Technical Information Release

Summary of the STS-108 Flight of the Avian Development Facility
Experiment for the Biospecimen Sharing Plan Tissues

This Technical Information Release provides a summary of the pre-flight,
in-flight, and post-flight operations for the Avian Development Facility
(ADF) experiment for investigators requesting tissue from the
Biospecimen Sharing Plan (BSP). It provides technical information and
data concerning the handling and processing of the ADF, including the
Japanese quail eggs and embryos.

A\) Avian Development Facility

The ADF was a Small Business Innovative Research contract managed by the
Space Station Biological Research Project (SSBRP; NASA-Ames Research
Center, Moffett Field, CA). The ADF was developed by SHOT, Inc
(Greenville, IN) as a precursor to the Egg Incubator, a system capable
of supporting long-term egg incubation experiments in microgravity. The
ADF is designed to provide a "window" for the study of embryogenesis in
space. It provided facilities for investigators to better understand and
then mitigate or nullify the forces of altered gravity upon embryo
development. The ADF allows the incubation of eggs after the Orbiter has
achieved orbit and then fixation of the specimens while on orbit. The
ADF minimizes the effects of launch and re-entry in order to isolate and
preserve the effects of the experimental variable(s) of the microgravity
environment.

The ADF houses 36 egg holders designed for Japanese quail eggs and
provides pre-programmable control of the interior environmental
temperature, humidity, CO~2~ concentration, and O~2~ concentration, thus
providing optimal conditions for embryo development. It also provides an
automated fixative injection system that can be programmed to fix eggs
at specific times during incubation. The ADF allows for turning the eggs
(similar to turning in a natural environment) once an hour and provides
two separate carousels, which each carry 18 egg holders. Both carousels
are capable of spinning and accommodating centrifugation controls in a
microgravity environment.

The primary objective of the ADF flight was to validate the function of
the ADF subsystems for risk reduction for the development of the Egg
Incubator hardware. As part of the ADF flight, two NASA Research
Announcement peer-reviewed science experiments were selected. One study
examined the effects of microgravity on the development of the
vestibular system (D. Dickman, Ph.D.; Washington University, St. Louis,
MO). The other study examined bone development in microgravity (S. Doty,
Ph.D.; Hospital for Special Surgery/Cornell University, New York, NY).

**B) ADF Quail Colony Selection Requirements**

**1) Selection Criteria**

The criteria for selecting a quail colony to furnish the eggs for the
ADF flight were developed by a committee that included John Vellinger
(SHOT, Inc., Greenville, IN), Dr. Patricia Hester (Purdue University,
West Lafeyette, IN), Dr. Ronald Hullinger (Purdue University, West
Lafeyette, IN), and Dr. Bernard Wentworth (University of Wisconsin,
Madison, WI). The accepted criteria are compiled in following table.

**Japanese Quail Colony Selection Criteria**

+------------------+---------------------------------------------------+
| **Criterion**    | **Explanation**                                   |
+------------------+---------------------------------------------------+
| 1\) The colony   | Insures a high quality of husbandry for the       |
| is located at    | colony and consistency of fertile egg quality     |
|                  | over time. Neither is obtainable from a           |
| an academic      | commercial source.                                |
| Institution      |                                                   |
+------------------+---------------------------------------------------+
| 2\) Close Colony | No breeding with an outside source.               |
+------------------+---------------------------------------------------+
| 3\) Isolated     | Isolated from bacterial cross-contamination from  |
| Colony           | feral or other avian species.                     |
+------------------+---------------------------------------------------+
| 4\) Egg          | The fertility of the provided eggs cannot be      |
| Fertility \> 85% | pre-determined. Colony must have ability to       |
|                  | produce a high fertility rate for supplied eggs.  |
+------------------+---------------------------------------------------+
| 5\) Egg Weight   | This egg weight is associated with eggs that have |
| (8.8g-9.9g)      | the physical dimensions that will fit into the    |
|                  | egg holder.                                       |
+------------------+---------------------------------------------------+
| 6\) Quantity     | Number of eggs required per day prior to launch.  |
| (300-400 eggs)   | Ensures enough eggs from the same lay-date for    |
|                  | scrubs and ground controls.                       |
+------------------+---------------------------------------------------+
| 7\) Frequency    | Ability to supply the large quantity of eggs for  |
|                  | flight. Can collect all of the eggs in a single   |
|                  | day for shipment.                                 |
+------------------+---------------------------------------------------+
| 8\) Certified    | Specific Pathogen Free certification. The colony  |
| SPF              | and eggs are free from specific bacterial         |
|                  | pathogens (Salmonella, Shigella, Klebsiella,      |
|                  | Edwardsiella, and Mycoplasma                      |
+------------------+---------------------------------------------------+
| 9\) Cost         |                                                   |
+------------------+---------------------------------------------------+
| 10\) Direct      | Eliminates the potential of an unscheduled delay  |
| commercial       | during the lay-over stop. Decreases the amount of |
| flight           | egg handling due to changing planes.              |
|                  |                                                   |
| to Orlando, FL   |                                                   |
+------------------+---------------------------------------------------+

The quail colony selection committee included John Vellinger, and Dr.
Doty, Dr. Dickman, Dr. Wentworth, Dr. Hester, and Dr. Hullinger. The
committee selected the colony at the University of Wisconsin, Madison,
which is maintained by Dr. Wentworth, as the primary colony.

**2) Specific Pathogen-Free Testing**

Flight qualification of the Japanese quail colony that supplied the eggs
for the Avian Development Facility required a Specific Pathogen-Free
certification. Receipt of this certification required testing the colony
for the presence of Salmonella, Shigella, Edwardsiella, Klebsiella, and
Mycoplasma species. The following information describes the independent
diagnostic test laboratories that conducted the tests and the procedures
that were use for the testing. For all test cycles, all microbiological
results came back negative.

**a) Salmonella, Shigella, Klebsiella, and Edwardseilla**

The Wisconsin Veterinary Diagnostic Laboratory (Madison, WI) tested 6
randomly selected quail eggs for the presence Salmonella, Shigella,
Klebsiella, and Edwardsiella. Testing was repeated every 6 months.

Prior to opening the egg, the shell was sterilized with an
alcohol/iodine. Once the internal content of the egg was obtained, the
following test procedures were performed.

**For Salmonella identification:**

The internal contents of an egg was be inoculated into the each of the
following enrichment liquid media:

Selenite Broth

Rappaport-Vassiliadis R10 Broth

After incubation, aliquots of the internal content-inoculated enrichment
broth was streaked onto the following selective medium agar plates to
test for the presence of Salmonella:

EMB

XLT4

Brilliant Green with Novobiocin

**For Shigella, Klebsiella, and Edwardsiella:**

The internal contents of an egg was streaked onto the following plates:

TSA Blood Agar

EMB Agar

In parallel, the internal contents of the same egg was inoculated into
the following enrichment media:

Selenite Broth

Rappaport-Vassiliadis R10 Broth

Following incubation, an aliquot of the inoculated broth was plated onto
the above plates to test for the presence of the pathogens.

**b) Mycoplasma**

The Diagnostic Services Laboratory (University of Georgia, Athens, GA)
tested the serum derived from 40 randomly selected adult quail for the
presence of Mycoplasma species *M. gallisepticum* *and M. synoviae*. The
laboratory was under the direction of Dr. Steven Thayer. Testing was
conducted once a year.

Blood was drawn from the quail by Dr. Bernard Wentworth (Director of the
Quail Colony) and then he perform a clotting procedure to isolate the
serum. The serum was shipped to Dr. Thayer on ice packs via Federal
Express mail. The serum was tested for the presence of Mycoplasma using
the plate agglutination test method.

**C) Pre-Flight and Flight Operations for the ADF:**

Prior to the need date of eggs for flight, Dr. Wentworth began to build
up the colony so that it would be populated with hens of appropriate age
that could lay a high percentage of fertile eggs (greater than 90 % egg
fertility rate). During pre-flight operations, a fresh set of newly laid
eggs (160 to 180 eggs) was shipped every day from the flight colony to
Kennedy Space Center to allow for potential flight delays and scrubs
and, later, ground controls. The quail eggs were collected at the colony
the day prior to shipping and then the eggs were stored at 13 ^o^C and
65 % relative humidity (RH) until shipment. The eggs were shipped
overnight to Kennedy Space Center at ambient temperature and then stored
at 13 ^o^C and 65 % humidity in a Percival refrigerator until loaded for
flight.

The eggs that were flown in the ADF on STS-108 were received on Dec 3,
2001 (approximately at 1:00 am EST). Upon receipt, the eggs were placed
into a Percival Refrigerator set at 13 ^o^C and 65 % RH. On the day of
egg receipt, the team conducted the flight egg selection procedure. The
flight selection criteria were as follows: 1) No observable cracks based
on candling and 2) a weight greater than or equal to 8.2 grams and less
than or equal to 9.9 grams. Eggs were numbered using a black Sharpie
pen. After the eggs were examined and weighed, a random number of 36
flight quality eggs were chosen and then these eggs were placed into
separate ADF egg holders followed by attachment of the holder to one of
the two ADF incubator carousels. The carousels were placed into the
Percival refrigerator (13 ^o^C, 65 % RH) until loading into the ADF. The
day after egg selection, the carousels were placed into the ADF and then
the ADF was set to Chill Mode (13 ^o^C and 65 % RH) to continue
suspension of embryo development. At approximately L-23 hours, the ADF
was turned over to the Kennedy Space Center Utilization Team for
insertion in to the Middeck of the Orbiter Endeavour. Due to weather
conditions on launch day, the first launch attempt on Dec. 4, 2001 was
scrubbed. The ADF remained in the Orbiter for the second launch attempt
on Dec. 5, 2001 On Dec. 5, 2001, Endeavour launched at 4:19 pm EST. As
previously described, the environment in the ADF was 13.5 ^o^C and 65 %
relative humidity at launch. Three hours and 3 minutes after launch, the
crew activated the incubation mode of the ADF. At this point, the ADF
transitioned from a refrigerator to an incubator. The ADF was
pre-programmed to allow 3 hours for the specimen environment to come to
thermal equilibrium at 37 ^o^C and 65 % RH. Once the ADF reach thermal
equilibrium, the upper carousel began to spin to provide a 1 G
centrifugal force (internal control 1 G eggs). The lower carousel
remained stationary. Also, the egg holders were turned approximately
180^o^ once an hour to simulate the egg turning by the hen. During the
incubation cycle, the upper carousel was pre-programmed to stop and then
start at two time points to allow automated fixative (4 %
paraformaldehyde in 50 mM Cacodylate buffer, pH 7.4) injection into a
pre-programmed number of eggs. These injection operations occurred on
incubation days 4 and 7.

On flight day 5, the crew began pre-breath operations for the EVA
scheduled for the next day. This operation included a reduction in the
total Orbiter cabin atmospheric pressure to 10.2 psi, which correlated
to a reduction in oxygen partial pressure to below 2.7 psi but no less
than 2.5 psi. Due to the air exhaust port in the top of the ADF
incubation chamber, the chamber was sensitive to atmospheric changes of
the Orbiter cabin. Therefore, the oxygen partial pressure in the ADF
incubation chamber reduced to between 2.5 psi and 2.25 psi. All other
environmental parameters in the incubation chamber remained nominal. On
flight day 6, after 19 hours and 20 minutes of atmospheric pressure
depress, the Orbiter cabin atmospheric pressure was repressurized to
14.7 atm and then the ADF incubation chamber oxygen partial pressure
quickly returned to set-point.

On the day of Orbiter landing, five hours and fifteen minutes prior to
de-orbit burn, the crew placed the ADF into "Idle Mode", which maintains
nominal environmental parameters but the rotating carousel is stopped
per NASA safety requirements. The 5 hours and 15 minutes length of time
is the duration of time that the 1 G control live embryos were exposed
to microgravity. De-orbit burn was initiated 1 hour and 15 minutes
before landing and represents the time the live embryos, which developed
on the stationary carousel, first began to experience a non-microgravity
G-force.

**D) ADF Ground Control and Standard Laboratory Incubator Control**

A ground control series of eggs were incubated in the back-up ADF unit.
The initiation of ADF Ground Control egg handling operations started the
day after the start of flight egg handling operations. The handling of
the eggs and selection process was as described above for the flight
ADF. The temperature conditions of the stored eggs in the Percival
refrigerator and ADF Ground Control, prior to Incubation Mode
activation, were as described for the flight eggs. The day prior to
"launch" of the ADF Ground control, all ADF pre-flight turnover
operations were simulated. The "launch" of the ADF Ground Control was
initiated 27 hours after the launch of the flight ADF. Launch vibration,
acoustics, and G-force loads were not simulated. However, the timing of
Incubation Mode activation was initiated 3 hours and 3 minutes after
"Launch". At this point, the ADF transitioned from a refrigerator to an
incubator. The ADF was pre-programmed to allow 3 hours for the specimen
environment to come to thermal equilibrium at 37 ^o^C and 65 % RH. Once
the ADF reach thermal equilibrium, the upper carousel began to spin to
provide a 1 G centrifugal force (internal control 1 G eggs). The lower
carousel remained stationary. Also, the egg holders were turned
approximately 180 ^o^C once an hour to simulate the egg turning by the
hen. The ADF was run continuously, as per the flight timeline, in the
ADF Hardware Laboratory (Rm 185) of the Kennedy Space Center Hangar-L
Life Science Support Facility. In order to position the eggs in the
orientation observed in a nest, the ADF was placed onto its side so that
the long axis of the eggs were perpendicular to the gravitational force
vector of the earth. Therefore, the rotating upper carousel was spinning
in an orientation similar to a merry-go-round. As a result of this
orientation, the eggs on the spinning carousel were experiencing a
variable centrifugal force that varied from approximately 2 G
(centrifugal force vector in the same direction as the gravitational
vector) and approximately 0 G (centrifugal force vector opposite the
gravitational force vector)the internal carousel. The ADF Ground Control
incubator chamber was maintained at nominal environmental set-points
throughout the "flight", and the change in Orbiter cabin atmospheric
pressure (EVA condition) was not simulated. During the incubation cycle,
the upper carousel was pre-programmed to stop and then start at two time
points to allow automated fixative (4 % paraformaldehyde in 50 mM
Cacodylate buffer, pH 7.4) injection into a pre-programmed number of
eggs. These injection operations occurred on incubation days 4 and 7.

On the ADF Ground Control "landing" day, the timelined in-flight
operations were simulated for the ADF. This included placing the ADF
into Idle mode and then rotating the ADF Ground Control back onto its
base. The duration of time between landing and receipt of the ADF at the
Life Science Support Facility was performed. Landing vibration,
acoustics, and G-force loads were not simulated.

A second ground control was conducted in a standard laboratory egg
incubator (Lyon Incubator). The eggs used for this control were received
at Kennedy Space Center on Dec. 6, 2001. The "pre-flight" handling of
these eggs were as described for the flight eggs, except the ADF egg
holder and carousel operations were not conducted. Twenty four hours
after the initiation of the ADF Ground Control, a random set of flight
quality eggs were removed from the Percival refrigerator (13 ^o^C, 65 %
RH) and then the eggs were placed into a Standard Laboratory Egg
Incubator (37.5 ^o^C, 66 % RH) The incubator thermal conditions were
monitored using a Hobo Temperature and Relative Humidity Sensor. During
incubation, the eggs were rocked once an hour. On Incubation Day 4 and
Incubation Day 7, a set of eggs were removed from the incubator and then
injected with 30 ml of flight fixative using an 18 gauge needle attached
to a 60 ml syringe. After injection the egg was placed into a specimen
vial containing 100 ml of flight fixative and then stored at 4 ^o^C
until post-flight processing.

**F) Processing of BSP Specimens**

Except for processing of the shell, the BSP dissection procedure was
performed after all primary tissue were collected for the ADF Validation
and primary ADF PI science.

1)  **Live Embryo Specimens**

Embryos were placed into a petri dish containing warm saline (37 ^o^C).
Embryos were maintained at 37 ^o^C until decapitation. After the head
was removed, the torso was placed into a petri dish containing ice cold
saline. The torso was maintained in ice cold saline through out the
primary dissection procedure and then passed to the BSP Workstation.

At the BSP Workstation:

a\) The torso was dissected for the following tissue: 1 kidney, 1 lung,
liver lobe, and ventral body with sternum (right side) was removed and
then snap frozen in liquid nitrogen. Transferred to --80 ^o^C freezer
for storage followed by overnight express shipment on dry ice and then
final cryostorage at --70 ^o^C.

b\) All remaining tissue that was dissected was fixed for 12 --24 hours
in 20 ml of 4 % paraformaldehyde in 50 mM Cacodylate buffer, pH 7.4 at 4
^o^C. These tissues included the kidney, liver lobe, lung,
proventriculus, thymus, spleen, bursa, gall bladder, gonads, and
remaining torso. After fixation, the specimens were transferred to
separate vials containing 7 mls of 50 mM Cacodylate buffer, pH 7.4.
These specimen were stored at 4 ^o^C followed by overnight express
shipment on ice and then final storage at 4 ^o^C.

2)  **In situ Preserved Embryo Specimens:**

These tissues were from eggs that were injected with fixative (4 %
paraformaldehyde in 50 mM Cacodylate buffer, pH 7.4) on flight day 4 or
flight day 7.

The torso was transferred to an empty petri dish. After the ADF Primary
PI removed his tissue, he transferred the torso to the BSP dissection
station. No chemical solutions were added to the petri dish prior to
transfer to the BSP Workstation.

At the BSP Workstation:

a\) In situ fixed 7 Day Embryos: The kidney, liver lobe, lung,
proventriculus, thymus, spleen, bursa, and gonads were dissected out and
then these tissues and the remains of the torso were placed into
separate vials containing 7 mls of 50 mM Cacodylate buffer, pH 7.4.
These specimen were stored at 4 ^o^C followed by overnight express
shipment on ice and then final storage at 4 ^o^C.

b\) In situ fixed 4 Day Embryos: The remains of the torso were placed
into separate vials containing 7 mls of 50 mM Cacodylate buffer, pH 7.4.
These specimen were stored at 4 ^o^C followed by overnight express
shipment on ice by an overnight express courier and then final storage
at 4 ^o^C.

**3) Chorioallantoic Membrane**

From live embryos: the CAM was removed by blunt dissection and then one
portion was placed into a cryotube and then snap frozen in liquid
nitrogen. Following freezing, the tissues were stored at --80 ^o^C until
shipment. For shipping the vial containing the cam was shipped on dry
ice by overnight express courier and then finally storage at --70 ^o^C.

The second portion of CAM was placed into a vial containing 20 ml of 4 %
paraformaldehyde in 50 mM Cacodylate buffer, pH 7.4. This portion of CAM
was incubated at 4 ^o^C for 12 to 24 hours and then transferred to a
vial containing 7 mls of 50 mM Cacodylate buffer, pH 7.4 followed by
storage at 4 ^o^C until shipment. These specimen were shipped on ice by
overnight express courier and then finally storage at 4 ^o^C.

b\) From In situ fixed embryos: The CAM was placed into a vial
containing 20 ml of 4 % paraformaldehyde in 50 mM Cacodylate buffer, pH
7.4. This portion of CAM was incubated at 4 ^o^C prior to overnight
express shipment on ice and then final storage at 4 ^o^C.

**F) General Information for the Avian Development Facility (ADF)**

**1) Duration of Flight and Incubation aboard STS-108**

> 1\) Duration of Flight: 11 Days 16 hrs 36 mins
>
> 2\) Duration of Incubation: 11 Days 14 hrs 44 mins, including the
> duration of ground time from Orbiter landing to start of processing
> the first egg

**2) ADF Launch Set-Points (Chill Mode)**

> 1\) ADF Incubation Chamber Temperature: 13.5 ^o^C
>
> 2\) ADF Relative Humidity: 65 %
>
> 3\) ADF Partial Pressure O~2~: ≥ 2.7 psi
>
> 4\) ADF Partial Pressure CO~2~: ≤ 0.07 psi
>
> 5\) Carousels: Upper Stationary
>
> Lower Stationary
>
> 6\) Duration of Time Eggs Maintained at these Set-Points in the ADF:
> 50 hrs 22 min

**3) ADF In-Flight Incubation Set-Points**

> 1\) ADF Incubation Temperature 37.5 ^o^C
>
> 2\) ADF Relative Humidity: 66 %
>
> 3\) ADF Partial Pressure O~2~: ≥ 2.7 psi
>
> 4\) ADF Partial Pressure CO~2~: ≤ 0.07 psi
>
> 5\) Upper Carousel Rotation Velocity: 77 rpm (1 G centrifugal force)
>
> 6\) Lower Carousel Rotation Velocity: Stationary (microgravity)
>
> 7\) Egg Holder Turning Rate: Once per hour, alternating between 180^o^
> clockwise and 180^o^ counterclockwise)
>
> 8\) Incubation Mode Activated: 3 hrs 3 min post-launch
>
> 9\) Time to Reach Thermal Equilibrium: 3 hours
>
> (Post-Incubation Mode Activation)

**4) *Cortunix cortunix japonica* Eggs and Embryos**

> 1\) Total Number of Eggs 36 (18 per carousel)
>
> 2\) Live Embryos Recovered Post-Flight: 9 (microgravity); 9 (1 G
> centrifugal force)
>
> 3\) Embryos Treated with Fixative In-Flight: 6 at development age 4
> days

(3 - microgravity; 3 - 1 G centrifugal force)

12 at development age 7 days

> (6 - microgravity; 6 - 1 G centrifugal force)
>
> 4\) Flight Fixative: 4 % Paraformaldehyde in 50 mM Cacodylate Buffer,
> pH 7.4
>
> 5\) Volume of Fixative Injected into the egg: 30 ml

**5) Temporary Environmental Changes During EVA Operations:**

1)  Total Orbiter Depress Duration: 19 hrs 50 mins

Flight Day 5: Start Depress to 10.2 psi: 00:55 GMT

Reached 10.2 psi: 01:15 GMT

Flight Day 6: Start Repress to 14.7 psi: 20:35 GMT

Reached 14.7 psi: 21:05 GMT

2)  Orbiter Cabin atm Pressure: 10.2 psi

3)  Orbiter Cabin Partial Pressure O~2~: 2.5 psi ≤ X \< 2.7 psi

4)  ADF Partial Pressure O~2~: \~2.25 psi ≤ X ≤ 2.5 psi

5)  ADF Partial Pressure CO~2~: \< 0.04 psi

6)  ADF Relative Humidity: 65 % \< X \< 66 %

7)  ADF Incubation Temperature: 37.5 ^o^C

The environment in the ADF incubation chamber quickly returned to
nominal conditions when the Orbiter cabin was repressed back to its
nominal atmospheric pressure of 14.7 psi.

**6) Approximate Duration of Time that the Live Flight Embryos
Experienced Terrestrial 1 G (Post-Landing) prior to Dissection**

Duration of time from de-orbit burn to landing: 1 hr 15 min

**Duration of Time from Landing to Start of Post-Flight Egg Processing**

  ----------------- ----------------- ----------------- -----------------
  **Egg Number**    **In-flight       **Decapitation of **Receipt of
                    Gravity           Embryo**          Tissue for BSP
                    Exposure**                          Dissection**

  659               0 G               4 hrs 19 mins     4 hrs 27 mins

  758               0 G               4 hrs 27 mins     4 hrs 34 mins

  642               0 G               4 hrs 35 mins     4 hrs 41 mins

  729               0 G               4 hrs 41 mins     4 hrs 49 mins

  601               1 G               5 hrs 28 mins     5 hrs 35 mins

  673               1 G               5 hrs 31 mins     5 hrs 43 mins

  665               1 G               5 hrs 41 mins     5 hrs 48 mins

  663               1 G               5 hrs 44 mins     5 hrs 54 mins

  664               1 G               5 hrs 47 mins     6 hrs 02 mins

  629               1 G               5 hrs 51 mins     6 hrs 11 mins
  ----------------- ----------------- ----------------- -----------------

**G) Developmental Stage and Sex of Embryos**

**1) Flight Eggs**

+-------+---------+----------+-------------+---------+--------+-------+
| **Egg | **Type  | **Live   | **De        | **      | **Caro | **Sex |
| Num   | of      | or       | velopmental | Gravity | usel** | ^4^** |
| ber** | Incu    | Fixativ  | Stage**     | Exposu  |        |       |
|       | bator** | e^1,2^** |             | re^3^** | **Mo   |       |
|       |         |          | **(days)**  |         | tion** |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 642   | ADF     | Live     | 11.5        | 0 G     | Stat   | Male  |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 659   | ADF     | Live     | 12          | 0 G     | Stat   | F     |
|       | Flight  |          |             |         | ionary | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 729   | ADF     | Live     | 11.8        | 0 G     | Stat   | Male  |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 758   | ADF     | Live     | 11.7        | 0 G     | Stat   | Male  |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 601   | ADF     | Live     | 12          | 1 G     | Spun   | F     |
|       | Flight  |          |             |         |        | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 629   | ADF     | Live     | 11.9-12     | 1 G     | Spun   | Male  |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 663   | ADF     | Live     | 12          | 1 G     | Spun   | F     |
|       | Flight  |          |             |         |        | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 664   | ADF     | Live     | 12          | 1 G     | Spun   | F     |
|       | Flight  |          |             |         |        | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 665   | ADF     | Live     | 12          | 1 G     | Spun   | Male  |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 673   | ADF     | Live     | 12          | 1 G     | Spun   | Male  |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 638   | ADF     | Fixative | 4           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 679   | ADF     | Fixative | 4           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 714   | ADF     | Fixative | 7           | 0 G     | Stat   | N/D   |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 737   | ADF     | Fixative | 7           | 0 G     | Stat   | N/D   |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 746   | ADF     | Fixative | 7           | 0 G     | Stat   | N/D   |
|       | Flight  |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 757   | ADF     | Fixative | 7           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 640   | ADF     | Fixative | 7           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 649   | ADF     | Fixative | 7           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 675   | ADF     | Fixative | 7           | 1 G     | Spun   | N/D   |
|       | Flight  |          |             |         |        |       |
+-------+---------+----------+-------------+---------+--------+-------+

^1^4 % Paraformaldehyde/50 mM Sodium Cacodylate, pH 7.4, injected into
the egg using the ADF Automated Injection System.

^2^Live = embryos were recovered alive post-flight

^3^Carousel was spun at 77 rpm (1 G centrifugal force)

^4^N/D = not determined

**2) ADF Ground Control Eggs**

+-------+---------+----------+-------------+---------+--------+-------+
| **Egg | **Type  | *        | **De        | **      | **Caro | **Sex |
| Num   | of      | *Live^1^ | velopmental | Gravity | usel** | ^4^** |
| ber** | Incu    | or       | Stage**     | Exposu  |        |       |
|       | bator** | Fixat    |             | re^2^** | *      |       |
|       |         | ive^2^** | **(days)**  |         | *Motio |       |
|       |         |          |             |         | n^3^** |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 839   | ADF GC  | Live     | 12.3        | 1 G     | Stat   | F     |
|       |         |          |             |         | ionary | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 911   | ADF GC  | Live     | 12.2        | 1 G     | Stat   | Male  |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 917   | ADF GC  | Live     | 11.8        | 1 G     | Stat   | Male  |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 922   | ADF GC  | Live     | 12.2        | 1 G     | Stat   | F     |
|       |         |          |             |         | ionary | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 930   | ADF GC  | Live     | 12          | 1 G     | Stat   | F     |
|       |         |          |             |         | ionary | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 945   | ADF GC  | Live     | 11.8        | 1 G     | Stat   | F     |
|       |         |          |             |         | ionary | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 829   | ADF GC  | Live     | 12          | V       | Spun   | F     |
|       |         |          |             | ariable |        | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 841   | ADF GC  | Live     | 12          | V       | Spun   | F     |
|       |         |          |             | ariable |        | emale |
+-------+---------+----------+-------------+---------+--------+-------+
| 888   | ADF GC  | Live     | 12          | V       | Spun   | Male  |
|       |         |          |             | ariable |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 898   | ADF GC  | Fixative | 4           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 926   | ADF GC  | Fixative | 4           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 866   | ADF GC  | Fixative | 7           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 877   | ADF GC  | Fixative | 7           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 899   | ADF GC  | Fixative | 7           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 907   | ADF GC  | Fixative | 7           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 940   | ADF GC  | Fixative | 7           | 1 G     | Stat   | N/D   |
|       |         |          |             |         | ionary |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 882   | ADF GC  | Fixative | 7           | V       | Spun   | N/D   |
|       |         |          |             | ariable |        |       |
+-------+---------+----------+-------------+---------+--------+-------+
| 950   | ADF GC  | Fixative | 7           | V       | Spun   | N/D   |
|       |         |          |             | ariable |        |       |
+-------+---------+----------+-------------+---------+--------+-------+

^1^Live = embryos were recovered alive "post-flight"

^2^4 % Paraformaldehyde/50 mM Sodium Cacodylate, pH 7.4, injected into
the egg using the ADF Automated Injection System.

^3^Carousel was spun at 77 rpm. For the ADF Ground Control Unit, only,
the unit was placed on its side so that the gravitational vector of the
Earth was perpendicular to the longitudinal axis of the eggs, similar to
an egg in a nest. Therefore, the eggs on the spinning carousel were
exposed to a variable total centrifugal force (centrifugal force
vector + gravitation vector) that ranged from 0 G to 2 G.

^4^N/D = not determined

There is a BSP Archive data entry and a vial stored at 4 ^o^C for Egg
#822 (Embryonic Liver). Egg #822 was not a selected egg for use. The
origin of the dissected tissue is unknown.

3)  **Laboratory Incubator Control Eggs**

+-------+----------+---------+-------------+-----------+-------------+
| **Egg | **Inc    | **      | **De        | **Gravity | **Sex^3^**  |
| Num   | ubator** | Live^1^ | velopmental | E         |             |
| ber** |          | or      | Stage**     | xposure** |             |
|       | **Unit** | Fixati  |             |           |             |
|       |          | ve^2^** | **(days)**  |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A19   | I        | Live    | 11.7        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A22   | I        | Live    | 11.8        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A28   | I        | Live    | 11.7        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A32   | I        | Live    | 11.7        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A39   | I        | Live    | 11.7        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A52   | I        | Live    | 11.6        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A57   | I        | Live    | 11.8        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B05   | I        | Live    | 11.8        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B14   | I        | Live    | 11.5        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B15   | I        | Live    | 11.8        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B28   | I        | Live    | 11.6        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B30   | I        | Live    | 11.6        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B31   | I        | Live    | 11.6        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B36   | I        | Live    | 11.7        | 1 G       | Female      |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B37   | I        | Live    | 11.8        | 1 G       | Male        |
|       | ncubator |         |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A04   | I        | F       | 3.4         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A65   | I        | F       | 3.6         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A25   | I        | F       | 6.0         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A29   | I        | F       | \< 6.0      | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A45   | I        | F       | \< 6.0      | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A49   | I        | F       | 5.9         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A54   | I        | F       | 6.05        | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A74   | I        | F       | 5.9         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A87   | I        | F       | 6.5         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| A94   | I        | F       | 6.4         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B08   | I        | F       | 6.0         | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+
| B19   | I        | F       | \< 6.0      | 1 G       | N/D         |
|       | ncubator | ixative |             |           |             |
+-------+----------+---------+-------------+-----------+-------------+

^1^Live = embryos were recovered alive "post-flight"

^2^4 % Paraformaldehyde/50 mM Sodium Cacodylate, pH 7.4, injected into
the egg using an 18-gauge needle attached to a 60 ml syringe. A 30 ml
volume was injected on a pre-determined incubation date.

^3^N/D = not determined

There is a BSP Archive data entry and a vial stored at 4 ^o^C for Egg
#A38 (Embryonic Liver). Egg #A38 was not a selected egg for use. The
origin of the dissected tissue is unknown.