PRODUCTION, MODELING, AND EDUCATION
Research Note
Field evaluation of the accuracy of vaccine deposition by two different
commercially available in ovo injection systems
C. J. Williams1 and B. A. Hopkins
Pfizer Animal Health, Third Avenue, MS 685-10-1, New York, NY 10017
ABSTRACT The location of injection and vaccine de-
position in ovo is known to be critical to the efficacy of
Mareks disease (MD) vaccine protection against MD
viral challenge. Vaccine deposition into the amniotic
sac or a s.c. or i.m. site of the embryo is required for
MD vaccine efficacy. Vaccine deposition into the air
cell or allantoic fluid results in chicks that are not ad-
equately protected against subsequent MD viral chal-
lenge. A study was conducted in 2 commercial broiler
hatcheries to evaluate the ability of 2 in ovo injection
systems, the Embrex Inovoject system (Pfizer Poultry
Health, Research Triangle Park, NC) and the Intelliject
system (Avitech, Salisbury, MD; distributed by Merial
Ltd., Gainesville, GA) to deliver a vaccine approved
for use in ovo accurately and properly. A standard MD
vaccine diluent mixed with a protein-staining dye was
delivered through each machine to simulate in ovo vac-
cination. The location of the dye within the egg deter-
mined whether the vaccine was delivered correctly. Each
egg was also evaluated for normal embryo development
(normal eggs). Correct vaccine delivery included eggs
Key words: site of injection, in ovo, vaccine deposition, embryo
INTRODUCTION
The first research on the in ovo administration of
vaccines was completed by Sharma and Witter (1983).
Subsequent work demonstrated that in ovo adminis-
tration of Mareks disease (MD) vaccines HVT, SB-1,
and CVI988 to late-stage chicken embryos was safe and
would induce earlier immunity than posthatch adminis-
tration (Sharma et al., 1984; Zhang and Sharma, 2001).
Because of these results, the practice of in ovo vacci-
nation was moved from the laboratory to commercial
poultry hatcheries (Gildersleeve et al., 1993; Sarma et
al., 1995). Today, in ovo vaccination occurs routinely in
2011 Poultry Science Association Inc.
Received March 10, 2010.
Accepted September 6, 2010.
1 Corresponding author: christopher.williams2@pfizer.com
223
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in which the vaccine was injected into the amniotic
sac or into s.c. or i.m. regions of the embryo. Incorrect
vaccine delivery was defined as delivery into the air
cell; allantoic sac; any combinations including air cell
or allantois; the abdominal, cranial, orbital, or thoracic
cavities of the embryo; or no vaccine delivery at all.
In hatchery 1 (Chick Master, Newton, MS) 1,171 nor-
mal eggs were processed through the Inovoject system
and 1,138 eggs were processed by the Intelliject system.
The Inovoject system correctly vaccinated 94.62% of
the normal eggs as compared with 61.16% delivery ac-
curacy of normal eggs with the Intelliject system. In
hatchery 2 (Jamesway Super J, Magee, MS) 926 normal
eggs were processed by the Inovoject system and 910
normal eggs were processed by the Intelliject system.
The Inovoject system correctly vaccinated 91.04% of
the normal eggs, whereas the Intelliject system correct-
ly vaccinated 71.98% of the normal eggs. The results
of this study clearly demonstrate that the Inovoject
system accurately delivered in ovo vaccine at a signifi-
cantly higher rate than the Intelliject system.
2011 Poultry Science 90:223226
doi:10.3382/ps.2010-00759
many commercial broiler hatcheries around the world,
particularly in the Americas, Japan, Australia, and
parts of Europe.
The process and technique used to administer in ovo
vaccines is critical. Delivering vaccine to an incorrect
in ovo site can lead to an ineffective vaccination, thus
reducing the benefits normally seen by vaccinating in
ovo. The complexity of the in ovo route is often under-
appreciated, and this is attributable in part to the 5
embryonic compartments that can be accessed by the
needle during the in ovo vaccination process: the air
cell (AC), allantoic sac (AL), amniotic fluid (AM),
embryo body (EM), and yolk sac. Furthermore, EM
injections can be s.c., i.m., intracranial, intraorbital, or
intraabdominal, with the latter 3 causing excessive em-
bryo trauma. The efficacy of a vaccine and the safety to
the embryo can be affected by the embryonic compart-
ment into which it is deposited.
 224
Wakenell et al. (2002) showed that efficacy against
MD virus challenge was dependent on the site of MD
vaccine delivery in ovo. The MD vaccine was not effec-
tive when delivered onto the AC or into the AL. The
highest efficacy was found in the AM, with a 94.4%
protective index, and in the EM, with a 93.9% protec-
tive index. Protective efficacy in chicks with vaccine
injected into the AC or AL was less than 50%. This
work showed that the vaccine must be injected deep
enough into the egg to be delivered into the amnion,
which includes the AM and EM. When vaccines are
delivered to the AM, the vaccine is imbibed, breathed
in, or both by the embryo before hatching. The more
developmentally mature embryos in the population are
injected in the right breast area. Embryo body delivery
is normal; however, penetration too deep into the egg
at any stage of development can cause excessive trauma
to the embryo.
MATERIALS AND METHODS
In Ovo Injection Equipment
Technical representatives from both in ovo equipment
manufacturers prepared their respective machines and
ensured proper function of the equipment during the
conduct of each trial. The equipment differed by site
because a specific injector design was required for each
incubation system. The trial conducted at the Chick
Master hatchery (Newton, MS) compared the Embrex
Inovoject Systems Vaccine Saver CM 108 design (se-
rial no. V 2954, Pfizer Poultry Health, Research Tri-
angle Park, NC) with the Avitech Intelliject CM 162
design (serial no. 00035, Avitech, Salisbury, MD). The
trial conducted at the Jamesway hatchery (Magee, MS)
compared the Embrex Inovoject Systems Vaccine Saver
JW 84 design (serial no. V 2252A) with the Intelliject
JW 168 design (serial no. 00063).
Eggs
Eggs from Cobb breeder flocks were selected by
incubator tray from the egg incubation racks in the
same format and position as they were incubated. In
the Jamesway hatchery, 4 incubator trays (84 eggs/
tray, 336 eggs/age group) were randomly selected from
3 separate breeder flocks of differing ages. Eggs were
injected in ovo at their normal transfer age (Jamesway
incubation system: d 18 + 7 h of incubation). A total
of 1,008 eggs were used per injection system.
In the Chick Master hatchery, 6 trays (54 eggs/tray,
324 eggs/age group) were randomly selected from 4
separate breeder flocks of differing ages. Eggs were in-
jected in ovo at their normal transfer age (Chick Mas-
ter incubation system: d 19 + 1 h of incubation). A
total of 1,296 eggs were used for the evaluation of each
injection system.
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Williams and Hopkins
Vaccination Procedure
The Inovoject systems used in these studies are de-
signed to deliver 50 L of vaccine per injection cycle to
each egg determined as viable, whereas the Intelliject
systems are designed to deliver 100 L to all eggs dur-
ing each cycle. The test eggs were injected immediately
before the beginning of the normal transfer and injec-
tion time. Each vaccine system was purged of alcohol
with sterile saline, and then test vaccine was added.
The sanitation system for both machines functioned as
normal, using each manufacturers recommended chlo-
rinated sanitizer solution. The test vaccine bag con-
tained standard Mareks vaccine diluent and a protein-
staining dye without the inclusion of any vaccine.
Embryo Euthanasia and Vaccine
Deposition Evaluation
After injection was completed, test eggs, while still in
the original incubation flats, were bagged by treatment,
killed using CO2 gas, and stored for 4 h at low temper-
ature. After euthanasia, eggs were carefully dissected
and vaccine deposition site was determined. Each egg
was also evaluated for normal embryo development.
Eggs were segregated by embryo development status
into abnormal eggs (upside down, early dead, mid dead,
late dead, cracked shell, malformed or malpositioned
embryo, rotten, and infertile) and normal eggs (normal
embryo development). Only normally developed embry-
os were included in the site of injection analysis.
Experimental Design
Two separate trials were conducted in 2 commercial
broiler hatcheries. One hatchery used the Chick Master
incubation system and the other used the Jamesway
Super-J incubation system. Because of differences in
the design of the incubation tray that nests the eggs
during embryonic development, specific egg injection
machines from each manufacturer were required for
each incubation system (Chick Master or Jamesway).
Additionally, other application differences existed be-
tween the 2 types of egg injection systems (Pfizers Em-
brex Inovoject system or Avitechs Intelliject system),
hence the purpose for this evaluation. Both hatcheries
used egg injection equipment specifically designed for
use by either the Chick Master or Jamesway incuba-
tion system. Other than the egg flat configuration and
difference in time of injection dictated by either the
Jamesway or Chick Master incubation system, the pro-
tocol for vaccine application and injection site determi-
nation were identical between the 2 sites.
Correct injections in ovo for optimal MD efficacy are
defined as delivery of vaccine into the AM and EM
or EM-AM injections (Wakenell et al., 2002). For the
purposes of this study, incorrect injections were defined
as AC, AL, combinations that included AC or AL, and
 RESEARCH NOTE 225
Table 1. Number and percentage of eggs with proper and improper vaccine delivery by in ovo injection system and hatchery
Chick Master hatchery Jamesway hatchery

Injection system Correct delivery Incorrect delivery Correct delivery Incorrect delivery

Embrex Inovoject system

Eggs (no.) 1,108 63 843 83
Delivery (%) 94.62a 5.38 91.04a 8.96
Intelliject system
Eggs (no.) 696 442 655 255
Delivery (%) 61.16b 38.84 71.98b 28.02
a,bNumbers within a column lacking a common superscript differ (P 0.05).

all incorrect embryo sites (intracranial, intraorbital,
intraabdominal). No attempts were made to quantify
or differentiate volume of vaccine delivered to multiple
sites in ovo; therefore, suboptimal doses or partial de-
livery to correct sites in ovo were considered incorrect
applications. Correct injections included only complete
delivery of individual vaccine dispensed into the correct
site in ovo. The egg was considered the experimental
unit in a generalized randomized complete block design
with flock as the blocking factor. Correct vs. incorrect
injections were analyzed by exact conditional logistic
regression, with flock as a stratifying variable and treat-
ment as a factor (SAS/STAT Version 9.1.3 Service Pack
3, SAS Institute Inc., Cary, NC). In terms of adjust-
ment for multiple comparisons, the overall treatment
effect for site of injection was evaluated using a Fishers
protected least significant difference type approach for
comparisons for each site without adjustment. Statisti-
cal significance was determined with P 0.05.
RESULTS AND DISCUSSION
Two separate analyses were completed, one for each
type of incubation system involved, because myriad ran-
dom effects were related to each hatchery and respec-
tive incubation system. In the Chick Master hatchery,
1,296 eggs were injected by each injection system. Of
those, 1,171 normal eggs were injected by the Inovoject
system and 1,138 normal eggs were injected by the In-
telliject system. Although randomized at injection, the
difference in the number of normal eggs between the
2 groups was primarily due to fertility variation from
tray to tray associated with eggs from the oldest breed-
er flock. Only live normal embryos at injection time
were included in the evaluation. The Inovoject system
correctly injected a higher percentage of normal eggs
(P 0.0001) than the Intelliject system (Table 1). The
same model was then applied to each specific category
(vs. all others). The Intelliject system was significantly
higher (P 0.0001; Table 2) in all categories of incor-
rect vaccine deposition (AC, AL, AC or AL combina-
tions, and EM incorrect), whereas the Inovoject system
performed significantly better in all categories of cor-
rect vaccine deposition (AM and EM proper) except for
the combination AM and EM injections, for which the
Intelliject system delivered significantly more than the
Inovoject system (P 0.0001).
In the Jamesway hatchery, 1,008 eggs were injected
by each injection system. Of those, 926 normal eggs
were injected by the Inovoject system and 910 normal
eggs were injected by the Intelliject system. As in tri-
al 1, the Inovoject system (Table 3) had significantly
more correct vaccinations (AM) in ovo (P 0.0001)
than the Intelliject system, except for injections of EM
proper, for which there was no significant difference (P
> 0.5984) and injections of AM-EM proper, for which
the Intelliject system had significantly (P 0.0114)
more proper injections than the Inovoject system. The
Intelliject system (Table 3) delivered significantly (P
0.0001) more incorrect vaccine placements (AC, AC or
AL combinations, and EM incorrect) when compared
with the Inovoject system, except for AL (P > 0.4308)
and the combination AM-EM incorrect injections (P >
0.9999), for which there was no significant difference.

Table 2. Number and percentage of eggs at each specific site of vaccine deposition1 at the Chick Master hatchery
AC AL AL AM-EM EM Total
Item AC combination AL AM combination AM incorrect AM-EM incorrect EM eggs

Injection system
Embrex Inovoject
system
Eggs (no.) 0 3 1 2 0 673 1 2 56 433 1,171
Delivery (%) 0.00b 0.26b 0.09b 0.17b 0.00b 57.47a 0.09b 0.17b 4.78b 36.98a
Intelliject system
Eggs (no.) 41 26 18 179 78 480 20 48 80 168 1,138
Delivery (%) 3.60a 2.28a 1.58a 15.73a 6.85a 42.18b 1.76a 4.22a 7.03a 14.76b
Total eggs 41 29 19 181 78 1,153 21 50 136 601 2,309
a,bNumbers within a column lacking a common superscript differ (P 0.05).
1AC = air cell; AL = allantoic sac; AM = amniotic fluid; EM = embryo body.

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 226 Williams and Hopkins
Table 3. Number and percentage of eggs at each specific site of vaccine deposition1 in the Jamesway hatchery
AC AL AM-EM EM Total
Item AC combination AL AL-AM combination AM incorrect AM-EM incorrect EM eggs

Injection system
Embrex Inovoject
system
Eggs (no.) 2 2 27 48 3 744 0 3 1 96 926
Delivery (%) 0.22b 0.22b 2.92a 5.18b 0.32b 80.35a 0.00a 0.32b 0.11b 10.37a
Intelliject system
Eggs (no.) 17 30 33 134 30 541 1 13 10 101 910
Delivery (%) 1.87a 3.30a 3.63a 14.73a 3.30a 59.45b 0.11a 1.43a 1.10a 11.10a
Total eggs 19 32 60 182 33 1,285 1 16 11 197 1,836
a,bNumbers within a column lacking a common superscript differ (P 0.05).
1AC = air cell; AL = allantoic sac; AM = amniotic fluid; EM = embryo body.

With respect to accuracy of vaccine deposition by the
Inovoject system in the Chick Master hatchery vs. the
JW hatchery, differences could be seen that were caused
by embryo age and egg setter tray design. The egg tray
configuration and stability of the egg in the tray varied
between the 2 incubation systems, with the Jamesway
incubation tray allowing eggs to deviate more than the
Chick Master incubation tray along the upright axis.
The design of the Chick Master incubation tray kept
eggs in a very tight, upright position and did not al-
low egg movement laterally while the eggs were being
turned in the incubator. In addition, embryos in the
Chick Master hatchery were 18 h older at transfer com-
pared with those in the Jamesway hatchery. The opti-
mal time to inject eggs, developmentally, is the physi-
ological stage between when the stalk of the yolk sac is
beginning its ascent into the abdomen and the head is
tucked under the wing up until external pipping is initi-
ated. The optimal target site for uniform automated in
ovo vaccination is the AM or right breast area of the
developing embryo. Injections into the cranium, ocu-
lar space, and abdomen of the embryo are considered
off-target injections and are a result of misalignment
of injectors with the eggshell surface. These delivery
sites in ovo are not desirable for MD vaccine delivery
to the late-stage developing embryo and are incorrect
applications. As the embryo increases in size late in in-
cubation, the likelihood of injecting the embryo proper
increases and, with correct injector-to-egg alignment,
results in targeted delivery to the right breast area.
Why the delivery results of the Intelliject system did
not reflect this is unknown.
This study shows the large variability in accuracy of
vaccine deposition, depending on the in ovo vaccination
system used. Previous work done by Islam et al. (2001)
and Wakenell et al. (2002) demonstrated that vaccine
protection against challenge was dependent on the vac-
cine delivery site. The vaccine must be injected deep
enough into the egg to be delivered into the amnion,
which includes the AM and EM. When delivered to
the AM, the vaccine is imbibed, breathed in, or both
by the embryo before hatching. The MD vaccination
delivered via the AL results only in significantly lower
viremia and does not adequately protect against chal-
lenge, whereas delivery to the AC shows no embryo
access to the vaccine, thus no vaccinal response. As
the embryo matures and begins the hatching process
(internal and external pipping), more embryos in the
population are injected either s.c. or i.m. into the right
breast area. Embryo body delivery during in ovo vac-
cination is normal; however, penetration too deep into
the egg and embryo during late-stage development can
cause excessive trauma to the embryo. Therefore, use
of an in ovo injection system that does not deliver vac-
cine to the correct embryonic site would compromise
embryo safety and vaccine performance. The objective
of commercial in ovo vaccination is to vaccinate every
viable embryo safely and uniformly. To accomplish this
objective, a commercial in ovo vaccination device must
administer vaccine to sites that result in the greatest
safety and efficacy for the vaccine delivered. In this
study, the Inovoject system was significantly better at
meeting that objective.
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