Method and apparatus for early embryonic in ovo injection

An apparatus for administering compounds to avian eggs through the shell thereof comprises a blocking member, an elongate shaft connected to the blocking member, an active agent depot connected to the elongate shaft, and an adhesive seal connected to the blocking member for sealably connecting the blocking member to the egg shell with the shaft penetrating through the shell. Methods of using the apparatus of the present invention are also described.

FIELD OF THE INVENTION 
The present invention relates to the administration of compounds to avian 
eggs, and in particular relates to an improved method and apparatus for 
early embryonic in ovo administration. 
BACKGROUND OF THE INVENTION 
Advances in poultry embryology have made possible the addition of various 
substances to the embryo or to the environment around the embryo within an 
avian egg for the purpose of encouraging beneficial effects in the 
subsequently hatched chick. Such beneficial effects include increased 
growth, prevention of disease, increasing the percentage hatch of multiple 
incubated eggs, and otherwise improving physical characteristics of 
hatched poultry. Additionally, certain types of vaccinations which could 
previously only be carried out upon either recently hatched or fully 
mature poultry can now be successful in the embryonated chick. In ovo 
administration techniques that replace the injection of very young hatched 
chicks can increase the efficiency of administration and reduce the stress 
on young chicks caused by injection. 
Many methods of adding compounds to avian eggs utilize the injection of 
fluids by syringe. One traditional method has been syringe injection of 
eggs by hand. A number of automatic egg injection devices have also been 
developed. These include U.S. Pat. Nos. 5,056,464 to Lewis; 4,903,635 and 
4,681,063 to Hebrank; 3,377,989 to Sandhage; and 4,040,388, 4,469,047, and 
4,593,646 to Miller. A review of all these patents and their associated 
systems reveals, however, that all require that fluid be delivered from a 
storage device to the egg through a system of pumps and tubing which 
carries the fluid to the syringe needle. 
Several injection devices seal the injection site after injection to 
prevent leakage and contamination. U.S. Pat. No. 4,040,388 to Miller 
describes heating the portion of the injection device which punctures the 
egg, allegedly sterilizing the exterior of the egg and also sealing the 
hole by heat coagulating a small portion of the egg albumin. U.S. Pat. No. 
4,593,646 to Miller et al. discloses sealing of eggs after injection by 
heating and coagulating the albumin located near the injection site. An 
additional sealant is then applied to the outer shell by dipping each egg 
in a bath of sealant. U.S. Pat. No. 2,477,752 to Kiss discloses a method 
of injecting fertile eggs for the purpose of producing chicks have colored 
down. The '752 patent describes manual injection of the egg and subsequent 
sealing of the injection site. 
Methods other than syringe injection to add compounds to eggs have been 
disclosed. U.S. Pat. No. 3,256,856 to Nicely discloses puncturing the egg 
shell over the air cell, applying negative pressure to draw air from the 
air cell, and then immersing the egg in a bath of liquid treatment 
material and returning to atmospheric pressure to draw treatment fluid 
into the egg. Treatment substances have also been administered by creating 
a pressure gradient across the shell or by immersion in a treatment bath. 
See also U.S. Pat. No. 4,928,628 to Gassman, 4,928,629 to Trampel, and 
2,734,482 to Seltzer. These techniques are generally cumbersome and 
difficult to apply on a commercial scale. 
For some applications it would be desirable to have a means for delivering 
substances into an egg other than by automated syringe injection. Other 
than hand injection with a syringe, however, few such techniques are 
available. 
In view of the foregoing, an object of the present invention is to provide 
methods and apparatuses for in ovo injection in which injection and 
sealing of the injection site is accomplished in one step. 
SUMMARY OF THE INVENTION 
A first aspect of the present invention is an apparatus for administering 
an active agent to a bird egg, comprising a blocking member connected to 
an elongate shaft, an active agent depot connected to the elongate shaft, 
and seal means connected to the inner surface of the blocking member for 
sealably connecting the blocking member to the egg shell. 
A further aspect of the present invention is an apparatus for administering 
an active agent to a bird egg, comprising a blocking member connected to 
an elongate hollow shaft, a septum transversely positioned within the 
elongate hollow shaft, and seal means connected to the inner surface of 
the blocking member for sealably connecting the blocking member to the egg 
shell. An active agent depot may be carried within the lumen of the hollow 
shaft, or delivered through the hollow shaft. 
A further aspect of the present invention is a method for administering an 
active agent to a bird egg through the egg shell, comprising the steps of 
providing an injection apparatus (comprising a blocking member connected 
to an elongate shaft, an active agent depot connected to the elongate 
shaft, and seal means connected to the inner surface of the blocking 
member for sealably connecting the blocking member to the egg shell) and 
inserting the elongate shaft through the egg shell and into the egg 
interior, so that the blocking member is sealably connected to the shell 
and so that the active agent is placed within the interior of the egg. 
A further aspect of the present invention is a method for administering an 
active agent to a bird egg through the egg shell, comprising the steps of 
providing an injection apparatus (comprising a blocking member connected 
to an elongate hollow shaft, a penetrable septum transversely positioned 
within the elongate hollow shaft, seal means connected to the inner 
surface of the blocking member for sealably connecting the blocking member 
to the egg shell, and delivery means for delivering an active agent to the 
interior of the egg, the delivery means having an active agent depot and 
being capable of penetrating the septum) and inserting the elongate shaft 
of the injection apparatus into the interior of the egg so that the 
blocking member is sealably connected to the shell, and then penetrating 
the septum with the delivery means to deliver the active agent into the 
egg. 
A further aspect of the present invention is a method for administering an 
active agent to a bird egg through the shell, comprising providing an 
injection apparatus (comprising a blocking member connected to an elongate 
hollow shaft, a septum slidably related to the hollow shaft and positioned 
transversely within the hollow shaft to create proximal and distal 
compartments within the shaft, seal means connected to the inner surface 
of the blocking member for sealably connecting the blocking member to the 
egg shell, delivery means for delivering an active agent to the interior 
of the egg, the delivery means capable of penetrating the septum, and an 
active agent depot placed within the proximal compartment of the hollow 
shaft) and inserting the elongate shaft of the injection apparatus into 
the egg so that the blocking member is sealably connected to the shell, 
and then forcing the septum and active agent into the interior of the egg 
using the delivery means. 
The foregoing and other objects and aspects of the present invention are 
explained in detail in the specification set forth below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The apparatus of the present invention comprises, in general, an elongate 
shaft designed to penetrate the shell and inner membranes of an avian egg, 
a blocking member, sealant, and an active agent depot such as a reservoir 
of active agent deposited in a lumen or opening in the shaft, an agent 
deposited in a solid biodegradable polymer which is carried by or adhered 
to the shaft, or simply by fixing the active agent directly to the shaft. 
As used herein, the term "active agent depot" refers to a quantity of 
active agent sufficient to achieve the desired physiological or biological 
effect in the bird, either alone or in combination with a carrier (e.g., a 
degradable polymer or other pharmaceutically acceptable excipient). 
A preferred embodiment of the present invention is shown in FIGS. 1 & 2. 
The injection apparatus comprises a disc-shaped blocking member (10) 
having an outer surface (13) and a concave inner surface (14), and an 
elongate shaft (11) having a proximal end (15), an intermediate portion 
(16), and a distal end (17). The elongate shaft is connected at its 
proximal end to the concave inner surface of the blocking member; the 
distal end may be tapered, planar or concave. The elongate shaft is of a 
length sufficient to extend, when the concave inner surface of the 
blocking member is seated against the exterior of an egg, into the 
compartment of the egg into which active agent deposition is desired. 
Sealant (12) is carried on the concave inner surface of the blocking 
member and completely surrounds the elongate shaft at the point it 
connects to the blocking member. The active agent depot (18), in this 
embodiment, is carried on the surface of the elongate shaft. 
In use (FIG. 2) the elongate shaft (11) of the injection apparatus is 
inserted through the shell (20) and the inner membranes (21) of an avian 
egg, so that the shaft (11) extends into the compartment of the egg into 
which active agent deposition is desired (as shown in FIG. 2, the shaft 
extends into the albumin). As shown in FIG. 2A, the blocking member (10) 
is seated firmly against the exterior of the egg shell, and the sealant 
(12) both adhesively bonds the blocking member to the egg shell and seals 
the opening formed in the shell by the shaft. The active agent (22) is 
deposited into the interior of the egg. 
The active agent depot may be contained on the surface of the shaft, which 
may have ridges, pores, depressions or other surface irregularities formed 
therein to facilitate carrying the active agent. Alternatively, the 
elongate shaft may have a lumen formed therein and be open at the distal 
end, and carry the active agent within the lumen. Alternatively, the shaft 
may be made of a composite material consisting of the active agent to be 
delivered combined with a material which degrades while in the inner egg 
environment to release the active agent. In another embodiment of the 
present invention, a hollow elongate shaft extends through the blocking 
member, the shaft having a lumen formed therein and being open at both the 
proximal and distal ends. At the time active agent delivery is desired, 
the active agent depot is delivered to the interior of the egg through the 
lumen of the hollow shaft. 
The blocking member performs two functions: (1) limit the depth to which 
the shaft penetrates the egg, and (2) carry sealant in an amount and a 
position such that the opening created in the egg shell by the shaft is 
sealed when the blocking member is bonded to the egg by the sealant. Use 
of a sealant prevents the leakage of egg components and the ingress of 
contaminants and air into the egg. 
Suitable seal means for use in the present invention are those materials 
capable of adhesively bonding the egg shell to the blocking member, so 
that the combination of the blocking member and the seal means isolates 
the interior of the egg from the external environment and essentially 
prevents leakage of egg contents and entry of air into the egg. Examples 
of suitable seal means include sealants or adhesives, including but not 
limited to, silicone sealants (e.g., G.E..TM. Silicon II), mucilage 
adhesives (e.g., Carter's Mucilage.TM., Carters Ink Co., Waltham, Mass.), 
glues, hot-melt adhesives, and other essentially nontoxic glues, sealants 
and adhesives. Sealant is deposited on the blocking member in an amount 
sufficient to bond the blocking member to the egg shell and to seal the 
injection site in the egg shell. 
In an embodiment of the apparatus as shown in FIG. 3, the elongate shaft 
(11) has a lumen formed within it, but the elongate shaft does not extend 
through the blocking member (10). The active agent depot (18) is contained 
within the lumen of the hollow shaft and is separated from the egg 
contents by a septum (30) made of a biodegradable material. In use the 
injection apparatus is inserted into the egg, as above. Upon degradation 
of the septum, the active agent is released into the egg. Alternatively, 
the entire lumen of the elongate shaft may be filled with degradable 
polymer into which the active agent is mixed. The active agent is released 
over time as the polymer degrades. 
In another embodiment of the present invention, the elongate shaft of the 
injection apparatus is made entirely or partially of degradable polymer in 
which the active agent to be delivered is contained. The active agent is 
released over time as the polymer of the shaft degrades. In another 
embodiment of the present invention, the elongate shaft contains inserts 
of biodegradable polymer in which the active agent to be delivered is 
contained. Such polymer inserts may be of any configuration which does not 
substantially affect the ability of the shaft to penetrate and be inserted 
into the egg. Such inserts may, for example, be configured as a layer of 
polymer which surrounds the shaft or as small particles adhered to the 
shaft. 
In an embodiment of the apparatus as shown in FIG. 4, the elongate shaft 
(11) of the injection apparatus extends through the blocking member (10) 
and has a lumen formed throughout its length. A penetrable, self-sealing 
septum (31) is contained within, and blocks, the lumen. In use the distal 
end (17) of the shaft is inserted into an egg through the shell (20) and 
inner egg membranes (21) until the blocking member (10) is seated against 
and bonded to the egg shell. A syringe and hollow needle (32) provide a 
delivery means for delivering the active agent to the interior of the egg. 
The delivery means contains the active agent depot (18), and is inserted 
into the shaft (11) to pierce the septum (31) and deliver the active agent 
to the interior of the egg through the shaft (11). The syringe (32) is 
then withdrawn from the shaft (11). Materials which may be used to form a 
penetrable self-sealing septum include SILASTIC.TM. adhesive, silicone 
sealant, or rubber. Many suitable delivery means may be employed, 
including but not limited to hypodermic injection syringes and needles, 
eye droppers, pipettes, and bulb syringes. 
In an alternative embodiment of the injection apparatus as shown in FIG. 5, 
the elongate shaft (11) of the injection apparatus extends through the 
blocking member (10) and has a lumen formed throughout its length. The 
lumen is blocked by a septum (40) which is slidably related to the inner 
walls of the hollow shaft. The active agent depot (18)is carried within 
the lumen, such that when the shaft is inserted into an egg, the active 
agent depot is separated from the egg contents by the septum. In use, once 
the shaft is inserted in an egg, a stylet (41) is inserted into the shaft 
(11) and is used to push the active agent and slidable septum into the egg 
interior. The stylet is of a length and configuration sufficient to expel 
the active agent into the egg interior, and to essentially block the lumen 
of the hollow shaft, when inserted therein; the stylet is left within the 
lumen during any subsequent egg incubation. 
The blocking member of the injection apparatus may be modified to include 
labels or markable surfaces, or may be color-coded or otherwise 
identified. The blocking member may also be adapted for use as a handle or 
otherwise used as a means to move the egg to which it is attached (e.g., 
by attaching a graspable handle to the blocking member). FIG. 6 shows an 
embodiment of the injection apparatus having a handle (50) attached to the 
outer surface (13) of the blocking member (10), and having an elongate 
shaft (11) with a blunt distal end (17). The sealant (12) completely 
surrounds the proximal end (15) of the elongate shaft. 
The term "birds" as used herein, is intended to include males or females of 
any avian species, but is primarily intended to encompass poultry which 
are commercially raised for eggs or meat. Accordingly, the term "bird" is 
particularly intended to encompass hens, cocks and drakes of chickens, 
turkeys, ducks, geese, quail, ostrich and pheasant. Chickens and turkeys 
are preferred, with chickens most preferred. 
The term "in ovo," as used herein, refers to birds contained within an egg 
prior to hatch. Thus, the present invention may be conceived of as both an 
apparatus for, and a method of administering a compound to, an egg as well 
as an apparatus for, and a method of administering a compound to, a bird. 
The present invention may be practiced with any type of bird egg, 
including chicken, turkey, duck, goose, quail, ostrich and pheasant eggs. 
Chicken and turkey eggs are preferred, with chicken eggs most preferred. 
Eggs treated by the method of the present invention are fertile eggs which 
may be in any period of incubation, from early to late, but are preferably 
in the first half of incubation. Eggs may be treated prior to incubation 
(i.e., Day 0 of incubation). Eggs may be incubated to hatch following 
treatment. 
The active agent is, in general, an organic compound which produces a 
biological, physiological, and/or immunological effect in the bird, such 
as an antibiotic, vitamin, vaccine, hormone, enzyme inhibitor, peptide, 
protein, cell or DNA. 
The method and apparatus of the present invention may be utilized to inject 
substances into the amniotic fluid, allantois, albumin, or aircell of the 
egg. The injection site will vary depending upon the active agent to be 
injected and the effect desired; one skilled in the art will be able to 
readily select an appropriate injection site. 
Septums may be formed of any essentially nontoxic material having suitable 
physical properties. Examples of materials suitable for self-sealing 
penetrable septums include rubber, SILASTIC.TM. adhesive, and silicone 
sealants (e.g., G.E..TM. Silicon II). 
Erodible or biodegradable polymers useful in practicing the present 
invention include polylactide polymers, polylactic polyglycolic acid 
copolymers, erodible hydroxypropylmethyl cellulose, and methacylate 
polymers. A depot amount of an active compound can be incorporated into 
the polymer to allow delivery of the compound as the polymer erodes. Such 
polymers, and their use, are disclosed in U.S. Pat. No. 3,773,919, at 
column 2, lines 1-4, and column 7, line 12, et seq (the disclosure of 
which is intended to be incorporated in its entirety herein). The term 
"polylactide," as used herein, includes both the generic meaning of a 
polyester derived from an alpha-hydrocarboxylic acid, and the specific 
meaning for the polymer derived from lactic acid (alpha-hydroxypropionic 
acid). Thus the term polylactide, when herein used generically, 
encompasses lactide/glycolide copolymers. Particularly preferred 
polylactide polymers are polymers formed of polylactide or polyglycolide, 
and particularly copolymers thereof. Properties of these polymers, and 
methods of making them, are discussed in D. L. Wise, et al., 
Lactic/Glycolic Acid Polymers, in Drug Carriers in Biology and Medicine, 
Chapter 12 (G. Gregoriadis, Ed. 1979), and in T. R. Tice and D. R. Cowsar, 
Biodegradable Controlled-Release Parenteral Systems, Pharmaceutical 
Technology, page 26 (November 1984). 
Polylactides having a high molecular weight (greater than 10,000 daltons) 
can form films, and are therefore preferred for practicing the present 
invention. The specific lactide used, in a poly(lactide-coglycolide) 
copolymer or otherwise, can be poly(D-lactide), poly(L-lactide), or 
racemic poly(D,L-lactide). Generally preferred for drug delivery systems 
are poly (L-lactide) and poly (D,L-lactide). Best results with these 
copolymers are obtained with copolymers ranging in molar composition form 
about 15 to 85 percent poly(glycolide), with the remainder poly(lactide). 
The rate of copolymer biodegradation is adjusted by altering the 
lactide/glycolide ratio, as is known in the art. 
The biodegradable polymer having the active compound therein may be in any 
physical form suitable for deposition within an egg. Preferably the 
polymer serves as a matrix in which the active compound is distributed. 
Use of various shapes and compositions of these polymers in 
controlled-release drug delivery systems is discussed in Wise, et al,, 
supra, and T. R. Tice and D. R. Cowsar, supra. 
The following examples are provided to illustrate the present invention, 
and should not be construed as limiting thereof. 
EXAMPLE 1 
Injection of Agent into Albumin of Egg 
The injection apparatus used in this example comprised a stainless steel 
tack having a pointed shaft 5/16 inch in length and 1/16 inch in diameter, 
and a slightly concave discoid head 3/8 inch in diameter (Moore Solidhead 
Steel, No. 51, #2, Wyndmoor, Pa.). The shaft of each tack used was coated 
with a mixture of mucilage (Carters Ink Co., Waltham, Mass.), the 
antibiotic GARASOL.TM. (Schering Corp., Kenilworth, N.J.) and green 
vegetable food dye. The underside of the tack head (concave side from 
which the shaft extended) was coated with a thin layer of silicone sealant 
(G.E..TM. Silicone II). 
Six fresh, non-incubated eggs were used. Each egg was held upright (large 
end up) while a tack was inserted upwardly into the small end of the egg. 
Tacks were inserted until the tack head was seated firmly against the egg 
shell and bonded to the egg shell by the sealant. After 24 hours the eggs 
were broken open and the albumin visually inspected. The presence of green 
colored albumin indicated the deposition of dye and antibiotic into the 
albumin, and the dispersion of dye and antibiotic through the albumin. 
EXAMPLE 2 
Hatchability: Injection without Sealant 
The hatchability of non-injected eggs was compared to eggs injected using a 
stainless steel tack having a pointed shaft 5/16 inch in length and 1/16 
inch in diameter, and a slightly concave discoid head 3/8 inch in diameter 
(Moore Solidhead Steel, No. 51, #2, Wyndmoor, Pa.). No sealant was used. 
Fresh, non-incubated eggs were used; control eggs (n=127) were not 
injected. Injected eggs (n=132) were held upright (large end up) while a 
tack was inserted upwardly into the small end of the egg. Tacks were 
inserted until the tack head was placed against the egg shell. Injected 
and control eggs were then incubated to hatch. 
Hatchability of control eggs was 72.2%; hatchability of injected eggs was 
56%. These results indicate that the hatchability of eggs injected without 
the use of sealant is significantly reduced over non-injected controls. 
EXAMPLE 3 
Hatchability: Injection With Sealant 
The hatchability of non-injected eggs was compared to eggs injected using a 
stainless steel tack having a pointed shaft 5/16 inch in length and 1/16 
inch in diameter, and a slightly concave discoid head 3/8 inch in diameter 
(Moore Solidhead Steel, No. 51, #2, Wyndmoor, Pa.). Fresh, non-incubated 
eggs were used. Control eggs (n=56) were not injected. Injected eggs were 
held upright (large end up) while a tack was inserted upwardly into the 
small end of the egg. Tacks were inserted until the tack head was placed 
against the egg shell. Fifty-six eggs were injected without the use of a 
sealant. Fifty-six eggs were injected using tacks with the underside of 
the tack head (concave side from which the shaft extended) coated with a 
thin layer of DUCO.TM. cement (E.I. DuPont). All eggs were then incubated 
to hatch under similar conditions. 
Hatchability of control eggs was 77.8%; hatchability of injected eggs (no 
sealant) was 60.5%; hatchability of injected (and sealed) eggs was 68.5%. 
These results indicate that the presence of sealant on the injection 
apparatus increases hatchability of injected eggs. 
The foregoing is illustrative of the present invention and is not to be 
construed as limiting thereof. The invention is defined by the following 
claims, with equivalents of the claims to be included therein.