the
egg of the present invention is fertilized after it has been
oviposited in its shell; thus, an embryo developing in the egg of
the present invention will at some time during incubation have
fewer than 40,000 cells. In fact, at the moment of activation, the
embryo in the egg of the present invention will have one cell and
is a zygote. As the embryo grows within the egg, normal cell
division will occur and the number of cells will increase. Thus,
the activated, oviposited egg of the present invention will at some
time during incubation comprise an embryo having, for example, 1,
100, 1,000, 10,000, 20,000, 30,000 or 40,000 cells, including
between these numbers of cells. Two commercially preferred avian
eggs are chicken and turkey.
[0041] In another embodiment, the present invention provides an
avian egg in a shell comprising an embryo having fewer than 40,000
cells (e.g. 30,000; 20,000; 10,000; 1,000; 100 and 1 (zygote)),
wherein the embryo can develop into a live chick, and wherein the
shell has an opening of less than 4 centimeters. In another
embodiment, the opening in the shell is less than 2 centimeters. In
another embodiment, the opening in the shell is less than 1
centimeter or 0.5 centimeter. In one embodiment, the opening in the
shell is only large enough to accommodate a 22-gauge needle. Thus,
the opening can be any size between the smallest opening that will
permit injection of a sample or means to disrupt the ovum, up to
smaller than the hole required to place an in vitro fertilized
(i.e., outside the shell) ovum back into the shell. By "opening" is
meant a hole has been made in the egg at some point after
oviposition. "Opening" includes an egg where the hole has
subsequently been sealed. For example, an egg having a hole created
by a needle used to inject a sample and then sealed is, even after
sealing, within the definition of avian egg having an opening. The
embryo can be either native or non-native to the egg. "Non-native"
includes embryos developed from an ovum not native to the shell in
which it was oviposited. Two commercially preferred eggs are
chicken and turkey. In addition, the invention provides an
oviposited avian egg comprising an embryo and a native yolk wherein
the embryo has fewer than 40,000, 30,000, 20,000, 10,000, 5,000,
1,000 or 100 cells, including numbers in between 1 and 40,000. The
chicks which hatch from these eggs can have a normal karyotype and
normal development.
[0042] An egg of the present invention may, for example, be derived
from avian species selected from the group consisting of chicken,
quail, duck, turkey, pheasant, ostrich, emu, goose, peafowl,
grouse, rhea, parrot, cockatiel, cockatoo, parakeets, swan, dove,
and other commercially valuable birds. In a commercially preferred
embodiment, the egg is derived from avian species used in the
methods of the present invention and is selected from the group
consisting of chicken, turkey, goose, duck, quail, and pheasant. In
a more preferred commercial embodiment, the egg is derived from a
chicken. The method can also be effectively utilized on avian
species in zoos, e.g., to help preserve endangered species.
[0043] The methods of the present invention can also be used for in
ovo activation of reptilian eggs. Reptilian eggs, similar to avian
eggs, comprise a yolk and female pronucleus and are protected by a
shell when they are laid. An unfertilized, oviposited reptilian egg
can be activated in the shell according to the methods of the
present invention.
[0044] The in ovo activation methods described herein can also be
utilized in conjunction with other in ovo procedures. For example,
the embryo can be vaccinated after activation. Such vaccination
procedures are well known to those skilled in the art.
Alternatively, such vaccination could occur simultaneously with in
ovo activation, provided that the vaccine did not prevent
development of the embryo.
[0045] Additionally, in ovo activation can be automated such that
multiple eggs are simultaneously activated by, for example,
injection techniques. Thus, 50, 100, 200, 300 or more eggs could be
simultaneously activated.
[0046] The unfertilized oviposited egg can be activated by various
specific activation methods as set forth below. The activation
methods described above can be accomplished for example with
fertilization, parthenogenesis, and nuclear transfer. Thus, for
example, as described below, the sample delivered for activation of
the oviposited unfertilized egg could be a sperm comprising
sample.
Fertilization
[0047] The present invention provides a method of fertilizing an
avian egg in a shell, wherein the egg comprises a yolk enclosed by
a membrane, comprising obtaining a sperm sample comprising avian
sperm in a physiologically acceptable carrier, and delivering the
sperm sample into the egg, so as to fertilize the egg. The process
of fertilizing an oviposited egg in a shell is referred to herein
as "in ovo fertilization" (IOF).
[0048] The sperm in the sperm sample may be obtained from a bird by
methods known to a person skilled in the art, such as the abdominal
massage method which is well-known to those of skill in the art.
This method allows the collection of an ejaculate (semen)
comprising sperm, seminal fluid, and transparent fluid. Transparent
fluid is a lymphlike fluid that passes from the lymph channels to
the surface of the phallus during phallic tumescence. Avian sperm
may also be obtained from commercial sources that are well known to
those of skill in the art.
[0049] In one embodiment, the sperm in the sperm sample is from a
single bird. In another embodiment, the sperm in the sperm sample
is a mixture of sperm obtained from more than one bird. When a
mixture of sperm from more than one bird is used in the methods of
the invention, the probability of successfully fertilizing the egg
can increase, because if one of the birds from which the sperm has
been collected is infertile, it is possible that the sperm
collected from the other bird or birds will be capable of
fertilizing the egg.
[0050] In a preferred embodiment, the sperm sample comprises sperm
from birds which are members of the same species, and the sperm
sample is used to fertilize eggs oviposited by hens which are
members of the same species as the sperm donors. The present
invention also contemplates the use of sperm from one species and
an egg from another species, if the sperm is capable of fertilizing
the egg.
[0051] While it is typically preferred that the sperm be used
within 30 minutes of the time that it is collected, older sperm,
and even sperm which have previously been frozen or freeze dried
may be used in the methods of the invention, as long as the sperm
retain their ability to fertilize an ovum. Where the sperm are to
be used more than 30 minutes after collection, it is preferred that
they be combined with a sperm extender, as is described below.
[0052] As mentioned above, the sperm sample also comprises a
physiologically acceptable carrier. As used herein, a
"physiologically acceptable carrier" is a fluid in which sperm
remain motile and viable. Examples of a physiologically acceptable
carrier include, but are not limited to, unaltered semen, seminal
fluid (either original to the sperm or added), transparent fluid
(either original to the sperm or added), buffered saline solution,
sperm extender, and combinations thereof. Preferably, the carrier
includes sperm extender, also referred to in the art as a diluent.
As mentioned above, the use of a sperm extender is especially
preferred where the collected sperm will not be used for
fertilization within 30 minutes after collection. M. R. Bakst, In
Manipulation of the Avian Genome, R. J. Etches and AM. Verrinder
Gibbons, eds., CRC Press, Boca Raton, Fla., pp. 15-28 (1993). As
used herein, a "sperm extender" is a physiologically acceptable
carrier that is used to dilute a sperm sample to produce a sperm
sample of greater volume in which the sperm are less concentrated.
Preferably, the composition of the sperm extender will extend the
shelf life of the sperm, as well as diluting the sperm so as to
increase the number of eggs which may be fertilized by the quantity
of sperm which has been collected. Examples of sperm extender
compositions, suggested dilution rates, optimal storage times and
conditions, and commercial sources of extender may be found in
Bakst ("Preservation of Avian Cells In: Poultry breeding and
Genetics, R. D. Crawford (ed.) Elsevier, New York, pp 91-108
(1990)). Other diluents commonly used in the poultry industry are
Lago Formulation Avian Semen Extender by Hygeia Biological
Laboratories, Semaid Turkey Extender by Poultry Health Laboratories
in Davis Calif., Beltsville Poultry Semen Extender by Tri Bio
Laboratories, Inc. in State College, Pa. In a preferred embodiment,
the sperm extender is Avidiluent. Avidiluent is produced by IMB, 10
rue Georges, Clemenceau, BP 81, 61302 l'Aigle, France. Thus, in one
embodiment, the sperm sample may comprise sperm and seminal fluid,
i.e., semen. Moreover, the sperm sample may comprise sperm and
seminal fluid which is diluted with a physiologically acceptable
carrier, including but not limited to buffered saline solution and
a sperm extender.
[0053] The sperm sample can also be prepared by methods which will
be clear to one of ordinary skill in the art, such as washing semen
from one or more birds with a solution such as buffered saline
solution or sperm extender, centrifuging the resulting solution,
removing the supernatant, and resuspending the washed sperm in a
volume of a solution such as buffered saline or semen extender. One
of ordinary skill in the art will readily understand how to achieve
the desired concentration of sperm by resuspending the sperm in the
appropriate volume of solution. For example, following
centrifugation and removal of supernatant, the packed sperm may
then be weighed, and the number of sperm then estimated by using
known values for the weight of avian sperm. The sperm may then be
resuspended in the volume required to obtain the desired sperm
concentration. Alternatively, the centrifuged sperm may be
resuspended following removal of the supernatant, and then
recentrifuged, allowing the determination of the packed sperm
volume. (Johnston, 1998). Subsequently, the concentration of the
sperm may be calculated using the formula of Maeza and Buss.
(Poultry Sci. 55:2059 (1976)).
[0054] Typically, the concentration of sperm in chicken semen is
from 300 million to 800 million per milliliter, in turkey semen
from 800 million to 1.5 billion per milliliter, in Guinea fowl
semen from 400 million to 800 million per milliliter, in Pekin duck
semen from 20 million to 600 million per milliliter. The standard
number of sperm used for artificial insemination is 100 million in
a total volume of 50 microliters. In the methods of the present
invention, because sperm are placed directly adjacent to the female
pronucleus, far fewer sperm are required to fertilize the egg.
Thus, as few as one sperm can be used in the methods of the present
invention. In fact, a large range of sperm concentrations can be
used in the present invention. In one embodiment, chicken semen is
diluted with an equal volume of Avidiluent and approximately 0.01
milliliters of this sperm sample is injected into an egg. Thus,
approximately 1 million sperm would be deposited adjacent to the
female pronucleus.
[0055] In one method of the invention, fertilization of the avian
egg in the shell is accomplished by delivering the sperm sample
into the egg. Delivery of the sperm sample may be accomplished as
described above. A sperm is approximately 0.5 um at its widest
point and 100 um in length. Therefore, in a preferred embodiment, a
needle with an inner diameter of at least 10 um can be used for
injections. In one embodiment the needle can remain in the shell
after injection. Various needles and methods now used for injection
of vaccines into eggs could be used or adapted for delivery of
sperm.
[0056] The opening can be made anywhere in the shell that effects
viable fertilization, but is typically made in an area of the shell
that is near the germinal disc. While an egg may be manipulated so
as to place the germinal disc at different regions of the egg, the
germinal disc in a newly oviposited egg is typically located at the
large end of the shell, which overlies the air cell adjacent to the
yolk. Once an opening has been created in the shell, the sperm
sample is preferably delivered by introducing the needle, pipette,
etc., through the air cell and beneath a membrane lying below the
air cell (inner shell membrane). The sperm number can be increased
or decreased, depending on where and in what form the sperm are
administered. In a further preferred embodiment, the sperm sample
is delivered into the egg using a needle. In nature, the sperm
cells must penetrate the inner perivitelline membrane and fuse with
the oolema for successful fertilization to occur. With IOF, the
sperm cells must also penetrate the outer perivitelline membrane
before successful fertilization can occur. To increase the
fertilization efficiency, one can treat the OPL or yolk membrane.
Any treatment which rendered the OPL or yolk membrane more
permeable to sperm could be utilized, for example, a non-toxic
acid, a proteolytic enzyme or physical abrasion.
[0057] In one embodiment, the needle, pipette, etc., is advanced
through the shell at an angle of approximately 15E, penetrating the
membrane lining the shell. In a method of the invention, the
needle, pipette, etc., can be advanced through the air cell, until
it meets the inner shell membrane. A person practicing the method
of the invention will know that the tip of the needle, pipette,
etc., has encountered the membrane when slight resistance to
further advancement of the tip is felt. As the tip is gently
advanced, the resistance from the membrane gives way and the tip is
allowed to barely penetrate the membrane. The sperm sample can then
be delivered into the egg, adjacent to a region of the membrane and
that is adjacent to the germinal disc. Therefore, the sperm can be
delivered just under the membrane, a procedure called
intracytoplasmic sperm injection (ICSI). Typical volumes of the
sperm sample are as small as 0.005 ml or as large as 0.10 ml. A
typical volume of injected sperm sample is about 0.01 ml.
[0058] Preferably, to prevent contamination of the egg and death of
an embryo, the opening in the shell is sealed as described above.
As described above, the method of the present invention can be used
to fertilize oviposited eggs from avian species selected from the
group consisting of chicken, quail, duck, turkey, pheasant,
ostrich, emu, goose, peafowl, grouse, rhea, parrot, cockatiel,
cockatoo, parakeets, and other commercially valuable birds.
[0059] The present invention also provides a method of fertilizing
an avian egg in a shell, wherein the egg comprises a yolk enclosed
by a membrane, and hatching a live chick, comprising obtaining a
sperm sample comprising avian sperm in a physiologically acceptable
carrier, delivering the sperm sample into the egg, so as to
fertilize the egg, incubating the egg, and hatching the live chick
from the egg. As used herein, "obtaining" includes utilizing
pre-made and pre-delivered sperm samples.
[0060] After the sperm sample has been delivered into the egg
according to the methods of the present invention as described
above, the egg is incubated until the live chick is hatched as
described above.
[0061] As described above, the methods of the present invention can
also be used for in ovo fertilization of reptilian eggs. Reptilian
eggs, similar to avian eggs, comprise a yolk and female pronucleus
and are protected by a shell when they are laid. An unfertilized,
oviposited reptilian egg can be fertilized in the shell according
to the methods of the present invention. In particular, a sperm
sample, comprising sperm from one or more reptiles of the same
species, is delivered into the unfertilized, oviposited egg through
an opening created in the shell and onto the yolk adjacent to the
female pronucleus where fertilization occurs.
[0062] As described above, the in ovo fertilization methods
described herein can also be utilized in conjunction with other in
ovo procedures. For example, the embryo can be vaccinated after
fertilization. Such vaccination procedures are well known to those
skilled in the art. Alternatively, such vaccination could occur
simultaneously with in ovo fertilization, provided that the vaccine
did not prevent development of the embryo.
[0063] Additionally, in ovo fertilization can be automated such
that multiple eggs are simultaneously fertilized by, for example,
injection techniques. Thus, 50, 100, 200, 300 or more eggs could be
simultaneously injected.
Parthenogenesis
[0064] In one embodiment of the present invention, activation of
the oviposited unfertilized egg is induced by parthenogenesis. As
used herein, "parthenogenesis" is the production of embryonic cells
from a female gamete in the absence of any contribution from a male
gamete.
[0065] In a preferred embodiment, activation by parthenogenesis of
an unfertilized oviposited avian egg can be induced by penetration
of the membrane that surrounds the yolk (yolk membrane) and
germinal disc, for example, by directing a 25-gauge needle through
the shell and into the egg to penetrate the membrane surrounding
the yolk. Preferably rupture of the yolk is avoided. Thus,
penetration and disruption of the membrane surrounding the yolk can
initiate activation of the ovum. It is contemplated that other
mechanical means of disrupting the membrane surrounding the yolk
can be used. For example, lasers, including a non-thermal YAG
(yttrium-aluminum-garnet) laser, can be used to disrupt the
membrane surrounding the yolk, instead of using a needle in this
procedure.
[0066] There is evidence that it is the transient increase in
cytosolic Ca2+ that initiates the program of egg development. The
cytosolic concentration of Ca2+ can be artificially increased
either by injecting Ca2+ directly into the egg or by the use of
Ca2+ carrying ionophores such as A23187. This activates the eggs of
all animals tested so far (Alberts 1983). Preventing the increase
in Ca2+ by injecting the Ca2+ chelator EGTA inhibits egg activation
after fertilization. Because the increase in Ca2+ concentration in
the cytosol is transient, lasting only for 2 to 3 minutes after
fertilization, it is clear that it cannot directly mediate the
events observed during the later stages of egg activation including
DNA and protein synthesis. Instead, the rise in Ca2+ concentration
serves only to trigger the entire sequence of developmental events;
some more permanent change must take place in the egg while the
Ca2+ level is high.
[0067] While the mechanism of activation is not fully understood,
it is clear that the sperm serves only to trigger a preset program
in the egg. The sperm itself is not required. An egg can be
activated by a variety of nonspecific chemical or physical
treatments. These processes are also generally thought to raise
intracellular Ca2+ (Rickord and White, 1992). For example, pricking
with a needle can activate a frog egg. (Alberts, 1983). Mouse
oocytes have been activated by exposure to Ca2+-Mg2+ free medium
(Surani and Kaufinan, 1977), medium containing hyaluronidase
(Graham, 1970), exposure to ethanol (Cuthbertson, 1983), Ca2+
ionophores or chelators (Steinhardt et al., 1974; Kline and Kline,
1992), inhibitors of protein synthesis (Siracusa et al., 1978) and
electrical stimulation (Tarkowski et al., 1970). Activation of
bovine oocytes has been reported by ethanol (Nagai, 1987),
electrical stimulation (Ware et al., 1989), exposure to room
temperature (Stice and Keefer, 1992), and a combination of
electrical stimulation and cycloheximide (First et al., 1992; Yang
et al., 1992). These methods can be applied to unfertilized
oviposited avian eggs.
[0068] One application of in ovo activation by parthenogenesis is
the rapid production of pure inbred lines of breeding stock.
Current industry practice involves the use of different
combinations of homozygous parent lines to produce unique
commercial bird products. Much effort is put into obtaining,
improving and maintaining these pedigreed lines. With
parthenogenesis, a female gamete can be induced to develop into a
live chick without a genetic contribution from the male. The
resulting chick is entirely homozygous at every allele. Thus,
homozygous pedigreed lines can be created in one generation instead
of the multiple rounds of inbreeding required today. Furthermore,
instead of using populations of birds to derive the homozygous
line, a single superior individual can be used. Because there are
individual birds in a breeding population that greatly outperform
the average, this method is a way to rapidly get the best available
genetic traits into the final commercial product.
Nuclear Transfer
[0069] Nuclear transfer/cellular micromanipulation technologies can
be utilized to activate an unfertilized oviposited avian egg. Thus,
a separate contemplated embodiment of the present invention
involves the delivery of fluid suspensions containing cellular
nuclei or "nucleoplasts." Nucleoplasts can be generated on a large
scale using certain advanced centrifugation procedures. In many
species, such as sheep, cow and mice, it has been determined that a
nucleoplast isolated from one cell can be inserted into another
enucleated cell for the purpose of generating an identical, cloned
individual. As used herein, "nuclear transfer" is the insertion of
a nucleus, also known as a nucleoplast, (either in a cell or as a
nucleus independent from a cell), into another cell in which the
native nucleus is ineffective, e.g., by removal or ablation. The
procedures described herein allow producers to efficiently
propagate elite genetic pedigrees into commercial flocks and serve
as the basis for development of an automated nuclear transfer
instrument platform.
[0070] The primary steps required for the implementation of this
strategy are as follows: (1) development of suitable cell lines and
conditions for the cultivation of nucleoplast donors; (2)
visualization and removal or ablation of nuclear structures within
avian ova and/or zygotes; (3) isolation of donor nuclei (4)
transfer of donor nuclei into ova (cytoplasts). Activation of
reconstituted ova leading to embryonic development may also be
effected.
[0071] Nucleoplast Donor Cells. Embryonic stem cells and primordial
germ cells have been shown to remain totipotent in the chicken and
can be utilized in an avian NT procedure. Cloned chickens generated
in this fashion can be valuable for applications in certain poultry
breeding schemes.
[0072] Alternatively, somatic cell lines can be advantageous for
other poultry breeding scenarios. A factor in the selection of a
somatic cell type is its ability to undergo "reprogramming."
Reprogramming can be utilized in order for the nucleoplast to
contribute to all embryonic cell lines and thus lead to normal
embryonic development. Cultured Embryonic Fibroblasts (CEF) have
been widely utilized in mammals with good results due to their ease
of culture and genetic manipulations. CEFs can be used for somatic
cell nuclear transfer in the avian system. Alternatively,
embryo-derived blastodermal cells (BCs) have been cultured and can
be used. Yet another cell type that can be used with the avian
nuclear transfer platform is an avian B cell.
[0073] An advantage of using avian B cells is their ability to
undergo high levels of recombination. This ability can be important
because it makes the process of getting exogenous DNA incorporated
into the avian genome much more efficient. Modifications performed
in any of these donor cell lines in vitro are then incorporated
into the genome of the resulting bird. Tests can be performed to
identify the most advantageous cell type. For example, nucleoplasts
can be created from these cell types and transferred to the avian
cytoplast (recipient cell). Those cell types leading to the highest
rate of embryonic survival can be incorporated into the standard
technology platform.
[0074] Nuclear transfer in mammals has been successfully carried
out using both ova and zygotes, although ova are predominantly used
for this purpose. Standard nuclear transfer procedures typically
require the removal of or ablation of the existing cellular
nucleus, preferably before the introduction of the donor nucleus.
Avian eggs are large and the distance between a donor nucleus and
the nucleus of a recipient cell is great enough that removal of the
nucleus of the recipient cell is not strictly required. However, it
is presently preferred that enucleation occur.
[0075] The cell nucleus in an avian egg is obscured by dense yolk
granules making traditional light microscopy largely ineffective
for visualization. For this reason the current method of choice for
enucleating avian ova involves the use of fluorescent nuclear
labeling. Known fluorescent nuclear dyes such as Hoescht 33342
(bis-benzamide) and DAPI (4' 6'-diamidino-2-phenylindole,
hydrochloride) can be utilized for their ability to effect maximum
visualization of the nucleus and its associated DNA.
[0076] Two standard strategies can be utilized for enucleation; (1)
removal via glass micropippette or (2) ablation with lasers. Both
strategies for cellular micromanipulation are currently in use.
Laser type and wavelength can be determined empirically.
Elimination of the fluorescent area within the cytoplasm by either
method would indicate removal of the nucleus. It is preferred at
this phase to remove not only the nuclear DNA but also the spindle
forming machinery.
[0077] A number of factors influence development after nuclear
transfer including a requirement that the reconstructed embryo
maintain normal ploidy. When a nucleus is transferred from a cell
that has begun to differentiate, the pattern of gene expression can
be "reprogrammed" from that of a differentiated cell type to that
of an early embryo. Experiments in which cell cycle has been varied
also suggest that the efficiency of this process is influenced by
both the donor and recipient cell cycle stage.
[0078] From mammals it appears that the cell cycle stage of both
donor and recipient cells influence when DNA replication occurs in
the reconstructed embryo. Due to the influence of meiosis promoting
factor (MPF) in the cytoplasm, the recipient cell may have a
greater influence (Barnes et al., 1993; Campbell et al., 1993). MPF
activity during replication can increase at the time of formation
of the spindles and can remain high during metaphase II. Nuclear
transfer to a cytoplast with a high level of MPF can be followed by
nuclear membrane breakdown, chromosome condensation, reformation of
the nuclear membrane and DNA replication regardless of the cell
cycle stage of the donor nucleus. In contrast, the nucleus
determines whether DNA replication occurs following transfer to an
oocyte with a low level of MPF activity (Campbell et al., 1993,
1994). From this work it is believed that there are two effects:
(1) a greater opportunity for reprogramming of gene expression
during specific phases of the cell cycle; and (2) a benefit from
transfer to similar phases of the cell cycle.
[0079] It has been shown that embryonic development can be enhanced
when donor nuclei are in the G0 or G1 phase of the cell cycle.
Dolly the sheep developed from an enucleated oocyte fused with a
mammary-derived cell presumed to be in G0. Likewise, Cumulus cells
in the G0 and G1 states have been used to achieve somatic cell
nuclear transfer in mice (Wakayama, et al, 1998). However, cloned
calves have been produced using nuclei from non-quiescent donor
cells (Cibelli et al, 1998). This indicates that the requirements
for successful nuclear transfer in poultry will have to be
determined empirically. Cells in various cell cycle stages can be
utilized to determine the optimum protocol. Delivery of donor
nuclei has been performed both via electrofusion of donor cells to
enucleated ova/zygotes and via direct injection of donor nuclei
into enucleated ova. Given the large size of avian eggs and the
ease of performing injections, direct injection of donor nuclei can
be the optimal method of delivery. When nuclear transfer is
performed into metaphase II arrested ova, typically it is required
to artificially activate the ova. This can be performed at various
time intervals ranging from simultaneous with, to several hours
after nuclear transfer. These same parameters can be tested for the
optimum time of activation.
[0080] An automated high-throughput system can be designed to carry
out the process. This has the potential to eliminate an entire
layer of the poultry industry called "multiplication breeding."
Typically in multiplication breeding several generations are needed
to go from pedigreed lines through grandparent lines, parent lines
and finally to the commercial birds. Populations of birds are
required for this process to work. IOA with nuclear transfer can
produce the final commercial product from a single superior bird.
The net effect is to produce flocks of genetically superior birds
in an extremely efficient breeding operation. Furthermore, the
cells utilized for nuclear or cellular transfer can be stored
indefinitely until a particular commercial product is requested by
the end-user. An equally important benefit would be the ability to
produce entire flocks of unisex birds. For instance, broiler
producers could request all male flocks for increased growth
efficiency while egg producers would obviously prefer all female
flocks.
Transgenesis
[0081] The present invention also provides a method for producing
an avian embryo containing heterologous nucleic acid comprising,
activating, e.g., fertilizing, an avian egg by the methods of the
present invention disclosed herein, and introducing a heterologous
nucleic acid into the avian egg. As used herein, nucleic acids
include, but are not limited to, DNA, cDNA, RNA, mRNA and antisense
RNA. The nucleic acids may be single, double, or multiple-stranded.
Heterologous nucleic acids can include nucleic acids not native to
the avian species and nucleic acids not normally expressed in the
introduced location in the cell or nucleus of the avian
species.
[0082] After an avian egg has been activated by IOA, the developing
embryo can be accessed at any stage of development to manipulate
the genetic makeup of some or all of its cells. Of greatest
interest for creating transgenic birds is the early embryo. IOA
allows for germ line transmission of the heterologous nucleic acid
as it allows the earliest possible introduction of heterologous
nucleic acid into the early avian embryo. This genetic manipulation
of the developing embryo can, for example, produce transgenic birds
comprising genetic material which can be used to modulate
endogenous DNA and its expression and to create cells which can
manufacture commercially valuable proteins for commercial use.
[0083] Transfer of a nucleic acid into the avian genome can be
performed by a person skilled in the art according to several known
methods. (Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988; Lennette
et al., Manual of Clinical Microbiology, 14th Ed., Amer. Soc. for
Microbiology, Washington, D.C., 1985; Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989; Antisense RNA and DNA,
D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1988)) Freshney, Culture of Animal Cells, A Manual of
Basic Technique, 2nd Ed. Alan R. Liss, Inc., New York (1987);
Centers for Disease Control Laboratory Manual, U.S. Department of
Health, Education, and Welfare Pub. No. 79-8375, p. 75, Centers for
Disease Control, Atlanta, Ga.; Fundamental Virology, 2nd Ed.
Bernard N. Fields and David M. Knipe, Chief Eds., Raven Press: New
York, 1990). Examples of methods of transferring genetic material
into the avian genome include, but are not limited to, avian
leukosis virus (ALV) transduction mediated transgenesis, transposon
mediated transgenesis, blastodermal cell mediated transgenesis,
primordial germ cell mediated transgenesis and nuclear transfer.
The heterologous nucleic acid may be introduced into the embryo at
the same time that the activating event occurs, e.g., at the same
time the sperm sample is introduced into the egg, or at a later
stage of embryonic development.
[0084] A standard method now in use for producing transgenic birds
is to produce replication deficient ALV-derived transducing
particles comprising a heterologous DNA insert. Because the
particles are not capable of replicating in the bird, there is no
risk of causing viremia and illness in the bird or humans. The
transducing particles can be administered directly to the
oviposited egg comprising the early avian embryo using the methods
described herein. For example, a window can be generated just above
the embryo and transducing particles introduced into the
subgerminal cavity of the embryo. The window is then sealed, and
the eggs are placed into incubators for development.
[0085] Another method used to transfer heterologous nucleic acid
into the avian genome is transposon-mediated transgenesis.
Transposons are genetic elements that are able to translocate or
move about within the genome of their host species. There are no
known transposons that occur in avian species. However, it has been
shown that certain transposons, reconstructed from other sources,
are able to function within the avian genome. These transposons can
be engineered to serve as useful nucleic acid vector systems for
inserting genes into the avian genome.
[0086] Further, another method for introducing heterologous DNA
into an avian embryo is blastodermal cell transgenesis.
Blastodermal or other early cells can be taken from stage X embryos
(the embryonic stage at which a fertilized egg is laid) can be
injected back into recipient embryos where they colonize and grow,
giving rise to chimeric chickens. A "chimeric" chicken is composed
of cells from two different genetic lineages. Blastodermal cells
injected into irradiated host embryos soon after they are isolated
have the ability to contribute to all tissues of the resulting bird
including the germline (cells that give rise to sperm and eggs and
thus to all following generations). Blastodermal cells have been
cultured and transfected with DNA by various methods known to
persons skilled in the art in an attempt to generate transgenic
poultry.
[0087] Thus, blastodermal cells or embryonic stem (ES) cells can be
injected into an in ovo fertilized embryo to create chimeric
poultry. These chimeric poultry can then be crossed and, assuming
they have germ line transmission of the blastodermal or ES cells,
one can create a clone of the original injected cell.
[0088] Moreover, primordial germ cell mediated transgenesis may be
used to create transgenic birds. As the developing embryo grows,
certain cells are committed to the germline to give rise to the
sperm and eggs. These primordial germ cells (PGCs) migrate to the
genital ridge of the developing gonad where they lie dormant until
the bird reaches sexual maturity. One approach to avian
transgenesis is to isolate these PGCs, genetically manipulate them
and place them back into a developing embryo for continued
development. This technical approach is comparable to the
blastodermal cell approach in both the extent to which it is
developed and its potential applications. Primordial germ cells
have been shown to contribute to the germline when injected into
recipient embryos just as blastodermal cells have.
[0089] Still another aspect of the present invention involves
delivering liquid formulations to the avian egg that provide vital
information about the genotypic status or constitution of the
individual bird. For instance, DNA and/or RNA probes could be
delivered into the avian egg for the purpose of sorting embryos by
sex or any other genotype. These probes bind specifically to their
target sequences and provide specific information about the genetic
makeup of the bird. Labeling the probes with various fluorescent,
radioactive, or chemiluminescent molecules provides highly-reliable
technology for determining genotypic status. Alternatively,
antibodies or other proteins may be utilized in a similar fashion
as reporter molecules.
[0090] Use of the present invention incorporates nucleic acids into
single-celled oocytes and early embryos allows for the
incorporation of exogenous DNA into all or most of the cells of the
resulting bird. However, in an alternative embodiment, the present
invention may be adapted to deliver these nucleic acids and nucleic
acid vectors to later stage embryos. One current protocol for the
production of transgenic poultry involves the injection of viral
particles into the sub-germinal cavity of developing avian embryos.
Another application would be for the transient expression of a
particular gene product in the avian embryo. Nucleic acid
formulations could be delivered with the specific intent of
generating egg and/or animal based bioreactor systems. Proteins,
antigens and antibodies could also be delivered to the embryo in
order to affect gene expression.
[0091] The in ovo activation methods described herein can also be
utilized in conjunction with other in ovo procedures. For example,
the embryo can be vaccinated after fertilization. Alternatively,
such vaccination could occur simultaneous with in ovo fertilization
providing however that the vaccine did not prevent development of
the embryo.
Automation
[0092] IOA can be automated such that multiple eggs are
simultaneously fertilized by, for example, injection techniques.
Thus, 50, 100, 200, 300 or more eggs can be injected
simultaneously. It will be clear to those of ordinary skill in the
art that the methods of the present invention may easily be applied
to a large-scale industrial operation, using automation to activate
newly laid eggs. Accordingly, an apparatus which has previously
been used, for example, to immunize laid fertilized eggs can be
adapted to instead introduce a sperm sample, nucleus, or adapted
for parthenogenesis in order to automate the IOA process.
[0093] As one skilled in the art appreciates, automated egg
handling technologies have been developed simultaneously by many
independent inventors worldwide. The technologies existing today
involve high-throughput systems for injecting liquid substances
into avian embryonated eggs. Examples include the injection of
vaccines to improve the immune state of resulting chicks, injection
of viruses for the production of human and animal vaccines, and
injection of proteins to influence chick health, growth, and the
like. Another useful automated system for poultry production allows
detection of live versus dead chick embryos-based on either
candling with various light sources or temperature differentials
between neighboring eggs in an egg flat. These technologies, taken
together, represent the current state of development in this field.
However, there exists a need for a more advanced platform adapted
specifically to the needs of avian breeders, a need that the
present invention addresses and satisfies.
[0094] Recent progress in the area of sperm preservation, taken
together with the advent of IOF technology, have made it possible
to move towards a much more efficient management model for poultry
breeding operations. Several of the important genetic technologies
described below can be linked together to create a truly versatile
platform for the automation of poultry breeding technologies.
[0095] In order to perform IOA, e.g., IOF, in an automated fashion,
two steps are contemplated. The first step involves calculating the
number of sperm cells in the sperm sample, or sample containing
cells or nucleus containing material for transfer, that is loaded
onto the machine. This task can be accomplished through the use of
an integrated spectrophotometer unit (sometimes called a
densimeter) similar to those marketed by Animal Reproduction
Systems of Chino Calif. and others. This task can also be performed
by a flow cytometer. The information obtained from this analysis is
communicated to a central processing unit or other analyzing
system, in which optimal volumes of sperm and semen extender
(diluent), or cells or nuclear material, are determined. This
information is then communicated to a fluid dispensing mechanism,
resulting in the correct fluid volumes to be dispensed into a
central fluid reservoir of an egg injection mechanism or similar
system.
[0096] For the second step, the central processing unit activates
the egg injection mechanism, which delivers the proper amount of
the diluted sperm sample or cells or nucleus containing material
formulation into the unfertilized egg to the specific depth and at
the appropriate angle to accomplish IOA, e.g., IOF. The process of
injection includes perforation of the egg shell by a tubular punch
and insertion of an injection needle through the shell membrane and
possibly the yolk membrane for delivery of the sample
formulation.
[0097] The egg injection mechanism may be of a design similar to
those manufactured and sold by Embrex, Inc., Merck Inc., and others
in the industry. As an example, one design is disclosed in U.S.
Pat. No. 4,903,635, entitled "High Speed Automation Injection
System for Avian Embryos," which is incorporated herein by
reference. As described in the patent, the disclosed device is a
high-speed automated injection system for avian embryos, which can
inject eggs with fluid substances, specifically an inoculating
fluid. The machine includes suction devices which lift eggs out of
engagement with surfaces, rather than pushing them, before
injecting them. Thus, the machine provides separate mechanisms and
devices for first forming an opening in the egg shell and then
injecting the avian embryo or the surrounding environment with a
fluid substance, avoiding use of a single needle or punch to both
puncture the shell of an egg and deliver fluid substances to the
interior of the egg. As is also known in the art, the present
invention here also contemplates using a single needle both to
puncture the shell and to deliver fluid substances. Other relevant
patents that disclose injection of fluids into eggs include U.S.
Pat. Nos. 5,900,929, entitled "Method and Apparatus for Selectively
Injecting Poultry Eggs"; 5,722,342, entitled "Ovo Antibiotic and
Microbial Treatment to Diminish Salmonellae Populations in Avians";
5,699,751, entitled "Method and Apparatus for in Ovo Injection";
5,438,954, entitled "Method and Apparatus for Early Embryonic in
Ovo Injection"; 5,339,766, "Method of Introducing Material into
Eggs During Early Embryonic Development"; 5,176,101, "Modular
Injection System for Avian Embryos"; 5,158,038, "Egg Injection
Method, Apparatus and Carrier Solution for Improving Hatchability
and Disease"; and 5,136,979, "Modular injection system for avian
embryos," all of which are incorporated by reference. In the
simplest embodiment for IOF, sperm is substituted for antigen in
these machines and the depth of injection is adjusted to accomplish
IOF.
[0098] The process of IOA, e.g., IOF, makes it possible to design
systems capable of high-throughput operation for the activation,
e.g., fertilization, of avian eggs after they reach the hatchery.
Advantageously, the present invention for IOF requires much smaller
quantities of sperm for this direct fertilization approach, making
it possible to streamline operations by reducing dramatically the
number of males in the breeding scheme. The remaining males could
be centrally housed with enough fresh sperm being delivered
directly to the hatchery to fertilize the billions of eggs hatched
in the industry every year. The use of fewer males would allow
breeders to make more rapid genetic progress in improving their
lines by using only the very elite performers for inseminations. In
addition, the industry's infrastructure could be re-rationalized
based on the elimination or significant reduction of males. For
instance, a much more efficient commercial egg laying operation
could be directly substituted for the existing hatching egg
facilities since only unfertilized eggs are required. In artificial
insemination programs where females represent from approximately
ninety-five to ninety-eight percent (95-98%) of the flock, it will
be possible to eliminate the need for manual insemination, and thus
remove approximately over ninety-five percent (95%) of the current
labor requirement associated with these programs.
[0099] For automated activation by nuclear transfer, one can
further adapt the egg injection mechanism to include a means to
render the native nucleus ineffective. For example, the mechanism
can include a micropipette or laser to either remove or render the
native nucleus ineffective.
[0100] In addition to activation, e.g., via fertilization, it is
contemplated that the present invention also includes the processes
of genetic analysis, manipulation, and propagation. As one skilled
in the art appreciates, these tools are currently designed for
laboratory usage requiring highly-skilled technicians and,
accordingly, have been impractical to date for routine usage in the
low-margin poultry industry. As implemented in the present
invention, many of these technologies are amenable to incorporation
into a totally automated platform for use by production personnel,
as well as geneticists and other researchers.
[0101] The present invention can be designed to include additional
mechanisms and steps to deliver various liquid formulations
intended to (a) impact gene expression in the avian embryo
concurrently with and/or subsequent to In Ovo Activation, e.g.,
IOF, and/or (b) to provide vital information as to the genotypic
status of the embryo. The delivery apparatus may be similar to the
egg injection mechanisms for sperm delivery discussed above. The
present invention may employ a common fluid reservoir with the
sperm or a separate reservoir independent of the sperm, in which
separate needles are inserted into the egg for each fluid, or the
fluids are injected sequentially through a single needle inserted
into the egg.
[0102] The present invention is also contemplated to incorporate a
detection mechanism to aid in various types of genetic and protein
based analyses. One embodiment of this mechanism would involve the
use of a light source of the appropriate wavelength, such as, for
example, a laser, and a corresponding dye set differentially
expressed in the avian egg as an indicator of certain genotypic or
physiologic states. For instance, this embodiment could sort eggs
by sex utilizing fluorescence-labeled sexing probes, sex-linked
promoters and expression systems, fluorescence-labeled sexing
antibodies, and the like.
[0103] The detection mechanism may, for example, use a CCD camera
or other suitable detector of fluorescent signals to activate a
sorting and/or identifying mechanism. In another embodiment, the
detection mechanism could utilize a scintillation counter for
radioaction and/or chemiluminescence-based detection methods for
the same general purposes described above. In yet another
embodiment, the system could utilize "gene-chip," genetic
microarray and/or genetic macroarray technologies for detection
purposes, an example of which is that produced by the company
Affymetrix.
[0104] As one skilled in the art appreciates, classification of
birds according to genotype may be used in production operations.
Classification of birds by sex allows the optimization of
production capacity. That is, males are desired in the broiler
industry, while females are desired as layers. Also, the present
invention, providing an enhanced ability for geneticists to perform
genetic selections based on the automated high-speed identification
and genotyping of eggs, results in more rapid genetic progress
towards developing improved poultry lines.
[0105] Independent of which type of detection is utilized for
genotypic classification, another contemplated design involves
classifying each egg as "live" or "dead." Simple light and/or
temperature mechanisms are also contemplated for this procedure as
incorporated in existing systems by PAS Reform, Breuil, and Embrex.
For this aspect of the present invention, U.S. Pat. No. 5,900,929
assigned to Embrex is incorporated herein by reference.
[0106] Coupled with the sorting devices described below, the
present invention injecting the liquid formulation that provides a
predetermined indication and the detection thereof provides a
versatile platform for all manner of molecular detection
applications.
[0107] Still another embodiment of the present invention
incorporates a liquid sampling device for obtaining liquid samples
from the avian eggs. This design uses a vacuum line in
communication with a sampling needle, the tip of which is
reciprocated to be surrounded by the liquid portion of a respective
avian egg and then removed therefrom. Alternatively, the design may
use an electro-osmotic gradient, similar to that utilized in the PE
Biosystems 310 genetic analyzer, to draw fluid samples into a
sampling capillary.
[0108] In order to utilize maximally the above-described detection
mechanism(s), it may be necessary at certain times to amplify the
signal by various techniques. One embodiment for the amplification
of a detection signal could be the incorporation of an integrated
thermal cycling unit, such as those produced by PE Biosytems,
Hybaid MJ Research and others, for DNA amplification. This device
would be important--if not essential--for the use of gene chip,
micro-array, and macro-array based genotyping devices discussed
above.
[0109] To obtain more detailed information about the samples being
analyzed, it is also contemplated separating the sample molecules
based on size, molecular weight, electric charge or other
chemical/physical properties. One embodiment of this separations
mechanism is an electrophoresis unit. For example, an integrated
capillary electrophoresis unit such as or similar to the 310
Genetic Analyzer produced by PE Biosystems could be utilized to
separate both nucleic acids and proteins. This embodiment of the
present invention would, for instance, be useful for
high-throughput genotyping of eggs from a primary breeder's
pedigreed lines of poultry.
[0110] Ultimately, the data obtained from the above embodiments,
either singularly or collectively, can be processed by software in
a central processing unit or other device to evaluate the value and
status of eggs as they come off the processing line. The evaluated
eggs can be labeled, sorted, and transferred to hatching baskets or
trays accordingly. Labeling can be performed by an ink jet
mechanism, similar to that currently found in Hewlett Packard and
Epson printers. Sorting and transfer of the eggs can be performed
by automated suction cups and movable belts which transport trays
of eggs through the instrument body and to the waiting egg carts.
Mechanisms for this part of the instrument could use designs
similar to systems manufactured and sold by Breuil, Kuhl
Corporation, Pas Reform, and Embrex.
[0111] The instrument platform being described here would also
benefit from certain existing genetic/protein analysis
capabilities. By directly incorporating the analysis capabilities
into the platform these procedures could be performed at high-speed
and at an industrial scale. Examples where these analysis
capabilities would be directly applicable to the commercial poultry
industry are described.
EXAMPLES
[0112] The following Examples are set forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the methods claimed herein are performed, and is
intended to be purely exemplary of the invention and is not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.) but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. F. and
pressure is at or near atmospheric.
Example 1
[0113] 1. Forty-three freshly laid unfertilized Barred Rock chicken
eggs were disinfected by wiping the shells with 3% hydrogen
peroxide and placed in racks. 2. Sperm was obtained from 4 barred
rock roosters on the same morning and collected in Vacutainer.RTM.
vials less than 1 hour before the fertilization procedure was
performed. 3. Sperm was pooled from the 4 roosters and mixed with 1
ml of Avidiluent.RTM.. 4. Sperm mixed with Avidiluent.RTM. was
drawn into a 1 ml syringe through a 1'', 22 gauge needle to form a
sperm sample. 5. The needle created an opening in the large, blunt
end of the eggshell and passed through the opening at a 15.degree.
angle to the surface of the shell. 6. The needle was passed through
the air cell until the tip just penetrated the membrane enclosing
the yolk and germinal disc. 7. One drop, 0.05 ml, of the sperm
sample was injected onto the surface of the yolk adjacent to the
membrane. 8. The needle attached to the syringe was withdrawn from
the egg. 9. The opening created in the shell by the needle was
patched with a small piece of shell, and the patch was secured to
the shell with an adhesive such as Elmer's Glue.RTM.. 10. The eggs
were placed in commercial grade setters maintained at 99.5.degree.
F. and 80% humidity from day 1 to about day 18 of incubation. The
eggs were turned according to methods known in the art and used in
commercial setters. 11. On day 19, the eggs were transferred to
commercial hatchers and maintained at 98.5.degree. F. and 80%
humidity until hatching.
[0114] Ten days after the fertilization method of the present
invention was performed on 43 eggs, routine candling of the eggs
was performed to determine which eggs had been successfully
fertilized. Thirty-five eggs of the 43 eggs had been fertilized. Of
the 35 fertilized eggs, 32 were successfully brought to hatching,
and all but one of the chicks were healthy. Thus, 72% of the 43
oviposited eggs treated by the fertilization method of the present
invention produced a healthy live chick.
Example 2
[0115] Data on "Hy-Line Variety Brown" commercial brown egg laying
hens: 1. 270 freshly laid eggs were collected at 6:30 in the
morning. 2. Semen was immediately collected from Black Giant males
into diluent at a 50:50 ratio. 3. Eggs and semen were delivered to
the lab within 20 minutes of semen collection. 4. Eggs were divided
into two groups, experimental and negative control with 135 eggs
each. 5. Experimental eggs were injected as previously described
with 10 ul of the diluted semen preparation. 6. Negative controls
were not injected. 7. Injected eggs were sealed with silicone
sealer and placed in the incubator as previously described. 8.
Fertility was checked after 5 days and recorded. 9. 33 of 135 eggs
(24%) were determined to be fertile in the experimental group. None
of the negative control eggs showed signs of development.
Example 3
Parthenogenesis
[0116] 1. 270 freshly laid eggs were collected in the morning. 2.
Eggs were divided into experimental and negative control groups of
135 each. 3. An opening 2-3 mm was created through the eggshell and
shell membranes such that the yolk is visible. 4. The membrane that
surrounds the yolk and germinal disc was gently "pricked" and/or
penetrated, taking care not to rupture the yolk. (Alternatively,
creation of the opening and penetration of the yolk membrane can be
performed at the same time by simply inserting a 25 gauge syringe
needle through the shell and into the yolk). 5. Eggs thus treated
were then sealed with silicon sealer and placed in the incubator.
6. Eggs were checked for development at 5 days and development
rates recorded before returning to the incubator. 7. Eggs were
allowed to develop until hatching when hatch rates and health
conditions are recorded.
Results:
[0117] 1. Eggs with ruptured yolks were discarded, leaving 131
experimental eggs and 135 negative controls. 2. Development was
checked after 5 days with 7 of 131 (5%) pricked eggs showing
obvious signs of embryonic development. 3. Of the 7 eggs showing
embryonic development, 5 hatched producing normal healthy chicks.
4. None of the negative control groups showed signs of embryonic
development.
[0118] Throughout this application, various publications are
referenced. The disclosures of these publications, and the
references cited therein, in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art to which this invention
pertains.
[0119] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and example be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the claims.
* * * * *