Flight experiments in which fertilized Japanese quail eggs were allowed to develop to various ages in space, and the results of the following laboratory tests are described. Laboratory-based experiments concerned with the embryonic development of Japanese quail in gravity using simulated vibrations and G-force are reported. Effect of turning and ambient temperature at various days of incubation on the development of Japanese quail, and method to feed and water adult and newly hatched Japanese quail in microgravity using a gelatin-based diet as a solid water supply, are also described

Wentworth, A. L.

Wentworth, B. C.

English

NASA Technical Reports Server

- y d / Y A  
UNIVERSITY OF WISCONSIN-MADISON 
COLLEGE OF AGRICULTURAL AND LIFE SCIENCES 
POULTRY SCIENCE DEPARTMENT 260 Animal Sciences Building 
1675 Observatory Dnve 
Madison, WI 53706-1284 
Phone 608-262-1243 
A Tradition of Excellence in Avian Biotechnology 
FAX 608-262-6005 
TO: Beatrice Modes 
Grant OfEcer SLO:24OA-3 
NASA 
Ames Research Center 
Moffett Field, CA 9403 5- 1000 
FROM Bernard Wentworth 
DATE: April25,1996 0 1 19% 
RE: Grant# 2- 1009 
Please fmd the enclosetf&Geglip> "Fecundity of Quail in Spacelab 
Microgravity," and budget req&st to rollover unencumbered Year One funds, Year Two budget 
and a request for budget supplement to cover the unplanned expenses associated with the STS-76 
flight. 
September of 1995 with a Year One ending date of April 30,1996. Consequently there is 
unftnished laboratory work committed for Year One ($9,550) and we ask that the unencumbered 
h d s  rollover for use in Year Two. The Year Two budget ($42,504) has been adjusted to 
reflect the experience and shortage noted in Year One. The suppiemental budget ($3,630) is 
requested to cover the cost of quail colony management and shipping cost associated with the 
fertile eggs provided for STS-76. The original plans for this flight called for obtaining fertile 
quail eggs from another source. 
available May 1 ,  1996. 
The Year One firnding was not released to the University of Wisconsin Until mid 
We anticipate that these budget requests Will be approved and Year Two funding will be 
cc: Cheryl E. Gest, Research Admin IT of W 
Charles Winget, (ham Office NASA - Ames 
Gary Jahns, NASA Payload Mmager 
https://ntrs.nasa.gov/search.jsp?R=19960021793 2020-06-16T05:08:09+00:00Z
1 
Progress Report 
Fecundity of Quail in Spacelab Microgravity 
B.C. Wentworth and A. L. Wentworth 
Successfid regeneration in space microgravity depends on successfid gamete 
production, proliferation, Werentiation and union to form a viable developing 
embryo. Progress in this area during 1995 using the Japanese quail, Cotumix 
cotumix japonica, is summarized below according to the following phases of 
Russian-American cooperative investigation: A) Embryonic development in 
microgravity; B) Laboratory-based tests on G-force and Vibrations (Gaveet tests at 
Ames, California); C. Laboratory tests conducted at the Avian Physiology 
laboratory U.W. Madison, Wi; D) Active participation in the coordination of 
Russian-American projects in Moscow and in the US.; E) Future Directions. 
A. DeveloDment in Micromaviity. Two flight experiments were performed in which 
fertilized Japanese quail eggs from the Moscow-based colony were allowed to 
develop to various ages in space, (after 7,10,14, and 16 days of incubation) at which 
time they were fixed in either paraformaldehyde or ethyl alcohol. After the fixed 
eggs were returned to earth American and Russian scientists participated in 
separating the specific tissues of scientific interest from the experimental flight 
embryos and the various controls at NASA in the Ames, California laboratories. 
Temperature control may have been faulty in each of these experiments so that 
recovery of tissues from experimental and synchronous controls was minimal. 
(Tables 1 and 2). Transport control fertile eggs were taken to the launch site and 
were returned to the Moscow laboratory and set to hatch. These eggs showed a 
72.5% hatchability. 
Histoloizical Results: From the limited number of flight samples which were 
available for analysis, we did not observe an obvious effect of microgravity on the 
development and cellular differentiation of the reproductive organs of the male and 
female embryos. The data are recorded for the one 10-day-old embryo flight sample 
as well as in the few synchronous controls which were fixed at day 10 and 16. The 
gonads were developed normally into male or female and had an equivalent number 
of germ cells to that of the controls. The lungs showed that the viable 16 day bird 
from the synchronous control group had broken through the air sac and had 
commenced breathing. From these data we are encouraged that normal reproductive 
development of fertilized Japanese quail eggs is possible. 
2 
Promess report on Mir 21 (STS-76) Launched 3-22-96. 
Forty-eight fertile Japanese quail eggs were launched on STS 76 on 3-22-96 
to be incubated on Spacelab Mir and fixed after 0,3,7,10,14 and 16 days of 
incubation. The eggs were from the random bred wild type Japanese quail 
maintained at the Avian Physiology laboratory, UW- Madison. They were placed in 
the Mir incubator six days from the day of lay. They will be retuaed in August of 
1996 for subsequent tissue removal at NASA- Ames, California, and then specific 
analyses at the scientists’ respective laboratories. Control data to date is from the 
transport controls and various laboratory controls. The transport controls were the 
fertile eggs that were shipped to Kennedy and returned to Madison for incubation to 
hatch B17.2 days of incubation. The eggs were held for two days at 14-2 1 C prior 
to incubation). These eggs were 92% fertile, 89% viable at 16 days and 82% 
hatched. The laboratory controls (eggs from the same colony set as fresh eggs) 
showed a 94% fertility, 96% viability and 82% hatchability. (Table 3) 
A second set of laboratory controls was perfonned in which eggs fiom the 
same colony of Japanese quail were stored at 14C for either six ( N=56) or five 
(N48)  days and then set to hatch. The group held for six days had a 96% fertility, 
91% viability at 17.2 days and 85% hatchability of fertile eggs at 17.2 days. The 
group held for five days prior to incubation showed a 96% fertility, 94% viability at 
17.2 days and 91% hatchability of fertile eggs at 17.2 days. (Table 4). These data 
are very encouraging as is the preliminary report of viable embryos on the fixation 
days on the Mir Spacelab. 
B. Laboratow-based exDeriments were conducted in anticipation of problems 
encountered in microgravity which could hinder successful regeneration in sDace. 
1. Embryonic development of Japanese quail in gravity using simulated 
vibrations and G-force. Gaveet Tests were performed in duplicate at Ames, 
California by the NASA staff. 
the avian physiology laboratory at UW-Madison on 1-8-1996. No differences in 
development was observed in the four groups analyzed. Vibrations; Centrihge; 
Centrifuge +Vibrations and Controls. (Table 5a&b labeled: I‘ Gaveet egg data”). 
Additionally, the results of a sampling of gonadal tissues fiom the Gaveet test (€3) at 
Ames, California, showed that neither centrifugation which simulates G-force at lift 
off, nor vibrations which simulated those as lift off, affected the reproductive 
development of the embryonic quail after 16 days of incubation. 
Evaluations were conducted(a) by the PI’S at Ames on 12-30-96 and (b) at 
3 
C. Progress reports on various mound-based laboratow tests at the Avian 
Phvsiologv laboratow, UW-Madison. 
1 .. Effects of turning and ambient temperature at various days of incubation on the 
development of Japanese quail. Cotumix cotumix japonica. 
projects, two questions arose. The first was if eggs were incubated on earth for the 
first few days of development, a critical time of development, could they be held at 
ambient temperature for a day so that they could be transferred into the shuttle and 
resume incubation after lift off. Additionally avian eggs have to be turned a 
minimum of four times on earth to develop well. The question of the necessity of 
turning eggs in microgravity also arose. These two considerations were addressed 
in a 2 x2x 4 factorial design experiment which was replicated five times. The quail 
used in this study were from the same random bred colony that was used for the 
STS 76 Flight experiment and controls. Experiment 1 consisted of two different 
treatment groups, one turning and the other not. On different days of incubation, 
days 2,3,4,and 5 ,  the eggs were placed at room temperature, 21C, for 24 hours, and 
were set to be either turning or not turning. Each group consisted of 100 eggs. 
Twenty eggs were broken out daily for five consecutive days and evaluated for 
development. The total number of eggs used in this experiment was 1000. 
Experiment 2 was a hatchability trial of the same experimental groups turning and 
not turning and ambient temperature for 24 hours on days 2,3,4,and 5. Hatchability 
was recorded at day 17, 18 and 19 to determine late hatching. At day 19, eggs that 
were not hatched were opened and recorded as to the stage of development. These 
data are currently being analyzed. Preliminary conclusions indicate that turning of 
quail eggs in gravity increases viability and hatchability. Many of those which did 
not turn did hatch. Statistical significance of these data is being calculated. 
Additionally, many eggs held at ambient temperature for 24 hours on the various 
days, 2,3,4 and 5 did develop although at a slower rate. Again, interactions and 
statistical significance of these data are being analyzed. The total number of eggs in 
this experiment was 200. 
During the 1994 and 1995 telecoms among the scientists involved in this 
2. A method to feed and water adult and newly hatched Japanese quail, Cotumix 
cotumix japonica. in microgravity using a gelatin-based diet as a solid water supply. 
This experiment was designed so that adult quail and newly hatched chicks could be 
4 
maintained in space microgravity. 
Japanese quail, unlike mice, depend on gravity to get their water supply fkom drip 
waterers. Therefore, a means to water adult and newly hatched quail has to be 
devised. We found that a 70% water based, 5% gelatin, 25% quail diet could 
support the body weight and egg production rate of adult female as well as male 
Japanese quail fertility. (Table 6a and b). This preliminary trial lasted only one 
week. However, quail with insufEcient water(ge1 blocks cut up so that evaporation 
was rapid) stopped laying after three days. This experiment will be expanded at a 
later date. For the newly hatched quail, a 75% water, 2% gelatin and 23% quail 
starter ration could support normal growth of newly hatched chicks Tor 10 days. 
This experiment is being repeated with a modified starter ration as the feather 
development in these quail was slightly abnormal. 
D. Participation in interactive communication between Russian and American 
scientists at the Moscow and the American-based laboratories 
T r i ~  to Russia. A major part of the information exchange involved hture missions 
and technical details of logistics and required hardware. It was apparent fiom the 
meeting that the following things must be accomplished. 
Complete success in the incubator project STS76 - MIR21 
Make preparation for MIR 21. 
Make preparation for NASA 4 & 5 .  
Determine who will supply eggs for MIR2 1. 
What vehicle will be used to transport hardware and who's payload manifest 
(U.S.  or Russian) will provide space for hardware. 
My major contribution in flight programing to meet this accomplishment was 
associated with the incubator hardware requirements, general and specific 
knowledge concerning avian physiology, and a continuous source of quail eggs. It 
was agreed that Dr. Hester would provide the eggs for MIR 2 1. We agreed that 150 
sexually mature birds in a ratio of three females to one male would be adequate to 
meet the March flight. We maintained a colony of random bred wild-type quail at 
the UW - Madison Avian Physiology laboratory as a back up egg supply to the 
planned egg supply. 
5 
E. Future Directions 
1. Adult quail maintenance in space microgravitv. 
2. Hatching fertile quail eggs in space microgravitv and maintaining auail chicks in 
space microgravitv. 
The ground research which was performed on this project to prepare for future 
flights includes: 
Analysis of the Russian quail feed. 
Analysis of U W  Madison quad adult and quail chick starter rations. 
Effect of turning during incubation 
Effect of chilling during incubation 
Gel feed containing adequate water for day-old chicks, and adults 
Artificial insemination of quail 
Freezing of semen 
3. Cell-cell and cell-matrix interactions in miaation. momhogenesis. and 
differentiation. Gravity is postulated to play a critical role in embryogenesis. In the 
case of Xenopus oocytes, gravity appears to be essential for the determination of the 
dorso-ventral axis. Turning of avian eggs is essential for the normal development of 
embryo in gravity. However, the requirement for turning of embryonated eggs for 
proper development of the avian embryo in space microgravity has not been 
resolved. One could postulate that gravity mav not be the determining factor in 
embryogenesis. Rather cell-cell adhesion and cell-substrate interactions involving a 
variety of integrins, growth factors and extracellular matrix components may be the 
predominant contributing factors toward morphogenesis and tissue differentiation. 
Space microgravity provides the ideal environment for studies involved in cell- 
matrix interactions. The major focus of the avian physiology laboratory is the 
primordial germ cell. Because of its unique extraembryonic segragation, migration 
and differentiation processes the primordial germ cell is the ideal cell to explore 
mechanisms which regulate cell adhesion, migration, chemotaxis and differentiation. 
The primordial germ cell possesses integrins which are presumed to react with 
specific extracellular matrix proteins and specific growth factors along its migratory 
route. Insights in this system of cell-ECM interactions could provide insights into 
other systems of cell-cell, cell-matrix interactions involved in development and in 
several physiological and pathological processes. 
6 
7 
Day 14 (8) 
@TOW (16) 
Day 16 (8) 
Day 16 
Table 1: Results fi-om MJR 18 (STS 74);launched eggs ; synchronous and 
laboratory controls 
0 1 7  3 5 7 1 
0 8 1 4 3  8 
0 16 0 13 3 11 2 3 
8 
Table 2: Results from MIR 19 (STS 76) launched eggs ; synchronous and 
laboratory controls 
Age at Fixation/ 
Number fixed on 
Fixation Day ( ) I 
Day 16 
Legend: N = numbei 
Flight/ 
Number Set 
Synch Control/ 
Number Set 
48 I48  
Results I Results 
Lab. Control/ 
Number Set 
48 
Results 
N I I E D ~ L D  
;D = early dead; 
LD = late dead; ETOH: 16 eggs fixed at day 16 in ethyl alcohol. Flight = launched 
eggs;Synch. = synchronous controls= simulated temperature aboard M R ;  Lab 
controls = Controls set in Moscow laboratory standard conditions 
9 
Table 3. Results of transport controls and laboratory controls taken to hatch at 
Madison laboratory. 
Lab 45 3 0 2 1 37 6 94% 96% 
48 
Legend: F=Fertile;I=Idertile; C=cracked;ED=early dead; LD=late dead; 
H=hatched; P=pipped or alive and not pipped. LBG2 = launch back up group 2; 
Laboratory = fresh laboratory controls. 
Table 4. Laboratory test on hatchability of fertile Japanese quail egg storage at 
40F for 6 and 5 days prior to incubation. 
I 4 8  (5) I46 I42  196% 191 194% I 
Legend: F=Fertile;I=Idertile; ED=early dead; LD=late dead; H=hatched; P=pipped 
or alive and not pipped. DST=days in storage before incubation; %V=%viable but 
not hatched by 17.2 days. 
P 
Q 

11 
Table 6a. Body Weights of male and female adult Japanese quail maintained on a 
gelatin based diet for one week. Preliminary data. 
Control Females 
Experimental Females 
Control Males 
Experimental males 
I 
Table 6b. Egg production and egg weights fiom Japanese quail females for one 
week prior to being placed an experimental gelatin ration and for one week after 
being on the gelatin based diet. (Three birds) 
I I I 1 
~ Number of Eggs 
Control (prior to exp’t) I Experimental I 
17 I17 
I Average Egg weight 19.87 19.5 I 
* Birds fed limited water ( Le. gelatin diet cut up so that it evaportated stopped 
laying at day three of the experiment. 
Request r o l l  over  of 1995-1996 NASA g r a n t  (October,  1935 - A p r i l  3 0 ,  1996) as  i t  
w a s  an abbrev ia t ed  yea r  w i t h  un f in i shed  l abora to ry  work as a r e s u l t  o f - t h e  two 
f l i g h t s  dur ing  t h e  g r a n t  per iod .  NASA Grant # NAG 2-1009 
PWCIPK HMS;K~ATOWPFIOGFWA DIFiECT’OR: * ‘emworth m u  0 
mainta in  q u a i l  colony 
a i r  and f e d  ex charges  
S l i d e  sec t ion ing  (Sch. of V e t .  Xed.) 
1,000 
306 
I 1,500 
TOTAL DIRECT COSTS FOR RRST 12-uONTw B U O C n  PEFIlOO (k#m 4 
. W l P M M  (h#&8, LCIe 8d&ion8ltirral ffnudadf 
t r a n s i l l u m i n a t o r  (white  f i g h t )  
8 9,550 
I 375 
SUPPLIES (hamha by uragoy. we addiio~irherr I -dad) 
Expendab-le s u p p l i e s  I 1 , 204 
1 FOREIGN Russia 1 4,700 
250 I c u r r e n t  protocols m m  EXPENSES flramit. by ur8gar): use 8ddiddlW?taw&d) 
Continuat ion of NASA Grant I\ NAG 2-1009 
PRLNdPK HVESRCATOWPROOW DIRECTOR: m u  E 
FFioM THROUGH I 
J 
MTuLED BUOGR FOR lZ--uONTH 8U- PUUOO 
URECT c13sTTs ONLY 
OTHER EXPENSES (lfemito by uiegory. LU. 8ddirwdrhuZ S-d) c u r r e n t  P r o t o c o l s  
.~ 
Student  l a b o r  
465 
aSULTAJ4T COSTS 
TOTU DIRECT COSTS M R  HRST 1a-UOH1u BUDGET PERK)O @em 4 firm& 
s l i d e  s e c t i o n i n g  (Sch. V e t .  Med.) 1 3,500 
8 31,059 
ECUIPMEM (&Of&& LU. rddhional3roUtlnud.d) bar code Scanner 2,200 I 
expendable s u p p l i e s  I 4,860 
7,100 DOMESTIC Ames (4) Kennedy (1) Houston (1) fRAVEL 
I FOREGN 1 
HMAECT COSTS FOR FIRST 12-MONTH BUOGn P E W D  11,445 
Supplemental budget to cover an unplanned expense of providing 
eggs for the STS-76 flight. NASA Grant f EAS 2-1009 
B. Wentworth 
PRlNClPK NVESIGATOWPROGRAM DIRECTOR: 
DOMESTIC 
FOREIGN 
TRAVEL 
OTHm EXPaSES (itomitr by utogor)? UH a d & d 8 h u t # ~  
maintain quail colony 
air and f e d  ex charges 
TOTAL DIRECT mSTS FOR RRST 12-UOWH BUDCET PER100 @#m4 krm4 8 
0 
$ 306 
1,800 
894 
3,000 


Fecundity of Quail in Spacelab Microgravity

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