The Effect of Increased Calcium Delivery on In Vitro

Chicken (Gallus domesticus) Embryo Culture Using

Artificial Vessels

Micaela Mertens

Proposal for Senior Thesis

Bio. 389 Junior Seminar
Loras College
1450 Alta Vista
Dubuque, IA

November 2013

Introduction
In vitro is known as a process that takes place outside of an organism
or outside of its original living environment. In the case of chickens (Gallus
domesticus), a shell-less egg culture can be performed, allowing the embryo
to be viewed without the presence of the shell. The removal of the shell
allows for research and manipulation of the embryo. Through these studies,
there has been further research on the genetic improvement of species and
the production of biological substances, as well as recombinant proteins from
chicken eggs (Kamihira, et al., 1998). In a study done by Dunn, et al. (2005),
the chick chorioallantoic membrane of a developing chick embryo in a shellless egg culture was used to see the effects of different vascular agents on
angiogenesis. This membrane offers a large and visible surface area
containing many blood vessels that can be maintained and studied through a
shell-less egg culture. An additional study can be done to determine what
substances will induce the formation of blood vessels (Ribatti 2010). These
advantages enforce the importance of making these eggs viable to provide
for good prospects of research.
The development of the avian species like the chicken (Gallus
domesticus) is not the only species studied. Eggs of reptilian species like the
snapping turtle (Chelydra serpentine) have been examined to determine
effects of calcium on developing embryos and to characterize the pattern of

its mobilization (Packard, et al., 1984). This type of research in many

different species leads to connections that can produce new outcomes.
A chicken embryo can be viewed in a shell-less egg culture in two
different environments. Surrogate eggshells provide for a more natural
environment, with an eggshell from a different species becoming the new
protection of the embryo. This route has been more successful, but still
provides a limit for what can be seen, due to the small opening or window
that is maintained at the top of the egg. A surrogate eggshell also provides
additional nutrients, especially calcium, to the egg, which can be hard to
regulate and monitor. When looking at how the embryo responds to
supplements of nutrients, it is better to look at the embryo and its fluid
compartments with an artificial vessel. An artificial vessel provides an
environment very similar to an egg, but without the calcium that the
eggshell offers. This kind of vessel provides an open field for viewing every
angle of the developing embryo, as well as multiple areas for manipulation.
The fluid compartments of the embryo can be sampled and added to during
development.
As mentioned earlier, the development of the embryo in a shell-less
egg culture can alter normal development. The eggshell of an embryo
provides calcium for growth. Calcium is an essential nutrient to the embryo.
The majority of it is received from the eggshell, where the yolk only contains
approximately 20 25 mg of calcium (Ono & Tuan, 1986) of the total 120

140 mg in the hatchling. This calcium in the eggshell is transferred to the

embryo through the chorioallantoic membrane. Without the proper amount
of calcium that the embryo needs, the growth and development are reduced
and the embryo shows signs of skeletal calcification (Tuan, 1980). In a study
done by Kamihira, et al. (1998), the addition of calcium supplementations to
the embryos in a shell-less egg culture was studied. No additions resulted
with none of the embryos surviving. Fine eggshell powder added to the
vessel increased the amount of embryos hatched by 10%. This improvement
increased with the addition of calcium lactate as well, yielding a hatchability
of 43%. The hatched embryos still did not compare to embryos under normal
incubation, showing the importance of the shell.

Aims of the Proposed Study

The above observations have led to exploring the ability to increase
the viability and hatchability of chicken eggs in shell-less egg cultures by
increasing the available calcium offered to the embryo. The hypothesis is
that an increase in delivery of calcium to an embryo in shell-less culture will
provide for additional nutrients that the embryo can absorb and utilize to
survive. Hopefully a certain concentration of calcium will provide just the
right amount of nutrients to ensure the proper growth, without providing too
much. In addition, the embryo will absorb and utilize the injected calcium for
its growth and this will supplement for the lack of calcium that would be
given through the eggshell. This calcium will help the embryo grow and

move closer to hatchability to produce more viable offspring than without

these additions. There are other studies that have supplied increased
calcium to in vitro eggs, but I plan on using other techniques to administer
the additions.
Thus, the research proposed here should help provide ways of
increasing the growth and survival of chicken embryos in shell-less egg
cultures. The multiple techniques will serve as a basis for the best way to
provide calcium to a chicken embryo. These experiments are planned for the
coming spring semester.

Methods
Fertile chicken eggs (Gallus domesticus) will be obtained from different
hatcheries. The eggs will be transported and set in an incubator at 38C. All
embryos in ovo will experience these conditions, with turning platforms and
humidified air. The experimental eggs will be taken out on Day 3 from the
incubator and put into their artificial vessel. These eggs will also be manually
rotated while in another incubator, since they will not have the conditions of
the turning platform. The experimental eggs will receive supplementations
after Day 8 10.

The artificial vessel consists of a 9 oz. clear plastic cup, with three
ventilation holes halfway down the side of the cup, spaced evenly. This
allows for air flow to the embryo and for gas exchange to occur. Saran wrap
will be used to create a cavity for the embryo to set in, by pushing the saran
wrap down and securing it with a rubberband (Figure 1). This set up, allows
for easy visibility of all areas of the egg. Several centimeters of water will be
placed in the bottom of the cup to provide moisture to the bottom of the egg.
The lid to a petri dish will cover the artificial vessel, after the embryo is
placed inside. The covering allows for sanitation and prevention of infection
to the embryo. The whole apparatus will be sterile and disposed of if any
infection occurs.

Figure 1. Shell-less Egg Culture system. It consists of a 9 oz. plastic cup

with ventilation holes, a plastic saran wrap membrane secured with a
rubberband, several centimeters of water, and a petri dish lid.

The incubating eggs were removed and shells were sterilized with 70%
Ethanol solution to prevent any infections from outside sources. A small hole
was made an inch down from the side of the egg that tapers to a point. This
was done by using a sterilized razor blade. With a small amount of pressure,
the blade was slowly lodged into the shell. This small hole was utilized to
slowly begin to crack the top of the egg off by turning the blade and moving
it up and down to initiate the crack. The blade was moved around the entire
diameter of the egg until the top was able to be detached. This open end
allowed for the egg to slowly be laid into the artificial vessel. This was done
by slowly tilting the egg and putting it a couple centimeters above the saran
wrap cavity, to slowly allow the contents to pour out. The yolk was then
repositioned with a sterilized spatula, if needed, to place the embryo
upwards. Each day, distilled water was added to the bottom of the cups to
replace the water lost due to evaporation. Embryonic mortality was
determined by visibility of no heartbeat and decreased movement. Dead
embryos were removed from the incubator that day.
Addition of calcium will be delivered through different techniques after
Day 8-10 of the developing process, to discover which technique provides
the best results. Injection of calcium lactate through a needle may be used,
into either the yolk or into the amniotic fluid. This will allow the nutrients to
be closest to the embryo and have the best chance of being absorbed during
development. The concentration of calcium lactate will be determined
according to previous studies. According to Kamihira, et al (1998) a

concentration of 70 mg/ml of calcium lactate to albumen was used and

different volumes of this concentration were administered to the embryos.
This study will be considered, as well as others, including a study done by a
previous peer. In addition to injection, small fluid-filled open-ended tubes
placed directly on the chorioallantoic membrane surface will be used to
distribute the calcium. These plastic tubes will be about a half an inch tall
and will set on the membrane. A similar concentration of calcium lactate as
mentioned earlier will be placed inside these tubes and the volume of the
tubes will be watched to see if it decreases over time. The addition of
fluorescent dye to the solution may also be utilized to track the entry and
sites of movement of the calcium.

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