U.S. patent application number 11/768046 was filed with the patent office on 2008-01-31 for methods for injecting avian eggs.
This patent application is currently assigned to Embrex, Inc.. Invention is credited to Molly Bland, John Hebrank, Dipak Mahato, Nandini Mendu, Stephen Wolfe.
Application Number | 20080022931 11/768046 |
Document ID | / |
Family ID | 23209489 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080022931 |
Kind Code |
A1 |
Mendu; Nandini ; et
al. |
January 31, 2008 |
Methods for Injecting Avian Eggs
Abstract
The present invention provides improved methods of injecting an
avian egg containing an embryo, preferably an early embryo (e.g., a
blastoderm). The methods of the invention may be used to deliver a
substance to an egg, remove a sample from an egg, and/or to insert
a detector device into an egg to collect information therefrom. In
preferred embodiments, the invention is used to deliver a substance
to the embryo in ovo. In other preferred embodiments, the invention
is used to produce chimeric or transgenic avian embryos in ovo.
Inventors: |
Mendu; Nandini; (Chapel
Hill, NC) ; Bland; Molly; (Cary, NC) ; Wolfe;
Stephen; (Chapel Hill, NC) ; Hebrank; John;
(Durham, NC) ; Mahato; Dipak; (Raleigh,
NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
Embrex, Inc.
|
Family ID: |
23209489 |
Appl. No.: |
11/768046 |
Filed: |
June 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10216427 |
Aug 9, 2002 |
7249569 |
|
|
11768046 |
Jun 25, 2007 |
|
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|
60312015 |
Aug 13, 2001 |
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Current U.S.
Class: |
119/6.8 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 15/8509 20130101; A01K 45/007 20130101; A01K 2227/30 20130101;
A01K 67/0271 20130101; A01K 2267/03 20130101 |
Class at
Publication: |
119/006.8 |
International
Class: |
A01K 45/00 20060101
A01K045/00 |
Claims
1-60. (canceled)
61. A method of producing a chimeric bird, comprising the steps of:
orienting an avian egg containing a blastoderm in a predetermined
position; introducing a small opening into a shell of the egg;
extending a delivery device through the small opening in the egg
shell; piercing an inner shell membrane with the delivery device,
wherein the inner shell membrane is essentially intact prior to
inserting the delivery device therethrough; releasing an avian
donor cell through the delivery device and depositing the donor
cell into the blastoderm or in close proximity thereto under
conditions sufficient to result in a chimeric embryo; retracting
the delivery device from the egg; incubating the chimeric embryo to
hatch to produce a chimeric bird.
62. The method of claim 61, wherein the delivery device is extended
through the small opening in the egg shell to a location selected
from the group consisting of: (a) the subgerminal cavity, (b)
between the area opaca and the vitelline membrane, (c) between the
area pellucida and the vitelline membrane, (d) between the area
opaca and the area pellucida, (e) in the area pellucida, (f) in the
area opaca, and (g) any combination of (a) to (f) above.
63. A method of producing a transgenic bird, comprising the steps
of: orienting an avian egg containing a blastoderm in a
predetermined position; introducing a small opening into a shell of
the egg; extending a delivery device through the small opening in
the egg shell; piercing an inner shell membrane with the delivery
device, wherein the inner shell membrane is essentially intact
prior to inserting the delivery device therethrough; releasing a
substance comprising a nucleotide sequence through the delivery
device and depositing the substance into the blastoderm or in close
proximity thereto under conditions sufficient to result in a
transgenic embryo; retracting the delivery device from the egg;
incubating the transgenic embryo to hatch to produce a transgenic
bird.
64-65. (canceled)
Description
RELATED APPLICATION INFORMATION
[0001] This application is a divisional of U.S. application Ser.
No. 10/216,427, filed Aug. 9, 2002 (allowed), which claims the
benefit of U.S. Provisional Application No. 60/312,015 filed Aug.
13, 2001, which is incorporated by reference herein in its
entirety.
FILED OF THE INVENTION
[0002] The present invention relates to methods of manipulating an
egg containing an embryo and, in particular, to methods of
introducing material into or removing material from an egg
containing an avian embryo.
BACKGROUND OF THE INVENTION
[0003] There are a number of applications for which it is desirable
to inject eggs containing early avian embryos. For example, it may
be desirable to deliver a substance to an early embryo, such as a
blastoderm. To illustrate, it may be desirable in the poultry
industry to manipulate an early embryo in ovo to introduce a
foreign nucleic acid molecule (i.e., to create a transgenic bird)
or to introduce a foreign cell(s) (i.e., to create a chimeric bird)
into the developing embryo.
[0004] Likewise, improved methods of injecting eggs containing an
early embryo may be used to remove samples from eggs, including
samples of embryonic and extra-embryonic materials. Further, for
other applications it may be desirable to insert a sensing device
inside an egg containing an embryo to collect information
therefrom.
[0005] Current methods of manipulating bird eggs containing early
embryos may be undesirable because they may result in unacceptably
low hatch rates. It has been suggested that depressed hatch rates
are a result of the opening made in the egg shell, introduction of
air bubbles into the egg, damage to the extra-embryonic membranes
or to the embryo itself, or a combination of these factors.
[0006] Accordingly, there is a need in the art for improved methods
of manipulating avian eggs containing early embryos.
SUMMARY OF THE INVENTION
[0007] The present invention provides a method of manipulating
avian eggs containing embryos, in particular early stage embryos,
which may result in reduced trauma to the embryo and improved
viability and hatch rates. Accordingly, the present invention
provides improved methods of inserting or implanting a variety of
devices (e.g., a delivery device, sampling device, and/or a
detector device, and the like) into an avian egg containing an
embryo, in particular an early embryo (e.g., a blastoderm), which
may result in lower levels of morbidity and mortality among the
manipulated embryos. The methods of the invention are particularly
useful in methods of delivering vaccines, vitamins, growth
promoting hormones and growth factors, enzymes, cytokines, nucleic
acids, and/or cells to a bird in ovo. For example, the method may
be used to produce a chimeric bird (i.e., containing foreign cells)
or a transgenic bird (i.e., containing a foreign nucleic acid
sequence). The methods of the invention are also useful for
collecting samples or information from a bird in ovo, for example,
for use in methods of gender sorting, determining embryo viability,
and/or obtaining information about the genetic profile of the
embryo.
[0008] The methods of the invention may be carried out on a single
egg or a plurality of eggs. Moreover, the inventive methods may be
manual, automated, or semi-automated.
[0009] Accordingly, as a first aspect, the present invention
provides a method of injecting an avian egg, comprising the steps
of: orienting an avian egg containing a blastoderm in a
predetermined position; introducing a small opening into a shell of
the egg; extending a device through the opening in the egg shell;
piercing an inner shell membrane with the device, wherein the inner
shell membrane is essentially intact prior to inserting the device
therethrough; and retracting the device from the egg.
[0010] The device may be, for example, a delivery device, a
sampling device, or a detector device. Preferably, the opening in
the egg shell is made at the blunt end of the egg over the air
cell.
[0011] As a further aspect, the present invention provides a method
of injecting an avian egg, comprising the steps of: orienting a
blunt end of an avian egg in a predetermined position, the avian
egg containing (i) a blastoderm and (ii) an air cell; introducing a
small opening into a shell of the egg at the blunt end of the egg
over the air cell; introducing a small opening in an outer shell
membrane under the small opening in the egg shell; extending a
delivery device through the openings in the egg shell and the outer
shell membrane; piercing an inner shell membrane with the delivery
device, wherein the inner shell membrane is essentially intact
prior to inserting the delivery device therethrough; releasing a
substance through the delivery device and depositing the substance
in a location in the blastoderm or in close proximity thereto; and
retracting the delivery device from the egg. In particular
embodiments, the egg is oriented in a generally upward
position.
[0012] As a still further aspect, the present invention provides a
method of injecting an avian egg, comprising the steps of:
orienting an avian egg containing a blastoderm in a predetermined
position; introducing a small opening into a shell of the egg;
extending a sampling device through the opening in the egg shell;
piercing an inner shell membrane with the sampling device, wherein
the inner shell membrane is essentially intact prior to inserting
the sampling device therethrough; removing a sample from the egg
with the sampling device; and retracting the sampling device from
the egg.
[0013] As yet a further aspect, the present invention provides a
method of injecting an avian egg, comprising the steps of:
orienting an avian egg containing a blastoderm in a predetermined
position; introducing a small opening into a shell of the egg;
extending a detector device through the opening in the egg shell;
piercing an inner shell membrane with the detector device, wherein
the inner shell membrane is essentially intact prior to inserting
the detector device therethrough; detecting with the detector
device information from the interior of the egg; and retracting the
detector device from the egg.
[0014] As described in more detail below, the present invention may
be advantageously employed for any application in which it is
desirable to manipulate the contents of an avian egg containing an
early embryo (e.g., a blastoderm) or to obtain information
therefrom. In particular, the invention may be used to produce
transgenic or chimeric embryos or to carry out any chromosomal or
DNA based determinations (e.g., gender sorting or to evaluate the
genetic profile of the embryo). Alternatively, the invention may be
practiced to remove a sample of any embryonic or extra-embryonic
fluid or tissue from an avian egg containing an early embryo.
Additionally, or alternatively, the invention may be carried out to
obtain information from the egg or the embryo, which method may be
used in conjunction with delivery or sampling methods.
[0015] These and other aspects of the present invention are set
forth in more detail in the description of the invention that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a flow chart that illustrates methods of
manipulating eggs according to the present invention.
[0017] FIG. 2 is a flow chart that illustrates methods of preparing
an egg prior to injection according to embodiments of the present
invention.
[0018] FIG. 3 is a flow chart that illustrates methods of steering
or positioning an embryo in a desired location prior to injection
according to embodiments of the present invention.
[0019] FIG. 4 is a flow chart that illustrates methods of
introducing an opening into an egg according to embodiments of the
present invention.
[0020] FIG. 5 is a flow chart that illustrates methods of
determining the location of an embryo prior to injection according
to embodiments of the present invention.
[0021] FIG. 6 is a flow chart that illustrates methods of inserting
a device into an egg according to embodiments of the present
invention. The device may be, for example, a delivery device, a
sampling device, or a detector device, or a combination of the
foregoing.
[0022] FIG. 7 shows photographs of two-day old embryos from
windowed eggs. Panels: (a) Representative control embryo, from a
non-windowed egg. (b-h) Experimental embryos, from windowed eggs.
Experimental embryos are virtually identical in stage and
morphology as compared with controls.
[0023] FIG. 8 shows detection of fluorescently labeled cells
(green) in the somite of a 2-day incubated recipient embryo.
[0024] FIG. 9 represents a series of photomicrographs showing in
situ identification of cell death in stage X avian embryos. All
pictures shown are from the central region of the area pellucida of
stage X chick embryos. Embryos shown in the top row (panels a-d)
are photographed under brightfield-lighting and the middle row
(panels e-h) shows the same fields photographed under
epifluorescence illumination using a rhodamine filter-set. The
bottom row (panels i-l) shows the top two rows digitally merged, to
demonstrate the precise localization of cell death in the
blastoderm. The Fluorescence Pixel Counts (bottom row) indicates
the number of red pixels from the digitized fluorescence image,
allowing quantitative comparison of the amount of cell death.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention will now be described with reference
to the accompanying drawings, in which preferred embodiments of the
invention are shown. This invention may, however, be embodied in
different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
[0026] It should be noted that, in some alternative embodiments of
the present invention, the functions noted in the blocks of the
flow charts of FIGS. 1-6 may occur out of the order noted. For
example, two or more blocks shown in succession may in fact be
executed substantially concurrently or the two or more blocks may
sometimes be executed in the reverse (or otherwise different)
order. Furthermore, in certain embodiments of the present
invention, functions illustrated in FIGS. 1-6 may be performed in
parallel or sequentially. Moreover, in other embodiments, certain
blocks may be omitted altogether.
[0027] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0028] As used in the description of the invention and the appended
claims, the singular forms "a", "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0029] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0030] The term "avian" and "avian subjects," as used herein, is
intended to include males and females of any avian species, but is
primarily intended to encompass poultry which are commercially
raised for eggs, meat or as pets. Accordingly, the terms "avian"
and "avian subject" are particularly intended to encompass
chickens, turkeys, ducks, geese, quail, pheasant, parakeets,
parrots, cockatoo, cockatiel, ostrich, emu and the like. Chickens
and turkeys are the preferred avian subjects, with chickens being
most preferred. Alternatively, the avian or avian subject is an
endangered species of bird.
[0031] The present invention provides improved methods of
manipulating an egg containing an avian embryo. By "manipulating"
or "manipulation" it is meant that openings are made in the egg
shell, the outer shell membrane, and the inner shell membrane of
the egg and a substance or device is implanted or inserted
therein.
[0032] As used herein, the term "early embryo" refers to an avian
embryo from the time of lay (blastodermal stage) through about the
developmental stage where primordial germ cells (PGCs) are
migrating. With particular respect to chicken embryos, an "early
embryo" is generally about an embryonic stage 20 (H&H) embryo
or earlier. The developmental stages of the chicken embryo are
well-understood in the art, see e.g., The Atlas of Chick
Development, R. Bellairs & M. Osmond, eds., Academic Press,
1998. In particular embodiments, the early chicken embryo is about
a stage 4 to about a stage 18 embryo, or about a stage 12 to about
a stage 17 (H&H) embryo or, alternatively, about a stage 13 to
about a stage 15 (H&H) embryo (for example, for the delivery of
PGCs). In other particular embodiments, the early embryo is a
blastoderm stage embryo, as described below.
[0033] As used herein, the term "blastoderm" has its understood
meaning in the art. Generally, in the practice of the instant
invention, a blastoderm includes an embryo from the time of lay
through the end of gastrulation. The blastoderm is sometimes
referred to by the alternative designations "germinal disc" or
"embryonic disc" in the art. A blastoderm may be described as a
flattened disc of cells that forms during cleavage in the early
embryo and persists until the end of gastrulation. By the time of
laying two major regions of the blastoderm are visible, the
centrally-situation area pellucida and the peripherally-located
area opaca (The Atlas of Chick Development, R. Bellairs & M.
Osmond, eds., Academic Press, 1998). With particular respect to
chicken embryos, the blastoderm is typically characterized as an
embryo from the time of lay (i.e., Stage IX or Stage X EG&K)
through about stage XIII (EG&K). In particular embodiments of
the invention, the chicken embryo is about a stage VIII through
about a stage XIII (EG&K) embryo, or alternatively, about a
stage IX through about a stage XIl (EG&K) embryo. In other
embodiments, the chicken embryo is about a stage X to about a stage
XI (EG&K) embryo. In still other embodiments, the chicken
embryo is about a stage X (EG&K) embryo.
[0034] Currently used methods of manipulating eggs containing early
embryos may not be satisfactory for commercial purposes in that
they may result in unacceptably high levels of morbidity and
mortality. In particular embodiments, the avian embryo may be
transgenic (i.e., contain a foreign nucleotide sequence).
[0035] By injection or insertion of the device in "close proximity
to" the embryo or blastoderm it is meant that the device is
injected or inserted right above, below or adjacent to the early
embryo or blastoderm or in the surrounding structures, e.g., within
about 10, 8, 5, 3, 2 or 1 mm or less of the early embryo or
blastoderm. The term in "close proximity" to the blastoderm,"
encompasses injection or insertion of a device into the subgerminal
cavity of the embryo, between the area opaca and the vitelline
membrane, between the area pellucida and the vitelline membrane,
and/or between the area opaca and the area pellucida.
[0036] As used herein, the terms "injection" and "injecting"
encompass methods of inserting a device (typically an elongate
device) into an egg or embryo, including methods of delivering or
discharging a substance into an egg or embryo, methods of removing
a substance (i.e., a sample) from an egg or embryo, and/or methods
of inserting a detector device into an egg or embryo.
[0037] The terms "chimeric bird" or "chimeric embryo" refer to a
"recipient bird" or embryo, respectively, that contains cells
(i.e., somatic cells and/or gametes) from another bird or embryo,
referred to as a "donor." The resulting chimeric bird or embryo
will typically contain cells derived from both the recipient and
the donor. In particular embodiments, at least about 5%, 10%, 25%,
35%, 50%, 65%, 75%, 85%, 90%, 95% or more of the somatic cells in
the chimeric bird may be derived from the donor. Likewise, in other
embodiments, essentially all of the somatic cells are derived from
the donor. In yet other particular embodiments, at least a portion
of the gametes (e.g., at least about 5%, 10%, 25%, 35%, 50%, 65%,
75%, 85%, 90%, 95% or more) in the chimeric bird or embryo are
derived from the donor. In other particular embodiments,
essentially all of the gametes are derived from the donor.
[0038] The terms "transgenic bird" and "transgenic embryo" are used
herein in accordance with their generally understood meanings in
the art. A transgenic bird or transgenic embryo contains a foreign
nucleic acid sequence in one or more cells. The foreign nucleic
acid may from a different species (e.g., avian, mammalian, insect,
bacterial, protozoan, yeast, fungal, viral) or from the same
species. For example, an additional copy of a wild-type coding
sequence or a mutated form of a coding sequence from the same
species may be introduced. The foreign nucleic acid may encode a
polypeptide, an antisense RNA or other untranslated RNA, as
described in more detail below. The foreign nucleic acid is
generally stably transformed into one or more cells in the
transgenic bird or embryo, e.g., by stable integration into the
genome or by introduction of an episomal construct that is stably
maintained by the host cell.
[0039] As used herein, the term "predetermined location" indicates
a fixed position or depth within an egg. For example, a device may
be inserted into an egg to a fixed depth and/or fixed position in
the egg (e.g., the egg may be placed in a predetermined orientation
and the device is inserted into the egg at a fixed point to a fixed
depth). In alternative embodiments, the injection may be carried
out based on information obtained from the egg, for example,
regarding the position of the embryo (e.g., blastoderm) or a
compartment therein, the vitelline membrane and the like within the
egg (for example, the position of a desired compartment may be
determined using a sensor or probe and the device is inserted based
on that information).
[0040] The term "essentially intact" when used to describe the egg
shell or the inner or outer shell membrane indicates that there are
no significant perforations or tears therethrough. The term
"essentially intact" when used to describe the inner shell membrane
indicates that there are no significant perforations or tears in
the inner shell membrane. Typically, the only portion of the inner
shell membrane that will be visible is beneath the window or
opening formed in the egg shell, and the condition of the inner
shell membrane is determined with respect to this exposed portion
beneath the opening.
[0041] U.S. Pat. No. 5,897,998 (Speksnijder) describes a method for
manipulating the contents of an avian egg by making an opening in
the shell of an egg positioned in a horizontal position with
respect to its long axis, and wherein the opening is made in the
egg shell on the top side of the egg. A drop of liquid is then
placed over the opening in the egg shell "such that the opening is
completely covered" (U.S. Pat. No. 5,897,998; Abstract). The
underlying membranes are then cut away, and a solution may be
microinjected through the openings in the shell and membranes into
the egg, and the opening sealed.
[0042] The present inventors have found that it is not necessary to
insert a device into the egg through a drop of liquid. Moreover, if
liquid is deposited, it need not cover the entire surface of the
exposed membranes and the opening in the egg shell as taught by
U.S. Pat. No. 5,897,998. Further, the inventors have found that
survival of the manipulated embryos may be improved by leaving at
least the inner egg shell membrane essentially intact prior to
insertion of the device into the egg.
[0043] To illustrate, in a preferred and exemplary method of
introducing a substance into an avian egg, the egg is oriented in a
predetermined position, a small opening is made into the shell of
the egg such that the inner shell membrane is maintained
essentially intact prior to injection. A delivery device is
extended through the opening in the egg shell, and the inner shell
membrane is pierced with the delivery device (e.g., a micropipette
or microinjection needle) and a substance is released through the
delivery device and deposited into a desired location within the
egg. The substance will generally be released in a predetermined
dosage in a volume of from approximately one to twenty microliters
(typically, less than 5 or 10 microliters). The volume to be
delivered is not critical as long as it does not unduly harm the
embryo and is effective for delivery.
[0044] By a "small opening" in the egg shell, it is meant an
opening that is sufficiently small that it does not unduly harm the
developing embryo and can be sealed or otherwise closed if so
desired. In particular embodiments, the "small opening" in the egg
shell may be about 35mm or less in diameter, for example, from
about 10 mm to about 30 mm in diameter or about 1 5 mm to about 25
mm in diameter. In still other embodiments, the "small opening" is
less than about 10 mm, less than about 5 mm, or less than about 3
mm, 2 mm or even 1 mm in diameter.
[0045] Typically, the delivery device will be retracted from the
egg, unless the delivery device is an implantable device that is
left within the egg. Generally, the opening in the egg shell is
sealed, unless a self-sealing material (i.e., a sealant) has been
applied to the egg shell prior to introducing the opening therein.
The manipulated egg may then be placed in an incubator or otherwise
stored until hatch or whatever other desired endpoint.
[0046] The opening in the egg shell may be made in any suitable
location, e.g., in the side of the egg near the equatorial axis or
at either end of the egg. In a particular preferred embodiment of
the invention, the opening in the egg shell is introduced at the
blunt end of the egg over the air cell. According to this
embodiment, a small opening is also introduced into the outer shell
membrane. The inner shell membrane, which is separated from the
outer shell membrane by the air cell, is left essentially intact
prior to injection. The inner shell membrane is pierced by the
delivery device and a substance is released therethrough into a
desired location in the egg.
[0047] A "small opening" in the outer shell membrane is essentially
as described above for a small opening in the egg shell.
[0048] Those skilled in the art will appreciate that the early
embryo (e.g., blastoderm) will typically locate itself in an area
at or near the uppermost portion of the egg, whether the egg is
positioned horizontally, vertically, or at an angle. Thus, the
opening in the egg shell will generally be made in the uppermost
portion of the egg near whether the early embryo (e.g., blastoderm)
is expected to locate unless measures are taken to steer the embryo
to a different position within the egg.
[0049] In particular embodiments, the opening in the egg shell is
made in the upper side of the egg (i.e., along the long axis of the
egg). According to this embodiment, the egg will typically be
oriented in a generally horizontal position, i.e., with the long
axis tilted at an angle of less than about 45 degrees from
horizontal. In particular embodiments, the egg is tilted less than
about 30, 20, 15, 10 or 5 degrees from horizontal with respect to
the long axis of the egg. In other embodiments, the egg is placed
in a substantially horizontal position, i.e., essentially without
tilting the egg along its long axis (e.g., tilted less than about
5% or 10% from horizontal).
[0050] When injecting into the side of the egg, the device will
generally be inserted concurrently through both the inner and outer
shell membranes. In other particular embodiments, the membranes may
be removed when the opening is made in the egg shell. In still
other embodiments, an opening is made in the outer shell membrane
prior to inserting the device through the inner shell membrane,
where the inner shell membrane is essentially intact prior to
inserting the device therethrough.
[0051] In particular preferred embodiments, the egg is oriented in
a vertical position with respect to the long axis of the egg, with
the blunt end of the egg oriented in a generally upward position.
By a "generally upward position" it is meant that the egg is
positioned vertically, and the long axis is tilted at an angle of
less than about 45 degrees from vertical. In particular
embodiments, the egg is tilted less than about 30, 20, 15, 10 or 5
degrees from vertical with respect to the long axis of the egg. In
other embodiments, the egg is placed in a substantially upright
position, ie., essentially without tilting the egg along its long
axis (e.g., tilted less than about 5% or 10% from vertical).
According to this embodiment, the egg is generally tilted an egg
such that the early embryo (e.g., blastoderm) is located beneath
the air cell at the blunt end of the egg.
[0052] The opening in the egg shell membrane may be made in a
predetermined position (e.g., in the center of the blunt end of the
egg or in the center of the air cell at the blunt end of the egg)
or may be made in a position that is determined, at least in part,
by the position of the early embryo (e.g., blastoderm) within the
egg.
[0053] The present invention may be advantageously used to deliver
or remove substances to or from an early embryo (as described
above) in ovo. In particular embodiments, the early embryo is a
blastoderm (also as described above). It is also preferred that the
early embryo is at a stage at which the air cell is detectable
within the egg.
[0054] The substance may be released into the embryo (e.g.,
blastoderm) itself (e.g., into the area pellucida or the area
opaca). Alternatively, the substance may be released in close
proximity to (e.g., right above, below or adjacent to) the early
embryo, e.g., within about 10, 8, 5, 3, 2 or 1 mm or less from the
embryo (e.g., blastoderm). According to this embodiment, the
substance may be released into the subgerminal cavity of the
embryo, between the area opaca and the vitelline membrane, between
the area pellucida and the vitelline membrane, and/or between the
area opaca and the area pellucida. In still other embodiments of
the invention, the substance is released into the latebra, and/or
into the nucleus of pander.
[0055] Methods of delivering PGCs are known in the art and are
generally carried out by injecting the PGCs into a blood vessel in
the embryo (e.g., the dorsal aorta) or any sufficiently large
vessel in the extra-embryonic membranes (see, e.g., patent
publication WO 99/06533; University of Massachusetts). Likewise, a
blood sample can be removed from the blood vessel in the embryo or
a vessel in the extra-embryonic membranes of a PGC stage
embryo.
[0056] In one particular embodiment, the present invention provides
a method of delivering a substance to an avian egg comprising an
early embryo (e.g., a blastoderm) and an air cell, the method
comprising: orienting the egg so that the blunt end of the egg is
in a predetermined position (e.g., in a generally upward position,
as defined above), introducing a small opening in the egg shell and
the outer shell membrane at the blunt end of the egg over the air
cell, where the inner shell membrane is maintained in an
essentially intact condition, extending a delivery device through
the openings in the shell and the outer shell membrane, piercing
the inner shell membrane with the delivery device, and releasing a
substance through the delivery device and into the blastoderm or in
close proximity thereto.
[0057] The step of forming the opening in the outer shell membrane
and piercing the inner shell membrane may be performed essentially
concurrently by inserting the device through the two membranes. In
particular embodiments, an opening will first be made in the outer
shell membrane, and then the device will be inserted through the
inner shell membrane to create an opening therein. Likewise, the
opening in the egg shell may be formed prior to (e.g., first an
opening is punched into the egg shell and then an opening is put in
the outer shell membrane) or essentially concurrently with the
formation of the opening in the outer shell membrane (i.e., as a
single step).
[0058] As described above, it is preferred that the injection
through the inner shell membrane is not made through a droplet of
liquid that has been deposited on the inner shell membrane. In
those embodiments in which injection is made through a liquid, it
is preferred that the liquid is an aqueous liquid (e.g., an albumen
solution) and that only a small volume (e.g., two to ten
microliters) is deposited onto the inner shell membrane.
[0059] Those skilled in the art will appreciate that the methods of
the present invention may be carried out on a plurality of bird
eggs, e.g., in a commercial poultry operation. When the inventive
methods are carried out on a plurality of bird eggs, an improved
hatch rate and reduced morbidity may be observed in the flock as a
whole, although there may still be morbidity and mortality in
individual birds that were subjected to manipulation in ovo.
[0060] Preferably, the hatch rate in the manipulated eggs is at
least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
higher. It is also preferred that the hatch rate in the manipulated
eggs is similar to that observed in control eggs that were not
subjected to manipulation (e.g., no openings made in the shell,
etc.), or the hatch rate is depressed by less than about 25%, 15%,
10%, 5%, 3%, 2% or even 1% as compared with the control eggs.
[0061] In particular embodiments, the invention may be used to
deliver a substance to an egg containing an early embryo (e.g., a
blastoderm) or to deliver a substance to the early embryo itself.
Any substance may be injected by embodiments of the present
invention, including but not limited to cells, vaccines,
polypeptides, growth-promoting agents, probiotic cultures such as
competitive exclusion media, antibiotics, heterologous nucleotide
sequences including gene transfer vectors, vitamins, and/or markers
such as dyes, etc. The substances may be injected alone, or in
combination (e.g., antibiotics may be included with the delivery of
other substances). As another illustrative example, a dye or other
marker may be included with other substances to be delivered to
provide a means of determining whether delivery was to the desired
location.
[0062] As used herein, a "polypeptide" encompasses both proteins
and peptides. Polypeptides to be delivered to the egg or to the
embryo itself include antibodies, antigens (e.g., for
immunization), growth factors, hormones, and other growth or
performance enhancing polypeptides, enzymes, cytokines, and the
like.
[0063] By "growth or performance enhancing" it is meant that the
rate of growth, the final size of the animal, feed to gain ratio,
egg production, meat production, and the like, is improved.
[0064] Vaccine organisms include dead, live or attenuated viruses,
bacteria, protozoa, and fungi, including the various life stages of
these different organisms.
[0065] The method may also be advantageously used to introduce a
nucleotide sequence of interest into the developing embryo
(preferably, the nucleotide sequence is stably transformed into the
embryonic cells), i.e., to create a transgenic bird (as defined
above). Those skilled in the art will appreciate that it is not
necessary that every cell of the resulting transgenic bird contain
the transgene. The nucleotide sequence may be DNA or RNA, and it
may encode any polypeptide of interest or may encode a
non-translated RNA (e.g., antisense RNAs or ribozymes). In those
embodiments wherein the nucleotide sequence encodes a polypeptide,
the polypeptide may be a reporter polypeptide (e.g., an enzyme such
as Green Fluorescent Protein or alkaline phosphatase), a
therapeutic polypeptide, an immunogenic polypeptide (i.e., for
vaccination), a growth or performance enhancing polypeptide, and
the like.
[0066] The nucleotide sequence may be introduced into the embryo
using any vector and method known in the art. For example, a viral
vector (e.g., retrovirus) or DNA vector may be used to carry the
foreign nucleotide sequence of interest. In particular embodiments,
a viral vector is not used to introduce the nucleotide sequence
into the embryo. Methods of viral transduction and introduction of
naked DNA vectors into cells (e.g., using liposomes or
electroporation) are known in the art. Suitable apparatus for in
ovo electroporation include the BTX ECM 2001 electroporation device
(www.genetronics.com).
[0067] In other preferred embodiments, the present invention is
used to introduce a foreign or "donor" cell into a recipient embryo
(i.e., to create a chimeric embryo and, optionally, a chimeric
bird, as defined above). The donor cell may be a transgenic cell or
have any other characteristic of interest for introduction into an
embryo. The donor cell will typically be an avian cell, as
described above.
[0068] In particular embodiments, the recipient embryo and the
donor cell are from the same avian species. Alternatively, the
donor cell may be from a different avian species from the recipient
embryo (e.g., putting a turkey cell into a chicken embryo). As a
further alternative, the embryo and donor cells may be from the
same avian species, but be from different strains or breeds (e.g.,
two different breeds or strains of chicken). The recipient or donor
cell may be from an endangered species of bird.
[0069] Furthermore, the donor cells may also be from a high
performing or elite pedigree bird. Typically, high performing or
elite pedigree birds are used as breeding stock to create lines
with desired traits. The present invention advantageously allows
for direct transfer of cells (and therefore, genetic material) from
a single high performing or elite pedigree bird to produce numerous
"progeny" in a single step rather than by the conventional process
of expanding the line through successive generations, which may
result in dilution or loss of some of the desired traits. The high
performing or elite pedigree birds may be improved or superior for
any desired trait (e.g., a commercially important trait), including
but not limited to increased muscle production, reduced fat
composition, increased egg production, improved disease resistance,
altered size (e.g., smaller birds), reduced feather reduction or
altered feather composition, and/or altered proportion of male
birds.
[0070] Typically, the donor cell will be selected from the group
consisting of a blastodermal cell, a stem cell, a cultured stem
cell, an embryonic stem cell, a primordial germ cell, and an
embryonic germ cell.
[0071] In some embodiments, it may be desirable to compromise the
cells in the recipient embryo prior to introduction of the donor
cells. Methods of compromising embryonic cells are known to those
skilled in the art. Suitable methods of compromising the embryo
include but are not limited to coring (removing cells from
recipient embryo), mechanical injury (e.g., tearing),
gamma-irradiation, microwaves, soft x-rays, chemical treatment
(e.g., ammonia gas), heat, laser treatment, and/or cryogenic
cooling, and the like. In particular embodiments, irradiation,
heating, chemical treatment, and soft x-rays may be applied to the
whole egg. Ultraviolet radiation, microwaves, heat, laser
treatment, cryogenic treatment, mechanical injury, and coring may
be used in other embodiments through a window in the egg shell to
compromise the embryo.
[0072] The extent to which the embryo is compromised may be
determined by any method known in the art, e.g., by using dyes, in
particular fluorescent dyes, that selectively stain necrotic (e.g.,
propidium iodide) or apoptotic cells (e.g., Hoeschst 333342 stain).
Alternatively, mRNA transcripts or polypeptides may be detected
which are indicative of a necrotic or apoptotic state as known in
the art.
[0073] Accordingly, in a preferred embodiment, the present
invention provides a method of producing a chimeric bird,
comprising the steps of introducing a small opening into the shell
of the egg, extending a delivery device through the opening in the
egg shell, piercing the inner shell membrane with the delivery
device, wherein the inner shell membrane is essentially intact
prior to inserting the delivery device therethrough, releasing an
avian donor cell through the delivery device and depositing the
donor cell into the blastoderm or in close proximity thereto (as
described above) under conditions sufficient to result in a
chimeric embryo, retracting the delivery device from the egg and
incubating the chimeric embryo to hatch to result in a chimeric
bird.
[0074] Successful delivery of the cells to the blastoderm and/or
chimera production may be assessed by any method known in the art.
For example, the donor cells may be contacted with a dye (e.g.,
fluorescent dye such as
carboxyfluorescein-diacetate-succinyl-ester), gold particles, or
any other marker known in the art that may be detected after
delivery of the cells to the recipient embryo. Alternatively, the
donor cells may carry particular epitopes or nucleic acid sequences
that may be detected using antibodies or standard nucleic acid
detection methods to identify the presence of the donor cell or
progeny thereof in the blastoderm, the embryo or the resulting
bird. Additionally, or alternatively, the donor cells may carry a
gene(s) conferring a particular phenotypic trait that may be
readily detected in the embryo or the bird after hatch (e.g.,
feather color).
[0075] The foregoing description of the invention has primarily
been with respect to methods of delivering a substance to an egg or
to an early embryo (e.g., blastoderm). The methods of injecting an
egg may also be carried out to insert a sampling device into the
egg to remove a substance (i.e., a sample) from the egg (e.g., from
the embryo, as described above) and/or to detect information from
the egg (e.g., from the embryo).
[0076] In one embodiment, a sampling device may be inserted into
the egg to remove a sample therefrom. The sample may be taken from
the extra-embryonic portions of the egg (e.g., the yolk or the
albumen). For example, a sample may be taken from the albumen to
determine the presence or absence of microbial contamination (e.g.,
Salmonella) therein. In other embodiments, the sample is taken from
the early embryo (e.g., blastoderm), such as from the area
pellucida, the area opaca, and/or the subgerminal cavity. In
embodiments of the invention, the sample contains blastodermal
cells. Typically, the sample will be removed to obtain information
therefrom. The sample may be removed, for example, in connection
with methods of sexing or determining the viability of the embryo.
To illustrate, a sample containing cells may be removed from the
embryo, and the cells may be analyzed (typically after removal from
the egg) to detect the sex chromosomes or sex-specific sequences on
the chromosomes, as known by those skilled in the art. The sample
may also be used for any other DNA based assay, e.g., to determine
the presence of a particular gene or allele of interest in the
embryo.
[0077] In another embodiment, a detector device may be inserted
into the egg to detect information therein (i.e., from the egg
and/or from the embryo). The detector may be inserted into an
extra-embryonic location of the egg (e.g., the yolk or the air
cell). Alternatively, the detector may be placed in close proximity
(as defined above) to the embryo. In other embodiments, the
detector may be placed into the area pellucida or the area opaca of
the embryo or into the subgerminal cavity.
[0078] The detector device may be used to collect information
including, but not limited to, the size of the embryo, the location
of the embryo, the developmental stage of the embryo, the sex of
the embryo, and/or the viability of the embryo. Preferably, the
detector device obtains information regarding the location of the
embryo and the subgerminal cavity. The information may be captured
by an instrument (e.g., a computer or other data processor) that is
connected to the detector.
[0079] For particular embodiments, the egg is identified from which
a sample will be removed or information will be detected with a
detector. Information regarding the sample or information obtained
by the detector may be stored in association with the
identification of the egg. Exemplary detector devices are discussed
in more detail below.
[0080] FIGS. 1-6 are flow charts that illustrate particular
embodiments of the invention.
[0081] As shown in FIG. 1, one particular method of injecting an
avian egg containing an early embryo (e.g., a blastoderm) according
to the invention comprises the steps of preparing the egg prior to
injection 1000, positioning the embryo within the egg 2000,
introducing an opening into the egg 3000, locating the embryo
position 4000, and inserting a device into a desired location
within the egg 5000.
[0082] As shown in FIG. 2, the egg is optionally prepared prior to
injection. The surface of the egg, at least around the site of
injection, is sanitized to reduce microbial contamination 1200
(e.g., with an alcohol or other sanitizing solution). The egg may
further be oriented in a predetermined position 1300 (e.g., with
the blunt end of the egg in a generally upward position). In
particular embodiments, the egg is stored 1100 prior to injection.
Typically, the egg is stored for a period of time that is
sufficient to assist in positioning or "steering" the embryo to a
desired position within the egg, but insufficient to result in an
unacceptable incidence of morbidity or mortality to the embryo. To
illustrate, the egg may be stored from about six, twelve,
twenty-four hours or longer. There is no particular limit to the
storage period as long as viability is not unduly impaired or the
embryo does not develop beyond a suitable point for the present
methods. Eggs may be stored for as long as about thirty or even
about sixty days prior to injection. In embodiments of the
invention, the egg is stored for about thirty days or less, about
14 days or less, about 10 days or less, or about 7 days or less
prior to injection. In other particular embodiments, the egg is
stored for about 1, 2, 3, 4, 5, 7, 8, 10, 14 or 21 days prior to
injection; alternatively, about 1-21 days, about 1-14 days, about
1-7 days, about 1-4 days, about 4-8 days, or about 6-12 days prior
to injection.
[0083] Generally, the egg is stored at conditions (e.g.,
temperature) that will not promote the development of the embryo
within the egg or will avoid development beyond the desired
developmental stage (e.g., the blastodermal stage). Those skilled
in the art will appreciate that some cell division may occur during
the storage period; however, in general, the development of the
embryo is suspended or significantly delayed during the storage
period.
[0084] The egg may be oriented in a horizontal or vertical position
(with respect to the long axis) or at an angle therefrom during the
storage period. In particular embodiments, the egg is stored in the
same orientation as used for injection. Further, the egg may be
held in a fixed position (e.g., within a device) in which both
side-to-side movement and rotation around the long axis of the egg
are restricted or prevented.
[0085] In particular embodiments, the embryo may further be
compromised prior to injection into the embryo, as described
above.
[0086] As indicated in the previous paragraph, it is often
desirable and advantageous to steer or position the embryo prior to
injection, in particular, for automated or semi-automated methods
of injecting the egg. Turning to FIG. 3, as described above, the
egg may be stored prior to injection 2100 to promote positioning of
the blastoderm on the top center surface of the yolk.
Alternatively, or concurrently, the egg may be agitated (e.g.,
shaken) 2200, rotated 2300, centrifuged 2400 and/or spun 2500 to
assist in steering the embryo. Alternatively or additionally, the
contents of the egg may be subjected to altered temperature
conditions by heating 2600 (e.g., to a temperature from about
55.degree. F. or 60.degree. F. and below about 75.degree. F.,
80.degree. F. or 95.degree. F.) or cooling 2700. A discussed above,
in heating the egg, it will be appreciated by those skilled in the
art that the egg is subjected to a sufficiently mild heat treatment
(time x temperature) such that the embryo does not develop beyond
the desired stage. As still further alternative or additional
treatments, the egg may be chemically treated (e.g., chemically
treating the albumen with ammonia gas) 2800 to facilitate steering
the embryo to a desired location. Additionally, or alternatively,
the relative humidity of the air surrounding the egg may be
controlled 2900 (e.g., to above or below about 75%).
[0087] In particular embodiments, eggs are subjected to treatments
(e.g., chemical or mechanical) that result in a thinning of the
albumen (i.e., loss of firmness or reduced Haugh Units in a broken
egg). It is believed that blastoderm positioning at the top center
surface of the yolk may be facilitated by a thinner albumen.
Alternatively, eggs from older flocks and/or larger eggs may be
used in the methods of the present invention. Eggs produced by
older layers and larger eggs are both more likely to have a
relatively thin albumen.
[0088] Ammonia gas has been reported in the literature to result in
a thinning of the albumen. The egg may be exposed to any suitable
level of ammonia gas that results in positioning of the blastoderm
in a desired location. Suitable concentrations of ammonia gas may
be less than about 100, 150, 250, 500, 750, 1000, 1500, 2000, 2500,
3000 or 3500, 4000, 5000, 6000, 7000, or 8000 mg/kg or higher for a
sufficient period of time (e.g., about 0.25, 0.5, 1, 1.5, 2, 3, 4,
5 or 6 hours). In particular embodiments, the eggs are exposed to
relatively high levels of ammonia gas (e.g., about 2000 to 4000
mg/kg) for a relatively short period of time (e.g., about 0.5 to
about 1 hour). In other embodiments, the eggs are exposed to lower
dosages of ammonia gas of about 250-1000 or about 500-1500 mg/kg
for periods of about 0.5 to 2 hours.
[0089] In particular embodiments, a combination of approaches is
taken to help position or steer the blastoderm to the top center
surface of the yolk. For example, in one particular embodiment, the
eggs are stored from about 2 to about 7 days at about 650.degree.
C. to about 750.degree. C. while concurrently turning eggs during
the storage period. Further, the eggs that are selected for this
treatment may be larger eggs and/or eggs from older layers.
[0090] Turning to FIG. 4, a small opening is introduced into the
egg shell 3300 and the outer shell membrane 3400. While these steps
may be carried out separately, they may also be carried out
essentially concurrently, particularly in automated or
semi-automated methods. As further optional steps, the position of
the air cell at the blunt end of the egg may be determined 3100,
typically prior to introducing the opening in the egg shell. As a
further optional step, a sealant may be applied to the egg shell
3200 prior to or after making a small opening therein, so that the
opening in the egg shell will be essentially self-sealing.
Alternatively, or additionally, a sealant may be applied to the
inner shell membrane prior to or after piercing the inner shell
membrane with the device.
[0091] In some embodiments of the invention, the device will be
inserted into the egg at a predetermined depth and location. In
other embodiments, the device will be inserted into the egg based
on the position of particular compartments or structures in the
egg, e.g., the embryo or the subgerminal cavity within the egg. The
position of the embryo or subgerminal cavity may be determined
concurrently with or prior to injection (or sampling). Preferred
methods of locating the embryo are shown in FIG. 5. For manual or
semi-automated injection methods, the embryo may simply be
visualized (with or without a magnifying device) 4200 through the
window in the egg shell. The egg may further be rotated 4100 to
bring the embryo into view. Contrast between the embryo and the
background may be enhanced by directing light of particular
wavelengths (e.g., blue) on the interior of the egg through the
opening in the egg shell or by adding a contrast agent (e.g., a
colored or fluorescent dye). Visualization of the early embryo may
also be facilitated by feeding a diet or compound to the hen that
will affect the color of the yolk (e.g., to a shade of purple or to
a different shade of yellow) using techniques known in the art.
[0092] In other embodiments, particularly automated or
semi-automated injection methods, information regarding the
position of the embryo or other compartment within the egg may be
detected with a detector device 4300. Detector devices are
discussed in detail below, and may be invasive or non-invasive
devices. Typically, however, a device will be inserted into the
opening in the egg shell and the outer shell membrane to collect
information relevant to determining the position of the embryo
within the egg. The detecting step may be carried out essentially
concurrently with a delivery and/or sampling step. Alternatively,
they may be carried out sequentially. Preferred apparatus for
concurrent in ovo injection and detection are as described in U.S.
Pat. No. 6,244,214 (Hebrank).
[0093] As described above, and shown in FIG. 6, the inner shell
membrane is maintained in an essentially intact condition (as
described above) prior to insertion of a device therethrough. A
small opening is made in the inner shell membrane 5100, typically
above or in close proximity to the embryo. Typically, the small
opening is made by piercing the inner shell membrane by insertion
of a device therethrough. The device may be a delivery device 5200,
a sampling device 5300, or a detector device 5400. In some
embodiments, the device may contain detector, delivery and/or
sampling functionalities.
[0094] In some embodiments, the steps of introducing an opening
into the shell and outer shell membrane and, optionally, piercing
the inner shell membrane are carried out concurrently, i.e., in a
single step. Typically, however, the opening will be introduced
into the egg shell as a separate step because the devices used
according to the present invention may be of too narrow a diameter
to punch through the egg shell, depending upon the functionality
(e.g., a microinjection needle). Likewise, although the invention
may be carried out by injecting through the outer and inner shell
membranes concurrently (in particular when injecting into the side
of the egg, where the membranes are fused), when injecting into the
blunt end of the egg, these steps will generally be carried out
separately.
[0095] The injection methods described herein may be fully manual,
fully automated, or semi-automated. For example, the steps of egg
preparation and positioning the embryo, as shown in FIG. 1, may be
more suited for manual procedures. The steps of introducing the
opening in the egg shell and outer shell membrane, locating the
embryo and inserting the device, may be manual, but are preferably
automated.
[0096] In preferred embodiments, a multi-site injection or sampling
device is used, for example, as described in U.S. Pat. No.
6,032,612 (Williams et al.). Other preferred delivery or sampling
devices include those described in U.S. Pat. No. 5,136,979 (Paul et
al.); U.S. Pat. Nos. 4,681,063 and 4,903,635 (Hebrank); and U.S.
Pat. Nos. 4,040,388, 4,469,047, and 4,593,646 (Miller).
[0097] In a further embodiment, an injection apparatus further
comprising a detector as described in U.S. Pat. No. 6,244,214
(Hebrank) is used to collect information regarding the position of
the embryo (e.g., blastoderm) or other compartment prior to or
concurrently with injection into the egg (for the purposes of
sampling and/or delivering a substance into the egg or embryo).
[0098] The timing of the detecting step will depend upon the
particular purpose of the method or feature being detected, and the
nature of the material being injected or the sample being
withdrawn. In general, the detecting step may be carried out
before, after, or concurrently with a delivering or sampling
step.
[0099] Electrical sensors, optical sensors, chemical sensors,
temperature sensors, acoustic sensors, pressure sensors, or any
other device for detecting a physical or chemical parameter may
serve as the detection means or detector in carrying out this
aspect of the present invention. The detector or sensor may be
connected to the outer side-wall of the injection device, or in the
case of an electrical detector, and where the device is formed from
a conductive metallic material rather than an insulative or
polymeric material, the side wall of the injection device itself
may serve as the detector, with suitable circuitry connected
thereto. The detector could be one or two (or more) electrodes
carried by a non-conductive injection device, or carried on an
insulated portion of a conductive injection device. It will be
appreciated that, for the purpose of sensing depth or location
within the egg anatomy, and for a variety of other purposes,
numerous different physical or chemical parameters may be sensed or
detected, as long as they provide a useful indication of whether or
not the egg should be injected, or a useful indication that a
particular depth or position has been achieved.
[0100] The sensor may be positioned at the tip of the injection
device, or at a predetermined position along a sidewall thereof,
and/or spaced apart from the tip of the injection device.
[0101] Biosensors may be used to carry out the present invention.
Numerous biosensors are known. See. e.g., U.S. Pat. Nos. 5,804,453,
5,770,369, 5,496,701, 4,682,895, 5,646,039, 5,587,128, and
4,786,396.
[0102] When an electrical detector is used, it may be desirable to
provide a second electrode in operative association with a first
electrode. Where two electrodes are employed, they may be both
connected to the injection device, or one may be connected to the
injection device and the other separately inserted through the same
opening in the eggshell. In a preferred embodiment, the second
electrode is contacted to the exterior of the egg. An electrical
signal may be passed through the two electrodes, and the presence
or absence of conduction between the two electrodes detected. When
the second electrode is simply contacted to the exterior of the
egg, the signal is preferably an alternating current signal so that
the second electrode is capacitatively coupled to the interior
contents of the egg. Preferably each egg (or a flat containing a
plurality of eggs) is placed on top of a conductive material prior
to detection using an electrical detector (see, e.g., U.S. Pat. No.
5,591,482 regarding conductive polyurethane foam).
[0103] When an electrical detector is used to sense the location of
a fluid-filled compartment such as the sub-germinal cavity, the
electrical detector senses the entry of the probe into the fluid
compartment and thus serves as a depth detector (the term "depth
detector" encompassing "position detector" herein). In one
embodiment of the present invention, the motion of the detector
and/or the associated injection device is halted as the detector
and/or the device enters the compartment. In this manner an
injection device can be halted just after penetrating the
subgerminal fluid, but prior to penetrating all the way through the
cavity.
[0104] An optical sensor may comprise a fiber optic fiber, and may
be connected to an external wall portion of the injection device. A
light source may be provided through a second fiber optic fiber
inserted concomitantly with the device into the egg, or an external
source of illumination may be directed at the egg. Light conduction
or transmission properties may be used to determine the position of
the embryo or the subgerminal cavity. Light may also detect a color
marker for a physiological measurement, a disease measurement, or a
gender measurement.
[0105] A chemical sensor may be provided in any of a variety of
manners known to those skilled in the art of biosensors. For
example, a chemical sensor, may be provided through BBL.RTM.
liposome technology available from Becton Dickinson Microbiology
Systems, Cockeysville, Ma. USA, or as described in U.S. Pat. Nos.
4,703,017 and 4,743,560. The results of such an assay, when the
components are mounted on the injection device, may be determined
by reading with a fiber optic fiber as discussed above. Other
chemical assays may be performed by electrochemical detection. Such
sensors may be used, for example, to determine the position or
gender of the embryo within the egg, and to detect potential
microbiological infection within the egg.
[0106] The chemical sensor may be a pH sensor mounted to the
injection device, with the pH measurement being used to detect
potential microbial contamination, distinguish live from dead eggs,
etc. Ion-specific electrodes to detect various anion or cation
species may also be used, as discussed further below. Ion and pH
probes sense movement between compartments within an egg by the
differences in chemistry of the biological fluids present in the
various areas and compartments of an egg.
[0107] A temperature sensor may be used to distinguish live from
dead eggs or the position of the embryo and/or subgerminal cavity
based on the temperature thereof, or for the gender sorting of
eggs.
[0108] An acoustic sensor can be used as a passive or active sensor
(i.e., coupled with an acoustic signal source such as a transducer
contacted to the external portion of the egg) to determine depth,
to distinguish viable from non-viable eggs, to determine the
position of the embryo or subgerminal cavity, etc.
[0109] A location or depth sensor can be implemented by any of a
variety of techniques. Electrical contact with the air cell
membrane can be used to control penetration of the device relative
to preselected compartments of the egg, e.g. to a predetermined
depth below the air cell membrane, to insure more accurate
injection into the subgerminal cavity, into the embryo, or into any
other desired location, etc. Alternatively, depth can be sensed
with a pressure sensor to assess pressure changes during transition
of the device from compartment to compartment within the egg (e.g.,
air cell to embryo; embryo to subgerminal cavity; etc.). One
suitable method of sensing the location of the sensor measures the
pressure exerted on the sensor by the egg media surrounding the
sensor. For example, the pressure required to emit a gas or liquid
into the media surrounding an exit aperture located in the sensor
can be measured using either the injection device or a hollow gas
or fluid-filled tube. The discharge pressure required increases as
the exit aperture moves from a gas-filled compartment (e.g., air
cell) into a liquid-filled compartment; and increases again as the
exit aperture moves from a liquid-filled compartment into the
embryo. Changes in pressure can be measured by a pressure
measurement device located outside of the egg.
[0110] A light sensor can be used in conjunction with an external
light source or a light source carried by the device to distinguish
whether the device is in an air-filled compartment such as the air
cell, a fluid-filled compartment such as the subgerminal cavity or
the yolk, or a cellular compartment such as the embryo.
[0111] The sensor may be a diagnostic sensor for the detection of a
bacterial contamination or other microbiological contamination of
the eggs, such as Escherichia coli Salmonella, or Listeria
monocytogenes contamination of eggs. The diagnostic sensor may be
implemented by any suitable means, typically a chemical sensor or
biosensor. Detection of a contaminated egg may be used to trigger a
signal for subsequent sorting of the contaminated from
uncontaminated eggs.
[0112] A plurality of sensors may be associated with the device.
For example, where it is desired to detect microbial contamination
of the egg, to determine the position of a structure in the egg, or
where it is desired to gender sort the egg, it may be beneficial to
provide two different or distinct types of data to provide a more
accurate indication of the desired condition.
[0113] The detection step may be carried out by withdrawing a
sample from the egg into a processing system in which subsequent
analysis is carried out. For example, a liquid sample may be
withdrawn and analyzed to obtain the desired information therefrom
in the same manner as available analytical systems for processing
small liquid samples (e.g., in which samples are separated by air
gaps in the liquid processing line). In such cases, it is
preferable to provide a way to identify the egg from which each
biological sample is withdrawn, for example by providing hardware,
software, or combinations of hardware and software for counting the
eggs and the relative position of each egg, in association with the
time of sampling and storing that information for a short or long
period of time until it is used in the manner desired (e.g., to
reject a particular egg, to sex the birds and sort by gender, or to
provide a large database of information about the quality or some
other parameter of the eggs injected).
[0114] It will be appreciated that the present invention may
provide a way to record and store large amounts of information
about eggs being injected. For example, population data can be
obtained that can be used for quality control programs, or to
modify the prior treatment of the eggs, or to modify selective
breeding programs. In such cases, the identity of the egg injected
may be its association with a particular batch of eggs, rather than
its identity as a particular individual within that batch of
eggs.
[0115] Accordingly, the present invention provides improved methods
of manipulating an avian egg containing an early embryo (e.g.,
blastoderm), and for manipulating the embryo itself. The invention
may be used, for example, to collect information from the egg, to
remove a sample from the egg, and/or to deliver a substance to the
egg.
[0116] Having now described the invention, the same will be
illustrated with reference to certain examples, which are included
herein for illustration purposes only, and which are not intended
to be limiting of the invention.
EXAMPLE 1
[0117] The following is an exemplary method used to inject fluid or
cells into recipient chicken blastoderms in ovo through the blunt
end of the egg: [0118] 1. Surface sterilize the entire egg surface
of Day E 0 eggs by wiping with 100% ethanol. [0119] 2. Candle the
egg to locate the air cell. The position of the air cell is marked
on the shell with a pencil. [0120] 3. A window (small opening) is
made at the blunt end of the egg (in center of air cell) by
dremeling away the shell. Care is taken to keep the outer shell
membrane intact. All of the following steps are preferably carried
out in a laminar hood or in a clean room: [0121] 4. The blunt end
of the egg, where the outer shell membrane is exposed through the
window in the shell, and the surrounding area are again surface
sterilized using 70% ethanol. [0122] 5. The outer shell membrane is
carefully removed with forceps and scalpel (#11 blade; Personna
Medical) taking care not to leave any jagged edges to the membrane.
[0123] 6. The blastoderm is visualized beneath the air cell
membrane (i.e., inner shell membrane) as the doughnut shaped
structure. [0124] 7. A glass micropipette (or needle) is used to
pierce through the air cell membrane and inject into the
subgerminal fluid of the blastoderm. Approximately 3-5 microliters
of fluid (containing Penicillin-streptomycin at 100 .mu.g/ml) is
injected. Micropipettes were pulled using a Sutter P-30 puller;
Temperature--800, Pull-950. [0125] 8. Following injection,
waterproof tape (Johnson & Johnson, 1 inch wide), cut to fit
the window is affixed over and pressed into place. [0126] 9.
Silicone (GE Silicone II, clear) seal is then spread over entire
surface of tape with emphasis on sealing the edges of the tape.
[0127] 10. Silicone is allowed to dry for approximately 30-45
minutes before eggs are transferred to the incubators.
EXAMPLE 2
[0128] A series of experiments was carried out to evaluate the use
of manual injection as described in Example 1 to inject either
fluid or cells into the subgerminal cavity of recipient chicken
blastoderms (approximately Stage X) in ovo. Donor cells were also
from Stage X chicken blastoderms. In some cases, the recipient
embryo was compromised prior to injection. In early experiments, a
small droplet (a few .mu.l) of an albumen solution was deposited on
the inner shell membrane above the blastoderm and injection was
performed by piercing the membrane through the albumen droplet. In
later experiments, this step was omitted. Eggs were either not
stored or stored from 4 to 8 days prior to treatment.
[0129] Hatch rates in control and manipulated eggs were determined.
The rate of successful chimera production was also evaluated. The
results are shown below in Table 1. TABLE-US-00001 TABLE 1 Percent
Chimera Obtained Recipient Compromise Donor No. of No. of
Hatchability of Hatchability Based on Based on Egg Recipient Egg
Eggs Cells Control Eggs of Injected no. of no. of chicks
Breed.sup.a,b Egg? Breed injected injected Recipient Donor Eggs
eggs set hatched R X R No -- 17 -- 27/30 = 90% -- 13/17 = 76% (drop
of albumen) R X R No -- 17 -- 22/30 = 73% -- 10/17 = 59% (drop of
albumen) R X R No -- 20 -- 27/30 = 90% -- 18/20 = 90% (no fluid) R
X R No -- 21 -- 30/30 = 100% -- 15/21 = 71% (no fluid) R X R No --
20 -- 23/25 = 92% -- 10/20 = 50% (no fluid) R X R No -- 21 -- 24/27
= 89% -- 13/21 = 62% (no fluid) R X R No -- 20 -- 21/25 = 84% --
9/20 = 45% (no fluid) R X R No -- 38 -- 17/20 = 85% -- 28/38 = 74%
(no fluid) R X R No -- 14 -- 14/14 = 100% -- 9/14 = 64% (no fluid)
R X R No BPR 15 625, 22/30 = 73% -- 625:27% 0% 0% (no fluid) 1250
1250:53% 0% 0% R x R No BPR 30 500, 80% 53% 500:47% 0% 0% (no
fluid) 1000 1000:40% 0% 0% R x R Yes BPR 20 500, 78% -- 500:60% 10%
(1/10) 16% (1/6) (no fluid) 1000 1000:60% 0% 0% R x R Yes BPR 20
500, 64% -- 500:50% 0% 0% (no fluid) 1000 1000:10% 0% 0% R x R Yes
BPR 24 500, 47% -- 500:17% 8.3% (2/12) 50% (1/2) 1000 (compromised)
1000:33% 0% 0% HyVac Yes BPR 25 500, 70% 57% 500:63% 7.6% (1/13)
12.5% (1/8) (SPF) 1000 (compromised) 1000:58% 16.6% (2/12) 28.5%
(2/7) R x R Yes BPR 25 1000 80% 76% 60% 8% (2/25) 13.3% (2/15) 64%
(compromised) R x R No BPR 39 1000 87% 69% 62% 10% (4/39) 16.6%
(4/24) R x R No BPR 30 1000 97% 20% 57% 0% 0% BPR No R .times. R 35
1000 11% 89% 63% 14.2% (5/35) 22.7% (5/22) Bovans No BPR 50 1000
78% 72% 68% 6% (3/50) 9% (3/34) Bovans Yes BPR 30 1000 70% 0% 0%
Bovans Yes BPR 20 1000 75% 60% 75% 0% 0% 80% (compromised) Bovans
Yes - for BPR 15 1000 87% 47% 73% (not 6.6% (1/15) 100% (1/1) one
treat- 13% compromised) ment group (compromised) 7% (compromised)
.sup.aAbbreviations: RxR = Rhode Island Red Cross; BPR = Barred
Plymouth .sup.bHyVac and Bovans are strains of Leghorn chicken.
EXAMPLE 3
[0130] Certain methods of physical compromise of the embryo, such
as ultraviolet (UV)-irradiation or laser ablation, may be
administered through a window in the egg shell and shell membranes.
We have developed a method to create a window in the side of the
egg, seal it, and incubate the egg under standard conditions (99 F,
rocking), that allows a very high percentage (>90%) of embryos
to develop normally at two days (FIG. 7). This method may be used
to evaluate the effects of compromise and chimera production at two
days of embryonic development. Further, this method may be used to
inject into the side of the egg. Briefly, the eggs were treated as
follows: [0131] 1. Eggs were stored at 60 F, 75% humidity for 0-7
days, on their long axis, to steer the blastoderm towards the
center of the side of the egg. [0132] 2. The position facing
upwards was marked with a sharpie pen. [0133] 3. Following storage,
a 7-mm hole was drilled at the side of the egg, at the location of
the pen mark. [0134] 4. The shell was removed. Usually, the inner
and outer shell membrane remained intact, and those were also
removed. [0135] 5. The blastoderm was identified, near or directly
underneath the opening made in the egg shell. For this particular
experiment, only those eggs were used where the blastoderm was
directly underneath the opening, allowing application of various
compromise treatments, such as UV-light. [0136] 6. The hole was
left open for 5-10 minutes. [0137] 7. The hole was sealed with GE
Silicon II and J&J waterproof tape. [0138] 8. The silicon was
allowed to dry for about 30 minutes. [0139] 9. Eggs were placed in
an incubator with the blunt end up, hole on the side, and incubated
at 99 F with turning every 3 hours, for two days. [0140] 10.
Embryos were collected from the eggs for photography.
[0141] Photographs of representative control and treated embryos
are shown in FIG. 7.
EXAMPLE 4
[0142] A method of monitoring blastodermal cell incorporation into
recipient embryos was developed. This method is applicable to
tracing freshly isolated blastodermal cells from any avian species
or strain, and should be easily applicable to monitoring the
incorporation of other cell, such as embryonic stem cells. To
summarize the method, freshly isolated blastodermal cells were
exposed to carboxyfluorescein-diacetate-succinyl-ester (CFDA SE)
prior to injection. CFDA SE is an amine-reactive compound, which
becomes highly fluorescent upon reacting with intracellular
proteins. Once it is incorporated into blastodermal cells, those
cells can be monitored in a recipient embryo by fluorescence
microscopy.
[0143] In the experiment illustrated below, freshly isolated
blastodermal cells were labeled with CFDA SE and microinjected into
Stage X blastoderms, by injection through the inner-shell membrane
at the blunt end of the egg (as described in Example 1). When
observed by microscopy after two days of incubation, fluorescently
labeled donor cells were found in a variety of tissues. FIG. 8
shows fluorescently labeled donor cells incorporated into the
somite of a 2-day incubated recipient embryo to create a chimeric
embryo. Fluorescent dye labeling may be an attractive alternative
or complement to using genetically modified cells to follow donor
cell incorporation in early embryos.
EXAMPLE 5
[0144] In order to optimize chick chimera production, recipient
embryos may be compromised or preconditioned so that they are more
receptive to the donor cells. The most widely accepted of method of
compromise involves exposing recipient eggs to 600 rads of
gamma-irradiation. In order to assess the effects of various
treatments on recipient embryos, we have developed techniques to
measure the amount of cell death in situ in the recipient
blastoderm.
[0145] To identify necrotic cells in situ, we have assessed
dye-labeling with Propidium Iodide (PI), a fluorescent DNA stain
which is primarily permeable to dead cells. It was found that with
a minimum amount of handling and washing the delicate blastoderm,
PI effectively labeled necrotic cells. These cells can be
identified and photographed by fluorescence microscopy, and the
relative amount of cell death determined by digital image analysis
(see FIG. 9). The precise location of necrotic cells in the
blastoderm can also be determined by digital image analysis (see
FIG. 9). To our knowledge, this is the first time that cell death
has been measured in situ in a living avian embryo.
[0146] In this experiment, the PI labeling technique was applied to
untreated, physically damaged, and heat-treated embryos. The
untreated blastoderm (FIG. 9, first column) shows the low level
background cell death. The physical damage inflicted in this
experiment (second and third columns) was unintended damage caused
by the blastoderm isolation procedure. Note that the cell death can
be precisely localized to areas surrounding tears in the blastoderm
(FIG. 9, panels j and k). The heat treatment (55 C for 15 minutes
in a water bath) was based on previous data indicating that this
treatment completely eliminates hatch. As shown in this experiment
(FIG. 9, fourth column), this reduction in hatch is likely due to
the extremely high level of cell death in the blastoderm. The
fluorescence pixel counts (FIG. 9, bottom row) can be used to
quantitatively measure and compare the amount of cell death between
treatments.
[0147] Detection of apoptotic cells (as opposed to necrotic cells)
may also be determined using the fluorescent dye Hoechst 333342,
which differentially stains living and apoptotic cells. Measurement
of apoptotic and/or necrotic cells may be used to determine the
extent of compromise of the recipient embryo.
EXAMPLE 6
[0148] The following studies were carried out to determine the
position and diameter of the blastoderm in eggs stored under
commercial hatchery storage conditions. White leghorn eggs (Bovans)
from a single flock are measured on a weekly basis; the flock is
studied to the end of its laying cycle. Eggs are received within 24
hours of lay. After the eggs are received, they are stored for 6 or
7 days at 60 F, and 75% relative humidity, in flats, similar to the
way they are stored in commercial hatcheries. Fifty eggs in flats
are measured each week. The average off-center distance of the
blastoderm is 5.24 mm for eggs stored for 6 or 7 days in flats,
with 3.05% of the embryos too far off-center to be seen.
[0149] In addition, a Hy-Line W-36 White Leghorn flock is studied.
These eggs are received up to 48 hours after lay. The eggs are
stored for 6 or 7 days at 60 F and 75% relative humidity. The
average off-center distance of the blastoderm is 5.86 mm for eggs
stored for 6 or 7 days in flats with 3.96% of the embryos too far
off-center to be seen.
[0150] The diameter of the blastoderm, including the diameter of
the area pellucida, is also measured at the time blastoderm
position is measured. The results are shown in Table 2 below under
commercial hatchery storage conditions. The eggs were stored for 7
days, and the embryos were typically seen as Stage X embryos.
TABLE-US-00002 TABLE 2 Diameter Area Pellucida Diameter Area Opaca
No of Eggs (mm) (mm) Flock Measured Average Std. Dev. Average Std.
Dev. Bovan 2146 1.99 0.26 3.68 0.36 Hy-Line 1351 2.11 0.25 3.87
0.36
EXAMPLE 7
[0151] Experiments were carried out to determine whether the
blastoderm may be passively steered by modulating storage
conditions. The eggs were stored either in flats as under
commercial conditions (controls) or in a device called a "fixture"
that holds the eggs in a fixed upright position (blunt end up)
where both side-to-side movement and rotation around the vertical
axis of the egg were essentially prevented.
[0152] In an ongoing study with a Bovan flock (the hens were in
their 69.sup.th week of lay; they lay up to 70 weeks approximately,
without a molt), a total of 2,766 eggs stored either in flats or in
a fixture for 7 days, at 60 F and 75% RH (30 eggs stored in flats
and 30 eggs stored in fixtures, weekly for 31 weeks), were measured
on an Acu-Gage optical Coordinate Measuring Machine (CMM) with a
blue fiber-optic light source to enhance the contrast of the
blastoderm. Based on the collected data, the average off-center
distance of the blastoderm was 5.24 mm for eggs stored for 7 days
in flats. The off-center distance improves to an average 4.58 mm
for the eggs stored for 7 days in a fixture device that holds the
eggs such that the long axis of the egg is vertical. In eggs stored
in flats, 3.05% of the embryos were too far off-center to be seen,
while in eggs stored in fixtures only 2.53% were too far
off-center. Hy-Line W-36 eggs from Elizabethtown, Pa. were also
measured. A total of 1,649 eggs were measured, where the eggs were
stored for 6 or 7 days at 60 F and 75% relative humidity. The
average off-center distance of the blastoderm was 5.86 mm for eggs
stored for 6 or 7 days in flats with 3.96% of the embryos too far
off-center to be seen. The off-center distance for the eggs stored
for 6 or 7 days in fixtures such that the long axis of the egg was
vertical was slightly better, an average 5.31 mm, and 4.81% of the
embryos were too far off-center to be seen.
[0153] Statistical analysis on the data (Bovans, 23-69 weeks;
Hy-Line, 26-42 weeks) was performed to determine whether holding
the egg in the fixture device influenced the position of the
blastoderm. Data from infertile or damaged eggs were excluded. If
the blastoderm was too far from the center to be measured, a value
of 13.716 was applied. An analysis of covariance was utilized that
considered position as the dependent variable and storage device
(in a fixture or in an egg flat) as fixed, with analyses conducted
separately by breed. The effect of storage in a fixture as compared
with flats on blastoderm position pooled across flock ages is
presented in Table 3 below. TABLE-US-00003 TABLE 3 Blastoderm
Position off-center (mm) Bovans Hy-Line Flat Mean 5.5370 6.2241 SD
3.1126 3.2125 N 1085 729 Fixture Mean 4.9579 5.7333 SD 3.0161
3.2439 N 1257 736
[0154] The effect of storage in a fixture was significant for both
the Bovans (p.ltoreq.0.001) and the Hy-Lines (p.ltoreq.0.004); eggs
stored in fixtures resulted in blastoderms significantly closer to
the center.
[0155] The dataset of eggs stored in a fixture was analyzed to
determine whether blastoderm position off-center was correlated
with flock age and /or egg weight. An analysis of covariance was
performed that considered position as the dependent variable and
flock age fixed, with egg weight as covariate. The analysis was
separated by breed. Bovans eggs show a highly significant
(p.ltoreq.0.001) flock age effect for eggs stored in fixtures,
while Hy-Line eggs did not show any significant correlation between
blastoderm position with flock age (p.gtoreq.0.05).
[0156] As expected, egg weight increased with flock age; an inverse
relationship was observed between egg weight and blastoderm
distance from the egg center. The Bovan data set showed egg weight
as a significant (p.ltoreq.0.05) covariate; while egg weight was
not a significant co-variate for the Hy-Line eggs (data not shown).
It may be that egg weight is not a significant covariate in the
Hy-Line analysis because the flock age did not exceed 42 weeks of
age and, thus, egg weight may not as yet be a factor. While
regression analysis of the Bovan dataset showed a significant
relationship between egg weight and blastoderm position, it
indicated that less than 1% of the variation in blastoderm position
could be explained by egg weight (R.sup.2=0.007).
[0157] The control group for the above experiments was made up of
eggs stored in flats. These are cardboard flats in which the eggs
are shipped; the eggs can move relatively freely about the vertical
axis while held in the flats. Hatcheries use plastic flats, in
which the egg has only a restricted level of movement about the
vertical axis. To determine the effect of storing eggs in plastic
flats versus cardboard flats or in fixtures, Bovan eggs were stored
in either plastic or cardboard flats as well as in fixtures for 6
days at 60 F, 75% RH. As shown in Table 4 below, blastoderm
position off-center was reduced in eggs stored in plastic flats,
but were much improved in eggs stored in fixtures. TABLE-US-00004
TABLE 4 Blastoderm position Blastoderms off-center too far Storage
Vessel (mm) off-center Cardboard Flats Mean 6.586 8% SD 3.460 n 45
Plastic Flats Mean 5.442 4% SD 2.947 n 47
EXAMPLE 8
[0158] Studies were carried out to determine the effect of
different storage time periods on blastoderm position. A previous
study demonstrated that eggs stored for 7 days (at 60 F and 75%
relative humidity) showed a significant improvement in the position
of the blastoderm (from 9.71 mm off-center on day 0 to 5.61 mm
off-center on day 7, significant at 0.0001 level) over day 0 eggs,
and storing eggs for 10 days did not significantly improve
blastoderm position any further.
[0159] One of the variables believed to affect blastoderm position
is albumen thickness; a thicker albumen restricts the free rotation
of the yolk. Albumen thickness is measured in terms of Haugh units.
In a separate experiment, albumen thickness was measured in eggs
stored for various lengths of time. Bovan eggs from a 55-week-old
flock were weighed and Haugh units determined after storage in
flats for 0, 3, 6, 10, 13, or 17 days. Albumen height or Haugh
units were shown to significantly decrease as storage time
increased.
[0160] An experiment was done to determine the effect of storage
time on blastoderm position off-center as well as albumen
thickness. Hyline eggs from a 37-week-old flock were weighed and
stored in fixtures (blunt end up) for 2, 6, 9, 12, or 15 days at 60
F and 75% relative humidity. The group stored for 6 days was
designated the control group. Analyses of covariance that
considered blastoderm position as the dependent variable, and
storage time (days) as fixed was performed. Separately, Haugh units
were considered as the dependant variable and storage time the
fixed variable. Mean blastoderm position or mean Haugh units for
each storage time were then compared by SNK test. As shown in Table
5 below, blastoderm position off-center significantly decreased as
storage time increased, with the mean position for 12 days and 15
days significantly lower than 2 days, with 6 days and 9 days
intermediate (the mean position for 6 was actually lower than 9,
and 12 lower than 15). Further, Haugh units also significantly
decreased with storage time, showing an even clearer effect than
blastoderm position. TABLE-US-00005 TABLE 5 Storage Time (days) 2 6
9 12 15 No. of eggs 47 46 45 47 44 Blastoderm Mean 6.6691 5.3541
5.6500 4.4831 4.7582 Position off- SD 2.9971 3.2487 3.9817 2.9454
2.9254 center (mm) Haugh Units Mean 73.8645 69.3088 66.8423 64.0056
61.8017 SD 5.2358 6.2709 8.9072 7.0988 6.1042
[0161] Overall, it appears that thinning the albumen allows more
freedom of rotation of the yolk and thereby positioning of the
blastoderm closer to the "top dead center" of an egg held in a
vertical, blunt end up, position.
EXAMPLE 9
[0162] Investigations were also carried out to determine the effect
of modulating egg orientation during storage on blastoderm
position. Preliminary experiments in which eggs were stored on
their side for 7 days did not improve blastoderm positioning.
However, these experiments had been performed by placing the eggs
on a flat surface and allowing them to freely orient themselves in
a sideways orientation.
[0163] In the present study, eggs were stored in various positions
while held in fixtures as described in Example 7, which limited
side-to-side and rotational movement to determine the effect on
blastoderm position. The fixtures held the eggs in a fixed
horizontal or vertical position. The various orientations in which
the eggs were stored, as well as the orientation in which the egg
was held for the measurement of the blastoderm position, were as
follows: [0164] 1. Sideways (Bovan and Hy-Line W-36), and measured
sideways [0165] 2. Upside down (i.e. blunt side down) for three
days (Hy-Line W-36), followed by blunt-end up for 3-4 days, and
measured blunt end up. [0166] 3. Upside down (i.e. blunt side down)
for 6-7 days and measured at the blunt end (Bovan). [0167] 4.
Upside down (i.e. blunt side down) for 6-7 days and measured upside
down (Hy-Line W-36).
[0168] The results are summarized in Table 6. TABLE-US-00006 TABLE
6 % Blastoderm blastoderms Storage Condition Position (mm) too far
off Comments 1 Sideways and measured Mean 3.937 6% Effect of
sideways storage of eggs sideways SD 3.441 on blastoderm position
is not n 45 significant. BOVANS Blunt end up, measured Mean 4.312
2% blunt end up SD 2.540 n 42 Sideways and measured Mean 4.783 10%
Effect of sideways storage of eggs sideways SD 3.709 on blastoderm
position is not n 47 significant. HYLINE Blunt end up, measured
Mean 5.213 4% blunt end up SD 3.829 n 45 2 Upside down for 3 days,
Mean 6.258 6.5% Effect of sideways storage of eggs and blunt end up
SD 2.801 on blastoderm position is remaining, measured n 40
significant at 0.057 level. blunt end HYLINE Blunt end up, measured
Mean 5.003 2.5% blunt end up SD 2.997 n 40 3 Upside down and Mean
11.451 70% Effect of upside down storage is measured right side up
i.e SD 4.140 significant to 0.001 level. blunt end n 47 BOVAN Blunt
end up, measured Mean 4.278 2% blunt end up SD 2.511 n 47 4 Upside
down, measured Mean 12.092 82% Effect of upside down storage is
upside down SD 3.798 significant to 0.001 level. n 49 HYLINE Blunt
end up 5.376 2% 2.845 43
[0169] Storage of eggs sideways did not produce significantly
improved results for either type of egg; although blastoderm
distance decreased, the number of blastoderms too far from the
center also increased. Storage of eggs upside down for 3 days
approached significance but actually increased blastoderm distance
from the center of the egg (p.ltoreq.057). Storage of eggs upside
down measured either right side up or upside down resulted in a
significant difference, but greatly increased blastoderm distance
from the center of the egg, for both Bovan and Hy-Line W-36.
EXAMPLE 10
[0170] The effects of storage temperature and relative humidity on
blastoderm position were also investigated. Temperature is believed
to be an important factor that affects albumen quality (i.e.
thickness), which in turn may affect the movement or mobility of
the yolk and hence the location of the blastoderm. Preliminary
experiments indicated that storing eggs at higher temperatures
might help "steer" the blastoderm towards the center of the surface
of the yolk.
[0171] Hyline eggs from a 47-week-old flock were stored for 7 days
at either 60 (commercial hatchery conditions) or 70 degrees F.
(15.5 or 21.1 degrees C.). Blastoderm position off-center of eggs
stored at 70 degrees were lower than those of eggs stored at 60
degrees (approached significance at p=0.063).
[0172] In a follow-up study, various permutations of temperature
and relative humidity conditions during egg storage were studied
for their influence on blastoderm position. Eggs stored in fixtures
as described in Example 7 for 6-7 days were subjected to varying
combinations of temperature and relative humidity, with 60 degrees
F and 75% relative humidity considered the control treatment. The
following different treatment conditions were evaluated: [0173] 1.
Normal temperature (60 F) with relative humidity (75%) lowered to
50% (Hy-Line W-36 eggs). [0174] 2. Normal temperature (60 F) and
relative humidity (75%) for first 5 days. followed by increased
temperature of 80 degrees (75% RH) for the last two days of storage
(Bovan). [0175] 3. Normal temperature (60 F) but relative humidity
set to 90% (Hy-Line W-36). [0176] 4. Temperature set to 70 F and
relative humidity set to 50% (Hy-Line W-36). [0177] 5. Temperature
set to 70 F and normal relative humidity (Hy-Line W-36). [0178] 6.
Temperature set to 70 F and relative humidity set to 90% (Bovan and
Hy-Line W-36). [0179] 7. Temperature set to 80 F with normal
relative humidity (Hy-Line W-36).
[0180] The data analysis is summarized in Table 7 below:
TABLE-US-00007 TABLE 7 Blastoderm Storage Condition Position (mm)
Haugh Units Comments 1 60 F.; 50% RH Mean 5.1306 71.33 Main effect
of RH on blastoderm SD 2.5212 5.21 position is not significant. n
45 45 60 F., 75% RH (control) Mean 5.2447 72.22 Effect of RH on
albumen thickness is SD 2.8391 5.74 also not significant n 43 43 2
Last two days of storage at Mean 3.2724 62.08 Main effect of
temperature on 80 F., 75% RH SD 1.7535 8.59 blastoderm position is
not significant. n 48 48 60 F., 75% RH - all days Mean 3.6235 66.73
Effect of temperature on albumen (control) SD 2.3655 8.30 thickness
significant (p .ltoreq. 0.01). n 47 47 3 60 F.; 90% RH Mean 5.3228
-- Main effect of Relative Humidity on SD 2.7827 -- blastoderm
position is not significant n 50 -- 60 F.; 75% RH (Control) Mean
5.7364 -- SD 3.1812 -- n 49 -- 4 70 F.; 50% RH Mean 4.8390 69.58
Main effect of temperature and relative SD 2.1712 5.30 humidity on
blastoderm position is n 46 46 significant to 0.04 level 60 F., 75%
RH (control) Mean 6.0275 71.89 Effect of temperature and relative
SD 3.1089 4.64 humidity on albumen thickness is n 48 48 significant
(p .ltoreq. 0.05). 5 70 F.; 75% RH Mean 4.4427 69.83 Main effect of
temperature on SD 2.6986 3.81 blastoderm position is significant n
49 49 to 0.02 level 60 F.; 75% RH (control) Mean 5.8881 75.34
Effect of temperature on albumen SD 3.0702 5.53 thickness is
significant (p .ltoreq. 0.001). n 48 48 6 70 F.; 90% RH Mean 3.5412
65.19 Main effect of temperature and relative SD 2.5177 6.20
humidity on blastoderm position is n 48 48 significant to 0.05
level BOVANS 60 F.; 75% RH (control) Mean 4.6323 68.22 Effect of
temperature and relative SD 2.5991 7.38 humidity on albumen
thickness is n 44 44 significant (p .ltoreq. 0.05). 70 F.; 90% RH
Mean 5.6043 65.31 Main effect of temperature and relative SD 3.6825
6.72 humidity on blastoderm position is not n 47 47 significant.
HY-LINE 60 F.; 75% RH (control) Mean 5.9906 72.50 Effect of
temperature and relative SD 3.0451 5.88 humidity on albumen
thickness is n 47 47 significant (p .ltoreq. 0.001). 7 80 F.; 75%
Mean 4.7258 55.42 Main effect of temperature on SD 2.8704 7.39
blastoderm position is significant at n 40 40 0.065 level. 60 F.;
75% RH (control) Mean 5.9530 69.16 Effect of temperature on albumen
SD 3.1971 7.24 thickness is significant (p .ltoreq. 0.001). n 47
47
[0181] In summary, a temperature of 70 F appeared in all cases to
improve blastoderm positioning and was significant to p.ltoreq.0.05
for all cases except for Hy-Line W-36 eggs at 90% relative
humidity. Relative humidity changes alone appeared to have little
effect on blastoderm position (note that in all cases regardless of
significance the control group had increased blastoderm distance
from center). Results were similar with Haugh units, which
decreased for all treatment groups as compared with the control
conditions (note that Haugh unit data was not collected for the
normal temperature and 90% relative humidity study). The Haugh unit
data showed more significance than the blastoderm position data,
with only the normal temperature and 50% relative humidity group
not showing a significant effect.
EXAMPLE 11
[0182] Modulating the external environment is one method to
influence blastoderm position. Exposure of eggs to ammonia has been
shown to thin the albumen. Benton and Brake (2000) have shown that
eggs exposed to high levels of NH.sub.3 (2747 and 6052 mg/kg) for 1
hour show significant reduction in the albumen height with a
concomitant increase in albumen pH when compared with the controls
(0 mg/kg NH.sub.3). Since a reduction in the albumen height has
been positively correlated to more fluidity of the albumen, it was
NH.sub.3 treatment to steer the blastoderm closer to the center of
the egg was assessed.
[0183] In the first experiment, eggs from 38 week-old Hyline birds
(egg type W 36; Flock I.D: PL 2506) were received and maintained
for 1 day at 60.degree. F. and 75% RH. Eighteen eggs (three batches
for a total of 54 eggs) were placed on their horizontal axis on a
porcelain plate in glass desiccators (250 mm in diameter, with
caps) (Fisher Scientific). One liter of NH.sub.4OH solution (14.8
M) (Sigma) or double distilled water (control) was poured into the
desiccators. The 14.8 M NH.sub.4OH is estimated to release 6052
mg/kg of NH.sub.3 (Benton and Brake, 2000). The eggs were exposed
for 1 hour to the respective levels of NH.sub.3. The eggs were then
removed, and the blastoderm position, albumen height and pH were
measured. These results are presented in Table 8: TABLE-US-00008
TABLE 8 Blastoderm Haugh Treatment PH Position (mm) Units NH.sub.3
6052 Mean 10.29 7.91 69.51 mg/kg for SD 0.50 4.02 6.47 1 hour n 42
42 42 No treatment Mean 9.23 7.72 74.98 SD 0.27 4.02 5.84 n 44 44
44
[0184] The results indicate that NH.sub.3 treatment caused a
significant increase in albumen pH when compared with their
respective controls. The albumen height in the NH.sub.3 treated
group was lower (i.e., had lower Haugh unit values) indicating a
thinning of the albumen.
[0185] In another experiment, the eggs were stored after exposure
to ammonia. The eggs (68-week-old Bovans) were subjected to two
concentrations of NH.sub.3 (2747 and 142 mg/kg of NH.sub.3). The
controls were exposed to 0 mg/kg of NH.sub.3. A total of 50 eggs
were used for the NH.sub.3 treatments and 48 eggs were used for the
control. Serial dilutions of one liter of NH.sub.4OH solution (14.8
M) (Sigma) were done to give 2747 or 142 mg/kg of NH.sub.3. For the
controls, double distilled water (1 L) was poured into the
desiccators. The eggs were exposed for 1 hour to the respective
levels of NH.sub.3. The eggs were then removed, placed in fixtures
with no rotational or side-to-side movement, and stored at 60 F and
75% RH for 6 days. After the 6-day storage, measurements were
carried out to determine the blastoderm position, albumen height
and pH. The results are shown in Table 9 below: TABLE-US-00009
TABLE 9 Blastoderm Haugh Treatment PH Position (mm) Units NH.sub.3
2747 mg/kg Mean 9.02 4.16 64.00 treatment for SD 0.11 2.72 9.56 1
hour n 45 45 45 NH.sub.3 142 mg/kg Mean 9.05 6.50 69.11 treatment
for SD 0.07 2.79 8.74 1 hour n 40 40 40 None (control) Mean 9.04
5.19 67.72 SD 0.07 3.15 7.57 n 36 36 36
[0186] The blastoderm position off-center in the eggs treated with
2747 mg/kg of NH.sub.3 is much closer to the top dead center of the
blunt end of the egg as compared with the controls. Interestingly,
the pH of these eggs is similar to the control. There were no
differences in the Haugh Units among the NH.sub.3-treated and
control eggs. The lower concentration of NH.sub.3 (142 mg/kg)
showed no changes in any of the measured parameters as compared
with the controls.
[0187] One of the changes that was observed with the embryos at the
2747 mg/kg NH.sub.3-treated groups is that they had a "cooked"
appearance and did not look alive. Thus, treatment with high levels
of ammonia may be useful as a method of compromising the
embryo.
EXAMPLE 12
[0188] A variety of approaches were evaluated to determine whether
the blastoderm could be actively "steered" to the center of the
egg.
Centrifugation.
[0189] Gravity is believed to play an important role in orienting
the blastoderm in the uppermost position in the egg. Centrifugation
is a way to create an artificial gravity or increase the body force
of gravity. Studies were conducted to determine whether the greater
force due to centrifugation would improve the position of the
blastoderm.
[0190] Hyline eggs from 27-week-old flocks were weighed, placed in
fixtures as described in Example 7, and stored for a total of seven
days. On day five of storage, the eggs were placed in Sorval RC 5C
centrifuge buckets and spun for 3 minutes at 500 rpm while held in
fixtures with the long axis of the egg aligned with the artificial
gravitational force throughout the spin. The eggs were then stored
for two more days, and then the position of the blastoderm from the
egg's center was measured. No significant change in blastoderm
position due to centrifugation was noted.
[0191] In a further study, eggs (Hy-Line) were weighed, stored in
fixtures for 5 days, spun for 30 minutes at 300 rpm (Beckman
Allegra 6KR centrifuge) and then stored for a day prior to
measuring the blastoderm position. An analysis of covariance was
utilized that considered position as the dependent variable and
centrifugation as fixed. As shown in Table 10 below, there were no
significant differences in blastoderm position, and in fact the
centrifuged eggs showed a blastoderm position slightly further from
center. TABLE-US-00010 TABLE 10 Blastoderm position Blastoderms
Treatment (mm) too far off Stored for 5 days, Mean 5.3005 2%
centrifuged, SD 3.0679 stored for a day. n 39 Not centrifuged Mean
5.1559 2% SD 2.7494 n 48
Turning.
[0192] The effect of turning the eggs during storage on blastoderm
orientation toward the top center of the egg was evaluated. Eggs
were placed in Hovabator turners at 60 F, 75% RH, for 6-7 days. The
turners moved the eggs plus and minus 45 degrees from vertical and
a complete cycle from 0 to +45 to 0 to -45 and back to zero took 4
hours. The no treatment eggs were stored in flats (cardboard) for
comparison (to match the eggs in the turners). The results are
shown in Table 11 below: TABLE-US-00011 TABLE 11 Blastoderm
position Blastoderms Treatment (mm) too far off Turned Mean 3.6036
0% SD 2.2245 n 46 BOVANS Not- Turned Mean 4.2386 0% SD 2.4452 n 42
Turned Mean 5.5419 0% SD 2.8708 n 60 HYLINE Not- Turned Mean 5.9126
0% SD 2.6295 n 60
[0193] Analysis of variance indicated that results between
treatment groups were not significant for either Bovan or Hy-Line
eggs, although the mean distance from the center was less for the
turned eggs.
Spinning.
[0194] The effect of spinning the eggs during storage on blastoderm
orientation toward the top center of the egg was evaluated. The
eggs were stored in fixtures as described in Example 7 and were
compared with eggs that were not spun but also stored in fixtures.
For this trial, the eggs were stored for 5 days, spun for 30
minutes at 300 rpm, and then stored for a day prior to measuring
blastoderm position. The spinning group had a slightly larger
blastoderm position from center, although the percentage of
blastoderms that were too far off center to be seen improved
slightly. The results are in Table 12 below: TABLE-US-00012 TABLE
12 Blastoderm position Blastoderms Treatment (mm) too far off Spun
Mean 5.7037 0% SD 3.4034 N 43 BOVANS Not- Spun Mean 5.9170 0% SD
3.4443 N 44
[0195] Analysis of variance indicated that the results were not
significantly different among treatment groups, although the mean
distance from center was slightly less for the turned eggs.
Shaking.
[0196] Shaking the eggs during storage to orient the blastoderm
toward the top dead center of the egg was investigated. The eggs
were stored in fixtures as described in Example 6 and were compared
with eggs that were not shaken but also stored in fixtures.
[0197] The diameter of the blastoderm was also measured while
blastoderm position was measured for each of the different
treatments described above. The results showed there was no change
in blastoderm size with any of the treatments.
[0198] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims and equivalents thereof.
* * * * *