Applications in In Ovo Technology1
P. A. JOHNSTON, H. LIU, T. OCONNELL, P. PHELPS, M. BLAND, J. TYCZKOWSKI, A. KEMPER,
T. HARDING, A. AVAKIAN, E. HADDAD, C. WHITFILL, R. GILDERSLEEVE, and C. A. RICKS
EMBREX Inc., P.O. Box 13989, Research Triangle Park, North Carolina 27709-3989
ABSTRACT By mid-August 1995, 55% of broiler
embryos in North America were vaccinated for Mareks
disease using the INOVOJECT system, with 201
INOVOJECT machines placed with 16 of the top 25
poultry producers, providing the industry with the
capacity to inject in excess of 400 million eggs per month
or about 5 billion eggs per annum. In ovo administration
of a bursal disease antibody-infectious bursal disease
virus (BDA-IBDV) complexed vaccine to specific-
pathogen-free (SPF) embryos was safer and more potent
than conventional IBDV vaccine alone because it
delayed the appearance of bursal lesions, produced no
early mortality, produced higher geometric mean anti-
body titers against IBDV, and generated protective
immunity against challenge. In ovo administration of a
BDA-IBDV complexed vaccine to broiler embryos gener-
ated antibody titers against IBDV sooner than conven-
tional virus vaccinates, and generated protective immu-
nity against challenge. Direct DNA injection of plasmid
DNA encoding b-galactosidase into breast muscle in ovo
and posthatch was an effective means to achieve both
(Key words: in ovo, vaccines, cytokines, development, sex)
INTRODUCTION
Over the past two decades, the rapidly growing U.S.
poultry industry has accepted labor-saving technologies
and improvements in genetic selection, management
practices, nutrition, and disease control. EMBREX was
founded in 1985 on the basis of an exclusively licensed
USDA patent Disease Control in Avian Species by
Embryonal Vaccination (Sharma and Burmester, 1984).
In the 10 yr from its inception, EMBREX has developed
and marketed the INOVOJECT (Gildersleeve, 1993;
Gildersleeve et al., 1993; Sarma et al., 1995), an auto-
mated egg injection machine that improves poultry
production efficiency. EMBREX has also initiated
research and development programs to evaluate novel
biological approaches compatible with this delivery
system. EMBREX intends to bring the benefits of in ovo
Received for publication August 13, 1995.
Accepted for publication August 7, 1996.
1All authors contributed equally.
165
gene transfer and expression, with potential for the
development of gene vaccines using plasmids encoding
protective antigens from poultry pathogens. In ovo
administration of 800 U chicken myelomonocytic growth
factor (cMGF), a chicken hematopoietic cytokine for cells
of the monocytic-granulocytic lineages, significantly
reduced mortality associated with Escherichia coli ex-
posure within the hatcher when compared to PBS
controls (6.1 vs 12.4, P 0.05), but not when compared
to a yeast expression control. A procedure was deve-
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loped enabling injection prior to the onset of incubation
without compromising embryo viability. This in ovo
injection process has opened up the window of embryo
development during incubation for intervention, as
illustrated by the 100% male phenotype produced in
chicks hatching from eggs injected with aromatase
inhibitor prior to incubation. These data illustrate some
of the in ovo applications presently in use by the poultry
industry, and under development or in research at
EMBREX.
1997 Poultry Science 76:165178
technology to the worldwide poultry industry and is
presently introducing the INOVOJECT to the Euro-
pean, Asian, and Middle Eastern markets.
In the poultry industry, hand inoculation of broiler
chicks at day of hatch is rapidly giving way to the
automated introduction of vaccines to the embryo by
injection through the eggshell at Day 18 of incubation,
when eggs are routinely transferred to hatching trays.
EMBREXs automated egg injection system, the IN-
OVOJECT, inoculates between 20,000 to 30,000 eggs
per hour, performs egg transfer, and eliminates the need
for posthatch vaccination (Gildersleeve, 1993; Gilders-
leeve et al., 1993). The INOVOJECT gently injects
compounds in precisely calibrated volumes without
causing trauma to the developing embryo, thereby
reducing chick handling, improving hatchery managea-
bility through automation, and reducing the costs of live
production. The INOVOJECT system works by gently
lowering an injection head onto the top of the egg, a
small diameter hollow punch pierces a little opening in
the shell, a needle descends through this tube to a
controlled depth (2.54 cm), a specific amount of vaccine
166 JOHNSTON ET AL.
is delivered, and then the needle is withdrawn and
cleansed in a sterilization wash. The site of delivery is,
therefore, a ratio of amnion:embryo injections, which
varies with the stage of development of the egg and
depends upon the length of egg incubation (Gilders-
leeve, 1993; Gildersleeve et al., 1993). Two Mareks
disease vaccines are presently delivered via the IN-
OVOJECT, and safety, potency, and efficacy studies
indicate that a novel infectious bursal disease vaccine,
which was developed by EMBREX and recently ap-
proved by the USDA (January 30, 1995) for at-hatch
administration, is also safe and efficacious for in ovo use,
and product registration for in ovo use will be pursued
(Gildersleeve, 1993; Gildersleeve et al., 1993; Sarma et al.,
1995).
A number of immune responses, including transplan-
tation reactions (splenomegaly) and antibody responses
to antigen (Solomon, 1962, 1966; Solomon and Tucker,
1963; Delhanty and Solomon, 1966; Jankovic et al., 1975),
can be induced in embryos at 12 to 14 d of incubation,
soon after the emergence of T cell and B cell progenitors
(Vainio and Lassila, 1989; Dunan et al., 1990; Houssaint
et al., 1991; Reynaud et al., 1992; Cormier, 1993; Dieterlin-
Lievre and Le Douarin, 1993). Because embryo vaccina-
tion was first proposed as a means of priming or
stimulating an early immune response that might
improve protection against poultry diseases (Sharma
and Burmester, 1982, 1984), efficacious embryo vaccines
against Mareks disease, infectious bursal disease virus
(IBDV), infectious bronchitis virus, and Newcastle
disease virus have been described (Sharma and Bur-
mester, 1982; Sharma et al., 1984; Sharma, 1985; Wakenell
and Sharma, 1986; Ahmad and Sharma, 1992, 1993;
Whitfill et al., 1992 a,b).
The purpose of the present paper is to update the
progress on INOVOJECT machine placement, briefly
review some field efficacy data on in ovo Mareks disease
vaccination, and provide some data on the products and
approaches to embryo intervention that are either under
development or are part of our discovery research
programs. We will discuss data on the efficacy of a
novel in ovo bursal vaccine comprised of a bursal disease
antibody (BDA) with virus neutralizing activity com-
plexed with IBDV virus, report on gene marking studies
from our gene vaccine program, present some prelimi-
nary in ovo recombinant cytokine efficacy data, and
provide data to illustrate the utility of our patented
early injection process, which provides embryo access
throughout the 21-d incubation period of development.
MATERIALS AND METHODS
Animals
Fertile White Leghorn or broiler (Gallus domesticus,
Peterson Arbor Acres) chicken eggs were obtained from
2Manufactured by Tri Bio Laboratories, State College, PA 16803.
3Sanofi Animal Health, Inc., Overland Park, KS 66210.
a commercial farm and incubated and hatched in our
facility. Embryonic age was designated as E1, E2, etc., E1
being the 1st d of incubation based upon the staging
system of Nicolas-Bolnet et al. (1991).
Egg Injection Machine
The development and operation of the automated egg
injection machine, the INOVOJECT, has been described
previously (Gildersleeve, 1993; Gildersleeve et al., 1993;
Sarma et al., 1995).
Mareks Disease Vaccine
A bivalent Herpes virus of Turkeys (HVT) (Serotype 3,
Strain FC-126)+ SB-1 (Serotype 2) vaccine2 was used as
described previously (Sarma et al., 1995).
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In Ovo and Conventional Vaccination
In ovo vaccination was performed according to the
vaccine manufacturers directions and in accordance with
the procedures described in the INOVOJECT operators
manual, as described previously (Gildersleeve et al., 1993;
Sarma et al., 1995). Conventional vaccination was per-
formed using standard chick vaccinators and in accor-
dance with the directions provided by the vaccine
manufacturer, as described previously (Sarma et al., 1995).
Field Efficacy Comparison of In Ovo
and Conventional Mareks Disease
Vaccine Administration
Data collected from a total of three consecutive field
trials, conducted by two separate commercial broiler
companies in North Carolina, were used for the compari-
son of Mareks disease vaccine administration efficacy
(Table 1), as described previously (Sarma et al., 1995).
Production and Measurement of BDA
Antisera containing BDA were produced in Hy-VAC
specific-pathogen-free (SPF) chickens as described previ-
ously (Whitfill et al., 1995). Neutralizing antibodies to
IBDV were measured using a viral neutralization bioas-
say, as previously described (Haddad et al., 1994).
Preparation of the BDA-IBDV Vaccine
The determination of the optimal formulation of the
BDA-IBDV complexed vaccine was as described previ-
ously (Whitfill et al., 1995). A vaccine constructed of 100
embryo infectious dose (EID50) of vaccine Strain 2512
(IBD-BLEN3 or a classical IBDV strain similar in
invasiveness) mixed with 24 U of BDA was used.
 SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 167
TABLE 1. Comparison of the effects of conventional and INOVOJECT HVT + SB-1 bivalent
Mareks disease vaccine administration on poultry production indicators1,2

Mareks vaccine administration

Production indicators Conventional INOVOJECT
Percentage hatchability 85.80a 85.23b
Percentage culled 0.39a 0.28b
Percentage mortality
1 wk 1.34a 0.88b
2 wk 1.68a 1.18b
Final 3.74a 2.87b
Average live weight, lb 4.73 4.76
Feed conversion 2.08 2.06
Condemnations
Total 1.02a 0.54b
Airsacculitis 0.40a 0.12b
Septicemia-toxemia 0.52a 0.34b
Leukosis 0.01 0.01
a,bValueswith no common superscript differ significantly (P < 0.05).
1Data compiled from Sarma et al. (1995) and is presented with permission from Avian Diseases.
2Represents pooled data from three independent trials with a total of 62,600 chicks placed per treatment.

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In Ovo Vaccination and IBDV Challenge
Eggs from SPF chickens were injected on E18 with
saline (nonvaccinates), 100 EID50 IBDV virus, or BDA-
IBDV (24 U BDA:100 EID50 IBDV). Eighty-five hatched
chicks were placed from each group, cumulative
posthatch mortality was observed through 5 d of age, and
five birds per group were euthanatized and evaluated for
gross bursa of Fabricius lesions at the indicated times. The
IBDV antibody titer was measured in 10 birds per group
on Day 28 of age by ELISA4 and the geometric means are
presented. Ten birds from each group were challenged on
Day 28 of age with 100 chick infectious doses (CID50) of
the highly pathogenic field strain DV86, bursas were
examined for gross acute bursal lesions 4 d postchallenge,
and protection was defined as the absence of edematous
or hemorrhaged bursas.
Eggs from broiler chickens were injected on E18 with
saline (nonvaccinates), 100 EID50 IBDV virus, or BDA-
IBDV (24 U BDA:100 EID50 IBDV). The IBDV antibody
titer was measured in 10 birds per group at weekly
intervals for 6 wk by ELISA and the geometric means are
presented. Twenty birds from each group were
challenged on Day 21 of age with 100 CID50 of the highly
pathogenic field Strain DV86 and the cumulative mortal-
ity over 14 d was observed.
NC 27695-2608). In the pMiwZ plasmid, lacZ expression is
driven by two eukaryotic transcriptional promoters
derived from the Rous Sarcoma Virus long terminal
repeat and chicken b-actin gene. Translation initiates
within an exon from the chicken d-crystallin gene, in-
frame with the lacZ, forming a chimeric gene. A series of
four experiments were performed to evaluate the expres-
sion of b-galactosidase activity in breast muscle tissues
excised at 1 to 6 wk posthatch after in ovo injection of 100
mg of pMiwZ DNA into the breast muscle of E19 embryos
(Table 4). Eggs were candled to determine the location of
the embryo within the egg and injection was angled at 2.5
to maximize penetration into muscle tissue on E19.
Typically, three to five embryos were injected per time
point in each experiment. DNA was injected along with
India ink to mark the site of injection. At 1 to 6 wk
postinjection, birds were euthanatized, muscle tissue was
excised around the area stained with India ink, and the
whole tissue was fixed in 2% formaldehyde, 0.2%
glutaraldehyde, 0.02% NP-40, 0.01% deoxycholate, 2mM
MgCl2 in 50 mM NaPO4 buffer pH 7.3, washed in 0.02%
NP-40, 2mM MgCl2 in 50 mM NaPO4 buffer pH 7.3, and
stained for the presence of b-galactosidase using X-Gal by
the technique of Sanes et al. (1986). Tissue was examined
for blue muscle fibers under a dissecting microscope
(Figure 2).

Direct DNA Injection In Ovo and Whole Expression of Recombinant Chicken

Tissue Staining for b-Galactosidase
Expression
The mammalian expression vector, pMiwZ (Suemori et
al., 1990), which contains the Escherichia coli lacZ gene as a
marker, was obtained from Jim Petitte (Department of
Poultry Science, North Carolina State University, Raleigh,
4IDEXX Laboratories, Inc., Westbrook, ME 04098.
Myelomonocytic Growth Factor
The cloning and expression of recombinant chicken
myelomonocytic growth factor (cMGF) will be described
in more detail elsewhere (Johnston et al., 1994, 1995, and
unpublished data). Reverse transcription (RT) and poly-
merase chain reaction (PCR) amplification of total RNA,
prepared from guanidinium isothiocyanate lysed Con-
canavalin A (Con A)-stimulated splenocytes isolated from
168 JOHNSTON ET AL.
6-wk-old Leghorns with cMGF-specific oligonucleotide
primers based on the published cMGF sequence (Leutz et
al., 1984, 1989), yielded a band of the expected size of 560
bp. The 560-bp band was subcloned into the Invitrogen5
pCRII vector, and DNA sequence analysis of three
clones confirmed that we had cloned the authentic cMGF
cDNA. The sequence varies from the published sequence
by one base pair (T C transition), which does not result in
a change in the amino acid level, indicating that it is
probably a naturally occurring genetic polymorphism
among the chicken lines.
The Pichia pastoris yeast expression system kit was
obtained from Invitrogen and was used according to the
manufacturers instructions. The cMGF clone was ream-
plified with PCR primers to engineer BamHI and EcoRI
sites, and subcloned into the expression vectors pPIC9 and
pHIL-S1, both of which produce secreted fusion protein
products with signal peptides from the a-mating factor or
acid phosphatase genes, respectively. Spheroplasts were
prepared from the GS115 (His) strain by treatment with
zymolase and transformed with BglII digests of cMGF
subcloned into pPIC9 and pHILS1. The transformed yeast
were plated onto selective media allowing the identifica-
tion of recombinants that have stably integrated the cMGF
gene into the yeast genome. Potential cMGF recombinant
clones were selected and grown in liquid culture in the
presence of methanol to induce expression and secretion
of the recombinant protein into the media. The culture
media supernatant from five candidate recombinant
clones was collected and analyzed for in vitro activity in
the bone marrow proliferation assay and were found to be
biologically active. To recover biologically active secreted
cMGF from the transformed yeast growth media, 3,000 g
centrifugation supernatants are diluted with 10% ethanol
in PBS and applied to Centripreps 10 kDa molecular
weight cut-off filters.6 By repeated operation of these
devices, cMGF supernatants were concentrated and the
media exchanged. The final product volume was 0.1 to 0.2
times the original supernatant volume and around 95% of
the ethanol and components < 10 kDa were replaced with
PBS. Processed and concentrated recombinant cMGF was
sterilized by passage through a 0.2 mM filter, aliquoted,
and stored at 70 C.
Bone Marrow Proliferation Assay
Bone marrow cells from the tibia and femur of E16
embryos were flushed using a 26 gauge 3/8 needle,
1-cc syringe containing RPMI 1640 supplemented with
gentamicin (0.5 mg/mL). Cells were pelleted by centrifu-
gation at 800 g for 5 min at 4 C, resuspended in RPMI
1640 plus gentamicin, and living cells enumerated on a
hemacytometer on the basis of trypan blue exclusion.
5San Diego, CA 92121.
6Amicon, Beverly, MA 01915.
7Costar, Boston, MA 02241.
Bone marrow cells were seeded at 5 105 cells/100 mL per
well in 96-well tissue culture plates.7 The cMGF was
diluted into RPMI 1640 medium plus gentamicin and 10%
fetal bovine serum at the concentrations indicated. Then,
100 mL was added to each well and the plate was incubated
at 41 C, 5% CO2 for 48 h. Bone marrow proliferation was
measured as the ability to convert MTT (3,[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
into a blue-colored product, MTT-formazan as described
previously (Nicolas-Bolnet et al., 1995).
Hatcher Contamination E. coli
Challenge Model
The hatcher contamination model involves placing
eggs (typically 36 eggs) that have been injected into the air
cell with a pathogenic E. coli 01 strain (obtained from J.
Barnes, North Carolina School of Veterinary Medicine,
Raleigh, NC 27695) (typically 104 cfu) in hatching trays
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evenly spaced throughout the hatcher. The chicks that
hatch from these infected eggs then transmit the pathogen
to the experimental chicks hatching from eggs incubated
in intervening trays within the same hatcher. The various
treatment groups are represented in two separate loca-
tions within the hatcher, and the locations are randomly
varied from trial to trial. On the day of hatch, chicks are
pulled, wing-banded, commingled, placed in floor pens,
and monitored for mortality twice daily for 2 wk. At 2 wk,
surviving chicks are counted.
Egg Injection on Day 0 Prior
to Incubation
One hundred eggs from each group were either not
injected (controls) or were inverted and injected into the
small end from the top followed by righting of eggs to the
correct orientation prior to incubation, or were directly
injected into the small end of the egg from below. Eggs
were injected with 100 mL of PBS into the small end of the
egg to a depth of 5.0 mm using a 22-gauge needle. Eggs
were then incubated under standard conditions and the
percentage hatchability relative to uninjected controls
presented.
Sixty eggs from each group were either not injected
(controls), or were injected into the small end while
upright and sealed with G.E. Silicone II silicone sealant
after injection, or were first sealed with silicone sealant
prior to injection into the small end of the egg while
upright. Eggs were sanitized by dipping into a solution of
5% H2O2 prior to injection. Eggs were injected with 100 mL
of PBS into the small end of the egg to a depth of 5.0 mm
using a 20-gauge needle. Eggs were then incubated under
standard conditions and the percentage hatchability
relative to uninjected controls presented.
The Day 0 prior to incubation egg injection procedure
entails the application of a liquid sealant material to the
site of egg injection, the curing of the sealant to form an
elastic seal thereon, insertion of the injection device
 SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 169
through the seal, delivery of the material into the egg, and
withdrawal of the injection device from the egg.
One hundred-and-twenty eggs from each group were
either not injected (controls), or were first sealed with
silicone sealant prior to injection into the small end of the
egg while upright with a variety of excipients,
2-Propanol8 C-18 Silica 10 mm particles,9 or 1-5%
Molecusol.10 Eggs were injected with 50 mL of the different
excipients into the small end of the egg through the sealant
to a depth of 5.0 mm using a 20-gauge needle. Eggs were
then incubated under standard conditions and the
percentage hatchability relative to uninjected controls
presented.

Effects of Day 0 Injection Prior to

Incubation of Aromatase Inhibitor on
the Sex Phenotype of Hatching Chicks

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Aromatase inhibitor (AI) (Elbrecht and Smith, 1992;
Wartenberg et al., 1992) was kindly provided by W. R.
Campbell (Ciba-Geigy, Greensboro, NC, 27419). In the
three experiments presented (Table 7), the indicated
number of eggs from Hubbard Hubbard feather-sexable
chickens11 were either not injected, or were injected on
Day 0 prior to incubation using the patented procedure
described above with 100 mL of propylene glycol alone, or
propylene glycol containing 0.1 mg of AI. Eggs were then
incubated under standard conditions and the percentage
hatchability relative to uninjected controls presented.
Genotypic sex ratios of hatching chicks were determined
by feather sexing and phenotypic sex ratios determined on
the basis of vent sexing.12
RESULTS
Placement of INOVOJECT
Since the first commercial placement of machines in
April 1992, embryo vaccination using the INOVOJECT
system has been accepted rapidly by the poultry industry
such that, at present (mid-August 1995), approximately
55% of all broiler birds in North America are vaccinated
for Mareks disease by this system (Figure 1). Currently,
201 INOVOJECT machines have been placed in North
America under the terms of a lease licensing agreement
with 16 of the top 25 poultry producers, providing the
industry with the capacity to inject in excess of 400 million
eggs per month, or about five billion per annum.
8Isopropanol Optima grade, Fisher Scientific, Raleigh, NC 27604.
9Waters, Marlborough, MA 01752.
10Cyclodextrins, Pharmatec, Inc., Alachua, FL 32615.
11Webber Hatchery, Goldsboro, NC 27530.
12Amchick, Inc., Lansdale, PA 19446.
FIGURE 1. INOVOJECT Placement. The number of INOVOJECT
systems placed under contract at the end of each quarterly period,
beginning in the last quarter of 1992 to the end of the second quarter of
1995.
In Ovo Mareks Disease Vaccination
Typically all commercial flocks in the U.S. are vacci-
nated against Mareks disease, and, as described above,
greater than 50% of these are injected using in ovo
technology. A number of comparison studies have been
conducted to evaluate the safety and efficacy of conven-
tional vs in ovo administration of an HVT + SB-1 bivalent
Mareks disease vaccine in commercial broilers under
field conditions (Gildersleeve, 1993; Gildersleeve et al.,
1993; Sarma et al., 1995). The pooled data from three
independent studies [Table 1, compiled from Sarma et al.
(1995)] indicates that in ovo Mareks disease vaccine
administration significantly reduced the number of culls,
condemnations, and both early and total mortality when
compared to conventional day of hatch administration.
However, in ovo Mareks disease vaccination in these trials
also produced a significant reduction in hatchability when
compared to at-hatch administration (Table 1), a trend not
observed in previous studies (Gildersleeve, 1993; Gilders-
leeve et al., 1993). Average live weights, feed conversion
ratios, and leukosis condemnations were not affected to
any statistically significant degree (Sarma et al., 1995,
Table 1).
In Ovo IBDV Vaccination
There are significant economic losses associated with
the well-documented immunosuppressive effects of IBDV
together with the emergence of highly virulent strains in
Europe, Japan, and the variant strains in the U.S.
(Faragher et al., 1974; Hirai et al., 1979; Sharma et al., 1989;
Saif, 1991; Nakamura et al., 1992). A novel vaccine that
170 JOHNSTON ET AL.
TABLE 2. Day 18 in ovo vaccination of specific-pathogen-free embryos1

IBDV antibody titer and

protection from Day 28
DV86 challenge
Day 5 Percentage
Bursal lesions2 mortality protection4
percentage IBDV 10 birds
Day Day Day Day Day Day (Cumulative antibody challenged
Treatment groups 1 3 5 7 9 11 n = 85) titer3 per group
(%) (%)
Nonvaccinated 0 0 0 0 0 0 0 121a 0
IBDV vaccine 5 5 5 5 5 5 15 87a 100
Bursal disease antibody-IBDV
vaccine 0 0 0 1 5 5 0 2,055b 100
a,bValues with no common superscript differ significantly using Duncans multiple range test (P 0.05).
1IBDV = infectious bursal disease virus.
2Presence or absence of visual signs of gross bursal lesions, n = 3 for nonvaccinated, and n = 5 each for IBDV and bursal disease antibody-IBDV
vaccinates, at each time.
3Day 28 geometric mean IBDV ELISA antibody titer, n = 10.
4Absence of edematous bursal infection on Day 32, 4 d postchallenge with the DV86 strain of IBDV on Day 28.

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consists of a complex between a measured amount of BDA
with IBDV neutralizing activity and a specific amount of
IBD virus has recently been approved by the USDA
(January, 1995) for at-hatch administration. A series of
studies were conducted to evaluate the safety and efficacy
of E18 in ovo administration of a BDA-IBDV complexed
vaccine to SPF (Table 2) and broiler (Table 3) embryos.
Chicks hatching from SPF eggs injected with conventional
IBDV vaccine had gross acute lesions of the bursa from
Day 1 posthatch and had a cumulative mortality of 15% by
Day 5 posthatch (Table 2). In contrast, BDA-IBDV complex
vaccinates did not develop visual signs of bursal lesions
until Day 5 posthatch, and no mortality was observed by
Day 5 posthatch, in the chicks placed. Although both
vaccinated treatment groups were protected from a Day
28 challenge with the virulent DV86 strain, BDA-IBDV
vaccinates had developed higher geometric mean anti-
body titers (P < 0.05) against IBDV than chicks hatched
from SPF eggs injected with the IBD vaccine virus alone
(Table 2). A similar evaluation of in ovo administration of
the two vaccines in high maternal antibody commercial
broiler embryos (Table 3) demonstrated that both groups
were protected from challenge. After maternal antibody
levels waned between Day 21 to 28 of age, the BDA-IBDV
vaccinates went on to develop higher geometric mean
antibody titers (P < 0.05) against IBDV by Day 42 than the
chicks injected with IBD vaccine virus alone (Table 3).
In Ovo Gene Transfer and Expression
in Muscle Tissue by Direct
DNA Injection
Gene vaccines based on the uptake of injected DNA by
striated muscle have been used to produce in situ
expression of genes encoding the protective antigens of
pathogens and thereby generate protective immunity
(Wolff et al., 1990, 1991; Cox et al., 1993; Robinson et al.,
1993; Ulmer et al., 1993; Wang et al., 1993). Gene marking
experiments using expression plasmids containing the b-
galactosidase reporter gene injected into breast muscle at
day of hatch (Figure 2), demonstrated that chicken breast

TABLE 3. Day 18 in ovo vaccination of broiler embryos

Posthatch infectious bursal disease virus (IBDV)

ELISA antibody titer
geometric mean titer (n = 10) Protection against Day 21
DV86 IBDV
Treatment group Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 challenge1 (n = 20)
(% mortality 7 d
postchallenge)
Nonvaccinated 2,990a 1,178a 1,245a 369a 0b 10c 20
IBDV vaccine 2,491ab 867a 688b 45a 6a 1,149b 0
Bursal disease
antibody-IBDV vaccine 2,361b 1,035a 621b 42a 41a 2,211a 0
acMeansin a column differ (P 0.05) level using Duncans multiple range test. Analyses are based on rank transformed data.
1On Day 21, 20 chicks from each group were challenged intraocularly with 100 embryo infectious dose of the DV86 strain of IBDV.
 SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 171

FIGURE 2. b-Galactosidase gene expression in breast muscle tissue excised 1 wk postinjection of the pMiwZ expression plasmid. Muscle tissues
were excised at 1 wk postinjection into the breast muscle of day-of-hatch chicks with either 1) PBS containing India ink or 2) 100 mg of pMiwZ DNA in
PBS containing India ink. Muscle tissue was excised around the area stained with India ink (I), and the whole tissue was fixed and stained for the

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presence of b-galactosidase (B) using 5-bromo-4-chloro-3-indolyl-b D-galactoside.

muscle is also capable of taking up and expressing early mortality associated with E. coli exposure within the
plasmid DNA. A series of four separate gene marking hatcher (Table 5). The 800 U cMGF treatment and the 0.2
experiments were conducted in which breast muscle mg gentamicin were efficacious when compared to PBS;
tissues (n = 3 to 5) were excised at various time points however, neither gentamicin or cMGF were efficacious
between 1 and 6 wk posthatch and evaluated for b- when compared to the YEA recombinant expression
galactosidase expression after E19 in ovo injection of 100 mg control. Eighty units of cMGF reduced mortality in Trials 1
of pMiwZ DNA (Table 4). Eggs were candled to locate the and 3 but not in Trial 2, and 8 U of cMGF did not
position of the embryo and the injection was angled at 2.5 significantly reduce mortality in any of the three trials,
to maximize breast muscle penetration. Of the 69% of perhaps indicating a dose dependency for cMGF efficacy.
injections (58/84) when embryo breast muscle penetration
was indicated by the presence of India ink stain when
removed posthatch, 91% (53/58) of these plasmid DNA-
injected muscle tissues also exhibited b-galactosidase
expression, and expression persisted through 6 wk of
growth (Table 4). These data indicate that gene transfer of
DNA to embryonic tissue can be used to produce gene
expression in tissues posthatch.

In Ovo Recombinant cMGF

Chicken myelomonocytic growth factor is an avian
hematopoietic cytokine that stimulates bone marrow cells
to proliferate and produce cells of the monocyte-
macrophage lineage (Leutz et al., 1984, 1989). Recom-
binant cMGF has been expressed in P. pastoris and shown
to stimulate the proliferation of bone marrow cells in vitro
(Figure 3). In ovo delivery of recombinant cMGF might,
therefore, accelerate the onset of myelopoiesis so that
chicks hatch with more monocytes-macrophages and
granulocytes, which may also be more functionally
activated and, therefore, better able to defend against
microbial pathogens. To test this hypothesis, a prelimi-
nary series of three separate E. coli hatcher contamination
challenge models were performed (Table 5). An E18 in ovo
injection of 0.2 mg gentamicin, yeast-expressed albumin
(YEA), and 800 U rcMGF demonstrated efficacy against
FIGURE 3. Recombinant chicken myelomonocytic growth factor
induced proliferation of Day 16 of incubation (E16) embryo bone marrow
cells. 5 105 bone marrow cells (E16) were incubated at 41 C, 5% CO2 for
48 h in RPMI 1640 plus gentamicin and 5% fetal bovine serum with the
indicated dilutions of supernatant derived from methanol induced
transformed Pichia pastoris clones; GS115-YEA, GS115-pHILS1-cMGF2,
GS115-pPIC9-cMGF1, GS115-pPIC9-cMGF2, and GS115-pPIC9-cMGF5.
3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
was then added, and after an additional 1 h of incubation the resulting
formazan product was solubilized and measured at 570 nm. A
representative experiment from three similar experiments is presented,
and the data are reported as the mean of triplicate wells ( SD).
172 JOHNSTON ET AL.
TABLE 4. Summary of in ovo breast muscle targeting and b-galactosidase gene expression data1,2

Time of muscle excision posthatch

Staining 1 wk 2 wk 3 wk 4 wk 6 wk
(%)
India ink 54 79 85 68 20
(7/13) (22/28) (11/13) (17/25) (1/5)
b-Galactosidase expression 54 71 92 52 20
(7/13) (20/28) (12/13) (13/25) (1/5)
1Total positives: India ink 69% (58/84), b-Galactosidase 63% (53/84). None of the no-DNA injected controls
(0/33, typically two to four per time point) exhibited b-Galactosidase staining.
2Data compiled from four separate experiments in which three to five embryos per time point were injected in
ovo with 100 mg pMiwZ on Day 19 of incubation.

In Ovo Injection Prior to Incubation development after reversion to the correct orientation and
subsequent egg incubation. Application of a sealant prior
In addition to egg transfer, eggs are also handled in to direct egg injection from below improved hatchability
commercial operations on E0 when they are set in to 5% above uninjected controls, whereas application of

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incubators, and in ovo intervention at this time would have
the potential to influence almost the entire window of
embryo development. To effectively access the avian
embryo at the onset of incubation, it was necessary to
develop an egg injection process that did not compromise
embryo viability (Table 6). In comparison to uninjected
controls, the effects of E0 injection of the bottom (small
end) of the egg into the albumin were dramatically
different if the egg was inverted first and injected
downwards into the small end of the egg from above
followed by returning the egg to its original orientation
prior to incubation, rather than direct injection upwards
into the small end of the egg from below (Table 6,
Experiment 1). Although both injection methods reduced
hatchability relative to uninjected controls, inversion and
top injection into the albumin followed by reorientation of
eggs to the upright orientation prior to incubation
dramatically decreased hatchability to 4.8%, compared to
the 95% with direct injection into the egg albumin from
below. Examination of injected eggs by breakout revealed
the entry of air during the inversion and top injection
procedure, which may have adversely affected embryo
the sealant after injection continued to result in reduced
hatchability, 14% below that of uninjected controls (Table
6, Experiment 2). On the basis of these and additional data
(data not shown), a E0 egg injection procedure was
developed that entails the application of a liquid sealant
material to the site of egg injection, the curing of the
sealant to form an elastic seal thereon, insertion of the
injection device through the seal, delivery of the material
into the egg, and withdrawal of the injection device from
the egg. Using this procedure, a variety of potential
excipients have been delivered to the albumin of E0 eggs
prior to the onset of incubation without affecting
hatchability (Table 6, Experiment 3).
Effects of E0 Injection Prior to
Incubation of AI on the Sex
Phenotype of Hatching Chicks
In three separate experiments utilizing the E0 injection
procedure described above, administration of either
propylene glycol or 0.1 mg of AI in propylene glycol,
reduced hatchability relative to uninjected controls by an

TABLE 5. Efficacy of in ovo administration of recombinant chicken myelomonocytic growth factor (cMGF) in the hatcher
contamination Escherichia coli challenge model

In ovo treatment Experiment 1 Experiment 2 Experiment 3 Mean SEM

(% Mortality)
PBS 14.3 8.3 14.5 12.4a 3.5
Gentamicin (0.2 mg) 5.7 5.8 0.83 4.1c 2.8
cMGF
8 U 14.3 7.5 12.5 11.4ab 3.5
80 U 4.3 10 10.8 8.4abc 3.5
800 U 5.7 2.5 10.1 6.1bc 3.8
YEA 5.7 6.7 7.5 6.6abc 0.9
10 min1 Hatcher cfu 280 247 208
Number of birds per treatment 70 120 120 310
acMeanswere partitioned by Duncans multiple range test. Means with no common superscript differ significantly (P 0.05).
1Number of colonies counted on TSB plates exposed for 10 min within the hatcher.
 SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 173
TABLE 6. Effects of Day 0 injection prior to incubation
on embryo viability measured by hatchability

Hatchability of
Experiment Injection uninjected controls Number of eggs
(%)
1 Uninjected 100 100
Bottom injected (small end) inverted 4.8 100
Bottom injected (small end) upright 95 94
2 Uninjected 100 60
Injected-sealed 86 60
Sealed-injected 105 60
3 Uninjected 100 120
Silica 5% isopropanol 98.7 120
Silica 20% isopropanol 103 120
Molecusol 1% 106.4 120
Molecusol 5% 103.5 120
Isopropanol 5% 102.6 120
Isopropanol 20% 94.2 120

average of 9 and 4.5% respectively. On the basis of vent handle breakdowns. Currently, 201 INOVOJECT

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sexing, the E0 injection of AI produced a male phenotype
in 100% of the hatching chicks, in contrast to their almost
50:50 male to female feather sex genotypic ratio (Table 7).
As expected, there was almost complete correspondence
between the genotypic and phenotypic sex ratios of
uninjected and vehicle-injected controls.
DISCUSSION
Commercial INOVOJECT placement typically in-
cludes a hatchery site survey to determine compatibility
with the INOVOJECT system, initial head-to-head
INOVOJECT trials to gain acceptance and demonstrate
benefit, installation with both operational and main-
tenance training of hatchery staff, bi-monthly preventa-
tive service and maintenance visits, refresher training
courses, and a 24-h technical and service support to
machines have been placed in North America, providing
the industry with the capacity to inject in excess of 400
million eggs per month, or about 5 billion per annum,
and approximately 55% of all U.S. broiler birds are
vaccinated for Mareks disease by this system.
Although INOVOJECT administration of Mareks
disease vaccine relative to conventional Mareks disease
vaccine administration provides benefits illustrated by
consistently decreasing condemnation rates, feed con-
version ratios, and settlement costs (Gildersleeve, 1993;
Gildersleeve et al., 1993), the other variables examined,
including leukosis condemnations, would seem to
indicate an equivalent field efficacy to posthatch vaccine
administration against Mareks disease (Table 1, com-
piled from Sarma et al., 1995). These field efficacy
evaluations of INOVOJECT and conventional Mareks
disease vaccine administration were subject to both the

TABLE 7. Comparison of genotypic and phenotypic sex ratio of hatching chicks

after injection of an aromatase inhibitor on Day 0 prior to incubation1

Feather sex ratio % Vent sex ratio %

Day 0 genotype phenotype
in ovo Hatchability
treatment % of control Male Female Male Female
(% of control) (%)
Experiment 1. (Hatchability n = 110/sex ratio n = 70)
NI 100 (68) 50 50 46 54
VH 91 50 50 46 54
AI 93 50 50 100 0
Experiment 2. (Hatchability n = 125/sex ratio n = all hatched chicks)
NI 100 (87) 50.5 49.5 51.5 48.5
VH 96.2 52.9 47.1 50.5 49.5
AI 94.5 60.2 39.7 100 0
Experiment 3. (Hatchability n = 150/sex ratio n = all hatched chicks)
UN 100 (76) 45 55 48 52
VH 83 57 43 57 43
AI 98.7 51 49 100 0
1UN = uninjected controls, VH = vehicle, propylene glycol, AI = 0.1 mg of aromatase inhibitor.
174 JOHNSTON ET AL.
level and timing of the environmental exposure to
virulent Mareks disease virus under standard produc-
tion conditions. Experimental comparisons of vaccine
administration efficacy against pathogenic Mareks dis-
ease virus have consistently demonstrated an in ovo
benefit in early (Days 0, 3, or 5 posthatch) challenge
situations (Sharma and Burmester, 1982; Sharma et al.,
1984; Sharma, 1985; Sarma, et al., 1995), because optimal
protection from vaccination requires several days to
develop, and embryo vaccination increases the time
between vaccination and environmental exposure to
Mareks disease virus. These data indicate that in ovo
administration of Mareks disease vaccine would likely
provide better efficacy against environmental exposure
to a virulent Mareks disease virus in the 1st wk
posthatch than conventional at-hatch administration,
while providing equivalent efficacy against later
Mareks disease virus exposure during growout. Addi-
tional benefits observed with in ovo administration may
stem from the fact that 100% of embryos are vaccinated
and that conventional vaccination systems do not
incorporate the needle sanitation technology found in
the INOVOJECT system.
Due to the significant economic losses associated with
IBDV, poultry producers have adopted a strategy of
hyperimmunization of breeder hens as the routine
practice to try to control IBDV (Faragher et al., 1974;
Hirai et al., 1979; Sharma et al., 1989; Saif, 1991;
Nakamura et al., 1992). However, a lack of uniformity in
the levels of maternal antibodies transferred to chicks
within the same flock and interference of IBDV vaccine
efficacy by maternal antibodies has limited the effective-
ness of this strategy (Weisman and Hitchner, 1978;
Winterfield and Thacker 1978). A novel bursal disease
vaccine has been developed that consists of a complex
between a measured amount of BDA with IBDV
neutralizing activity and a specific amount of IBDV
virus (Whitfill et al., 1991, 1992a,b, 1993, 1995; Haddad et
al., 1994). A E18 in ovo injection of SPF embryos that lack
maternal antibodies to IBDV with the BDA-IBDV
complexed vaccine was safer than the IBD vaccine virus
alone because it produced no early mortality (Table 2).
In broilers after the maternal antibody levels had waned,
the BDA-IBDV vaccinates went on to develop higher
geometric mean antibody titers (P < 0.05) against IBDV
than the IBD vaccine virus alone group (Table 3),
perhaps indicating an accelerated or more potent
humoral immune response. The BDA-IBDV complexed
vaccines have several features that make them superior
to conventional IBDV vaccines: 1) a single administra-
tion is sufficient to generate active immunity, 2) it
protects both low (SPF) and high (broiler) titer maternal
antibody chickens, 3) it is safe for administration to low
maternal antibody birds, 4) virus emergence is suffi-
ciently delayed to allow B cell population of secondary
lymphoid tissues, and 5) it is compatible with in ovo and
day of hatch administration (Tables 2 and 3; Whitfill et
al., 1991, 1992a,b, 1993, 1995; Avakian et al., 1993, 1994).
Gene vaccines based on the uptake of injected DNA
by striated muscle have been used to produce in situ
expression of genes encoding the protective antigens of
pathogens and thereby generate protective immunity
(Wolff et al., 1990, 1991; Cox et al., 1993; Robinson et al.,
1993; Ulmer et al., 1993; Wang et al., 1993). We have
demonstrated that gene transfer of DNA to embryonic
breast muscle tissue can be used to produce gene
expression in chicken muscle tissues posthatch (Figure 2,
Table 4). Embryo injection of plasmid DNA containing
genes encoding protective antigens via the IN-
OVOJECT system on E17 to E19, a time when
generation of immune responsiveness has already deve-
loped, coupled with an effective DNA delivery system
to maximize both gene transfer and expression of
protective poultry antigens might, therefore, lead to
effective vaccination and is currently under investiga-
tion. Gene vaccines have the ability to elicit both cellular
and humoral immunity providing advantages over
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killed, purified, or recombinant immunogens, which
tend to elicit a predominantly humoral response. Gene
vaccines may also be an advantage over live attenuated
vaccines that may have reduced immunogenicity due to
the attenuation process and have the potential to revert
to a virulent pathogenic forms (Cox et al., 1993; Ulmer et
al., 1993; Robinson et al., 1993; Wang et al., 1993).
Expression of protective antigens directed by recom-
binant viruses like Fowlpox or HVT have also been
developed as vaccines (Boursnell et al., 1990; Taylor et
al., 1990; Nazerian et al., 1992; Yanagida et al., 1992;
Heine and Boyle, 1993), however, recombinant viral
vaccines may also have associated limitations. Due to
the defined viral particle size, there are restrictions on
the size of the heterologous DNA that can be incorpo-
rated into the viral genome. Integration of the heterolo-
gous DNA into the host genome may disrupt the
replication of the virus, regulation of viral gene
expression, and infectivity of the recombinant virus.
Reversion to virulency, recombination events with other
pathogenic viruses, or the activation of tumor producing
oncogenes are all potential problems, and the generation
of strong immune responses to the viral backbone may
limit repeated use of the vaccine or suppress responses
to the antigen of interest (Ulmer et al., 1993). Harriet
Robinson and her colleagues have explored the use of
gene vaccines to generate protective immunity in
chickens against influenza virus (Fynan et al., 1993a,b).
Their data indicate that gene vaccines are an effective
approach for vaccinating chickens and that DNA
delivery to sites other than intramuscular injections
(intravenous and intratracheal) were also effective.
These authors also investigated gene-gun delivery of the
DNA to the epidermis of mice and found this method to
be very effective. However, as the target areas for
particle bombardment required considerable pretreat-
ment (shaving and depletion of remaining hair stubble),
subjects were anesthetized, and the force of the delivery
(electric spark discharge) is tightly controlled, it is hard
 SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
to see how this methodology could be applied in the
commercial hatchery environment. Although direct
injection into embryonic muscle employing the present
INOVOJECT system would be problematic, it is
possible to target specific embryo tissue sites by
capitalizing on the compartmentalized nature of the
embryonated egg. Effective drug delivery to embryos is
a function of embryonic age, the egg compartment
(embryo, allantois, yolk sac, air cell, amnion, etc.) into
which the drug is introduced, the desired site of
embryonic drug uptake, and the pharmacological fea-
tures of the drug itself. Coupling gene vaccine technol-
ogy and a variety of emerging gene transfer systems
with in ovo delivery should provide a powerful method
for improved disease prevention in the poultry industry.
The onset of complete immune competency in the
hatching chick is subject to a combination of develop-
mental and environmental influences. Although the
chick embryo possesses immunologically competent
cells from about E13 to E15, development of full
immunocompetence and increased disease resistance
appears delayed until 3 d posthatch and is preceded by
a burst of lymphopoiesis (Solomon, 1962; Solomon and
Tucker, 1963; Delhanty and Solomon, 1966; Solomon,
1966; Jankovic et al., 1975; Vainio and Lassila, 1989;
Dunon et al.,1990; Houssaint et al., 1991; Reynaud et al.,
1992; Cormier, 1993; Dieterlan-Lievre and Le Douarin,
1993). Stress associated with hatching, extreme tempera-
tures, handling of chicks, transport to the rearing house,
overcrowding, vaccination, and exposure to a large
number of antigenic insults can all compromise the
immune response of chickens for at least 3 to 5 d after
hatching (Gross and Siegel, 1973, 1980, 1983; Lombardi,
1993).
Cytokines regulate the amplitude and duration of the
immune response by controlling the numbers, lineages,
and functional activation of immune cells together with
their recruitment to the sites of immune localization and
infection (Balkwill, 1988; Kelso, 1989; Arai et al., 1990).
Chicken myelomonocytic growth factor is an avian
hematopoietic cytokine that stimulates bone marrow
cells to proliferate and produce cells of the monocyte-
macrophage lineage (Leutz et al., 1984, 1989). Recom-
binant cMGF has been expressed in P. pastoris and
shown to stimulate the proliferation of bone marrow
cells in vitro (Figure 3), and Day 18 in ovo injection of 0.2
mg gentamicin and 800 U rcMGF demonstrated efficacy
(P 0.05) against early mortality associated with E. coli
exposure within the hatcher (Table 5), when compared
to PBS controls. However, neither gentamicin or cMGF
were efficacious when compared to the YEA recom-
binant expression control.
It should be noted that in the conduct of these
preliminary challenge trials, variables including the dose
of cMGF, the route of administration, the number of
injections, the kinetics of administration relative to time
of challenge, and the formulation of cMGF have not
been optimized. In other preliminary studies, in ovo
175
cMGF injection, but not YEA injection, has produced
increased numbers of bone marrow cells recoverable
from the femurs of day-of-hatch chicks and increased
amounts of PMA inducible superoxide production from
adherence purified peripheral blood mononuclear cells
collected from chicks on Day 4 posthatch (data not
shown). In addition, YEA does not stimulate bone
marrow cells to proliferate in vitro (Figure 3). The YEA
was dosed in the challenge trials at an equivalent
protein concentration to the 800 U cMGF dose. The
mechanism of the yeast expression system control YEA
efficacy in this model is unknown. In addition to their
phagocytic anti-microbial effector function, macrophages
also initiate and control the immune response by their
ability to process or present antigens in the context of
major histocompatibility Class II molecules together
with their capacity for cytokine [interleukin (IL)-1,
tumor necrosis factor-a (TNF-a), IL-12, and IL-6]
production (Adams and Hamilton, 1984; Weaver and
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Unanue, 1990). Thus, cMGF administered in ovo may
significantly enhance overall immunity such that hatch-
ing chicks might be more resistant to early exposure to
pathogens in the hatcher and grow out environments. In
mammals, colony-stimulating factors (CSF), including
granulocyte-(g-CSF), macrophage-(m-CSF), and
granulocyte-macrophage-CSF(gm-CSF), together with
IL-3 (sometimes referred to as multi-potential-CSF), are
the dominant cytokines controlling the production,
differentiation, maturation, and functional activation of
granulocytes and monocyte-macrophages (Clark and
Kamen, 1987; Gregory et al., 1991; Metcalf, 1991). In
clinical trials, CSF treatments stimulate increases in the
granulocyte-macrophage populations resulting in the
partial or complete correction of preexisting
hematopoietic abnormalities, the more rapid regenera-
tion of hematopoietic cells after cytotoxic therapy, and
an overall reduction in the frequency of infections
(Gregory et al., 1991; Metcalf, 1991).
The preceding sections have focused on potential in
ovo applications targeted at improving or accelerating
the immune responses of hatching chicks by embryo
vaccination or by delivery of recombinant cytokines
during the last quarter of incubation, primarily on E18, a
time when eggs in a commercial hatchery are typically
transferred to hatching baskets. In addition to egg
transfer, eggs are also handled in commercial operations
on E0, when they are set in incubators, and in ovo
intervention at this time would have the potential to
influence almost the entire window of embryo develop-
ment. In the oviduct of the laying hen, egg fertilization
resulting from the union of the male and female
pronuclei is quickly followed by a series of cell divisions
(segmentation or cleavage) and embryo development
has proceeded to the multicellular stage with the
appearance of two of the three primary germ layers
(Romanoff, 1960). The stage of development of chick
embryos at the onset of incubation varies enormously
due to differences in the genetic background of the
176 JOHNSTON ET AL.
strain, the length of time between laying and incubation,
the length of egg storage in coolers, and differences in
the viability or vigor of embryos due to flock age,
seasonal, or environmental influences (Hamburger and
Hamilton, 1951; Romanoff, 1960). Egg injection immedi-
ately prior to incubation or in the first quarter of
incubation has typically resulted in reduced embryo
viability evidenced by decreased hatchability (Hargis et
al., 1989; Elbrecht and Smith, 1992; P. Phelps and T.
OConnell, unpublished observations). We have deve-
loped a E0 egg injection procedure that enables egg
injection immediately prior to incubation without com-
promising embryo viability (Table 6). The procedure
entails the application of a liquid sealant material to the
site of egg injection, the curing of the sealant to form an
elastic seal thereon, insertion of the injection device
through the seal, delivery of the material into the egg,
and withdrawal of the injection device from the egg.
This injection procedure should enable a more complete
evaluation of the effects of E0 in ovo injection of
substances on embryonic development without the
interference of embryo mortality associated with the
injection process.
The onset of incubation initiates a series of cell
division and cell movement events (gastrulation), which,
within a relatively short period of time (24 h), has seen
the development of the primitive streak, with the
appearance of ectoderm, mesoderm, and endoderm
primary germ layers in which organ forming potencies
have already been established (Hamburger and Hamil-
ton, 1951; Romanoff, 1960). Although embryo develop-
ment continues throughout incubation, many of the
formative events that may have an impact on further
embryo development or posthatch health and perfor-
mance are determined in the first half of incubation.
Potential targets for embryo intervention designed to
modulate some aspect of the developmental process
(commitment, proliferation, differentiation, maturation,
or function) might include tissues like the gonads,
endocrine tissues, muscle tissue, adipose tissue,
hematopoietic tissues, lymphoid tissue, gastrointestinal
tissues, vascular tissue, cartilage, and bone tissue.
The current understanding is that the genotype of the
chick embryo zygote determines the nature of the
gonad, which then determines the male or female
phenotype, and that in chickens males are homogametic
ZZ, whereas females are heterogametic ZW (Elbrecht
and Smith, 1992; Wartenberg et al., 1992). However,
during early development, chick gonads are bipotential,
sexual differentiation does not occur until approxi-
mately E5 to E8, and the ratio of male to female
phenotypes has been shown to be sensitive to the
administration of a variety of hormones, steroids, and
inhibitors during the bipotential stages of development
(Romanoff, 1960; Elbrecht and Smith, 1992; Wartenberg
et al., 1992). A single treatment of chicken embryos on
E0, E3, or E5 of incubation with an AI, which blocks the
synthesis of estrogen from testosterone, caused genetic
females to develop a male phenotype (vent sexing,
bilateral testis, comb, and wattle size) (Elbrecht and
Smith, 1992; Wartenberg et al., 1992). In one study
(Wartenberg et al., 1992), Day 3 injection of either the
vehicle alone or the vehicle plus AI produced < 50%
embryo survival, indicating problems with either the
choice of vehicle (Locke + 2mg/mL MYRJ53), the
injection process, or a combination of both. In another
study (Elbrecht and Smith, 1992), eggs injected on E0 or
E5 of incubation with either saline or AI also appear to
have reduced hatchability (34 to 49% reduction for E0;
10 to 24% reduction for E5) compared to uninjected
controls. A E0 injection of AI using our E0 injection
procedure produced a male phenotype in 100% of the
hatching chicks, in contrast to their almost 50:50 male to
female feather sex genotypic ratio (Table 7). In three
separate experiments, E0 injection of 0.1 mg of AI in
propylene glycol reduced hatchability relative to unin-
jected controls by an average of 4.5% (Table 7). If the
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male phenotype developed at hatching by genetic
females receiving Day 0 injection of AI was complete
and conserved throughout growout, these chicks might
be expected to perform like males and exhibit better
weight gain, leaner meat, and reduced feed conversion.
The development of an injection process for eggs prior
to incubation, which does not dramatically reduce
embryo viability, opens up the window of embryo
development for intervention.
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