Over the past 25 years, four independent advisory boards have been convened by the National Research Council (NRC) (1987, 1991, 1998, 2011) to guide the future of NASA’s Space Biology research. The corresponding reports have uniformly emphasized the fundamental importance of research on reproduction and development of mammals in space, and specifically that animals be studied within and across generations, completing at least two complete life cycles in space. The recent Decadal Survey published in 2011,
Spaceflight studies on rodent reproduction and development have revealed important, yet preliminary, findings and “lessons learned” (Maese and Ostrach, 2002; Ronca, 2003). Special requirements of reproducing and developing animals, restrictions on experimental design due to hardware limitations, short duration exposures to the space environment precluding analysis across multiple reproductive cycles or significant periods of development, have not permitted a comprehensive approach to life-cycle issues (Moody and Golden, 2000; NRC Report 2011). As ISS research capabilities expand to support studies of adult rodents (Cancedda et al., 2012; Mark I flight1), there is an opportunity and an expectation that well-designed and highly controlled studies of reproducing and developing animals will follow, addressing the recommendations of advisory panels. Such experiments will be of significant interest to the reproductive and developmental biology research communities and offer unprecedented opportunities to study developing and adult animals and specimens never exposed to Earth gravity. Finally, reproductive and developmental biology research using a mammalian model holds immense promise for advancing translational knowledge with major relevance to current and future space habitation by humans.
The major goal of the Rodent Mark III Habitat Workshop was to identify the top-level science requirements envelope suitable for meeting the research objectives of the science community. This is the first animal habitat specifically designed to support varying stages of reproduction and development of rodents for research on the ISS. Precedence is given to studies of rats and mice examining transmission across generations of structural and functional changes induced by exposure to the space environment. This directive encompasses key reproductive and developmental phases comprising the mammalian life cycle. There was strong consensus among workshop participants that, to attain this goal, spaceflight animal housing and hardware will need to support multiple neural, endocrine, and environmental requirements to maximize successful outcomes for: (1) mating, (2) conception, (3) pregnancy, (4) embryonic/fetal development, (5) birth, (6) lactation, (7) maternal care, and (8) offspring development through sexual maturity. Achieving these individual milestones and their repeating cycle will form the foundation for lifespan and multigenerational research success.
The Rodent Mark III Habitat Workshop was Co-Organized and Co-Chaired by Ken Souza and April Ronca. Josh Alwood served as the Executive Secretary, and Ruth Globus is the ISS Rodent Habitat Project Scientist. Approximately 20 participants met for two days at the Ames Research Center. Participants included experts in animal husbandry (rodents), animal behavior, reproductive and developmental biology, animal physiology, veterinary medicine, flight hardware development, and spaceflight operations (Table 1, Workshop Participants and Affiliations). In preparation for the workshop, participants were asked to review the NASA Rodent Research Science Requirements Envelope Document (SRED), Revision E, appropriate sections of the NRC’s recent Decadal Survey of the Life and Physical Sciences (2011), and other relevant material.
Participant | Affiliation |
---|---|
Jeffrey Alberts, PhD ( |
Indiana University, Bloomington |
Joshua S. Alwood, PhD Executive Secretary | Oak Ridge Associated Universities |
Ted A. Bateman, PhD | University of North Carolina |
Shawn G. Bengtson | McGowan Institute, University of Pittsburgh |
Allison Brown | LifeSource Biomedical, LLC |
Kristin D. Evans, PhD, DVM | University of California, Davis |
Charles A. Fuller, PhD | University of California, Davis |
Ruth K. Globus, PhD | NASA Lead, NASA Ames Research Center |
Mike Hines | NASA Ames Research Center |
Danny A. Riley, PhD | Medical College of Wisconsin |
April E. Ronca, PhD, Workshop Co-Chair | Wake Forest School of Medicine |
Stephanie Solis, DVM | LifeSource Biomedical, LLC |
Kenneth A. Souza, Workshop Co-Chair | Logyx, LLC |
Marianne Steele, PhD | Lockheed Martin |
Louis S. Stodieck, PhD | BioServe Space Technologies, University of Colorado at Boulder |
Joseph S. Tash, PhD | University of Kansas Medical Center |
During the workshop the participants: (1) Received an overview briefing covering the characteristics of the ISS Rodent Habitat (a.k.a., Animal Enclosure Module (AEM-X)) that is under development for flight in April 2014; (2) Considered and discussed what is known about rodent reproduction and development in space; (3) Discussed the current requirements and capabilities of the rodent habitat for transporting animals to the ISS, Animal Enclosure Module–Transfer (AEM-T), and the on-orbit habitat, AEM-X, and any additional or expanded requirements specific to the mating, birthing, nursing, and maturation of rodents on the ISS; (4) Determined any new research that is required to close gaps in knowledge needed to define the requirements necessary for habitat development; (5) Identified specific examples of developmental and reproductive research on the ISS that require a RH Mark III habitat; and (6) Observed the rodent habitat (AEM) flown on the Space Shuttle: the nursing insert for the AEM flown on the Space Transportation System (STS) STS-72 NIH-R3 mission in 1996 and a prototype Animal Development Habitat developed by STAR Enterprises, Inc. as a Small Business Innovative Research (SBIR) project in 1986.
Workshop products and follow-on activities include a detailed report, viewable at URL:
Specifically, the workshop report will be used to expand the current ISS Rodent Research SRED (Revision E) to include the requirements for Reproductive and Developmental Biology. In addition, it will provide the guidance necessary for a RH Mark III project team to develop more detailed science requirements and an engineering specification for the development of the RH Mark III habitat.
Prior to the workshop, participants were organized into five different science teams (Reproduction, Neuroscience and Behavior, Musculoskeletal, General Physiology and Immunology, and Commercial Interests). Each team was tasked with identifying key research hypotheses and problems requiring a RH Mark III habitat, and incorporating key phases of mammalian reproduction and development (Figure 1). Team leaders presented the findings (Table 2).
Multigenerational survival comprises a cycle of lifespan functional milestones at the multiple levels: Whole animal, organ and endocrine systems, and cell
A failure or deficit in any milestone compromises species survival |
Does male and female sexual definition and development proceed normally in the space environment?
Gonad development, maturation and health Gamete production, maturation and health |
Are patterns of social behavior and mating affected by the space environment?
Ex-gonad gamete maturation in the female reproductive tract Fertilization, conceptus, placentation, fetal development Support of pregnancy, birth, lactation, nursing, weaning Post-weaning growth, puberty, acquisition of sexual maturity |
Is the neural architecture of the brain, particularly the gravity sensing system, shaped by gravity?
Morphology of the neurovestibular system |
Are vestibular mediated behaviors shaped by gravity, and are these correlated with changes in vestibular morphology? |
Is development of the motor system dependent upon gravitational input?
Emergence of fine motor control of locomotion and gait may require gravitational input during development |
Are there critical periods during pre- and/or postnatal life during which gravity exerts formative effects? |
Does lack of gravitational input to the vestibular macular sensory organs, beginning prior to conception and continuing into adulthood, ‘developmentally program’ circadian and homeostatic processes across the lifespan and generations? |
Is there epigenetic (non-genomic) cross-generational heritability of early life programming by gravity?
Identifiable epigenetic changes in DNA methylation patterns may be associated with development in microgravity (a direct effect) distinct from indirect maternal contributions to epigenetic programming of offspring phenotype |
For animals born in space and undergoing development, growth and aging are there critical periods of development and growth that require gravity?
On-orbit, temporal, noninvasive bone density and shape measurements, body weight to assess growth, temporal muscle diameter and length measurements, tissue acquisition and preservation of bone, cartilage, and muscle at key time points. Intact sample return for earth-based analyses |
Is stem cell production (myoblasts, osteoblasts, osteoclasts, bone marrow cells) reduced during prolonged spaceflight?
Quantify stem cell 1G to evaluate reloading injury and the capacity for repair that is dependent on stem cell participation. 1G restores stem cell deficiency or reveals permanent deficiencies |
General Physiology & Immunology
Altered regulation (System to Genome) Adaptive capabilities (i.e., 0->1G) |
CNS Function
Covered by Neuroscience & Behavior Group Role in altered sensory signaling, esp. Central (Vestibular) sensing, in altering genome |
Musculoskeletal System
Covered by Musculoskeletal Group |
Growth, Body Size and Composition
Sex, Health |
Endocrinology |
Metabolism
Energetics, Nutrition, Gastrointestinal |
Cardiovascular & Blood (i.e., Oxygen transport) |
Temperature Regulation |
Circadian Biology |
Immune Function |
External Stimuli
Exercise, Centrifugation |
Number one application is drug or gene therapy testing (interpretations/contingency with off-target effects), in particular, where disease models would apply to young children (e.g., muscular dystrophy, myopathies) |
Use of transgenic models would be likely. |
Neonates: Model of severe disuse conditions for children |
Dam: Model of pregnancy and delivery during conditions of disuse |
Mammalian development has been studied in seven nascent, yet pioneering, spaceflight missions. To determine whether mammals can conceive in microgravity, the unmanned biosatellite Cosmos 1129, flown in 1979, provided rats the opportunity to mate in flight. However, no pregnancies were realized at recovery (Keefe, 1985; See Tash, Reproductive Biology, this review). In 1983, Cosmos 1514, led by Luba Serova, Dick Keefe, and Jeff Alberts, carried late pregnant rats on a 4.5-day mission, returning them to Earth prior to birth. This landmark mission provided proof-of-concept that mammalian pregnancy can proceed in the microgravity of space (Ronca, 2003). After a hiatus of more than a decade, NASA and NIH jointly sponsored two secondary payloads on NIH.R1/STS-66 and NIH.R2/STS-70. Sixteen international and domestic science teams analyzed the behavior, morphology, neurobiology, and physiology of ten pregnant rats and their offspring launched at mid-gestation for 11 and 9 days, respectively. Following recovery, dams had uncomplicated, successful vaginal deliveries and nursed their young. Notably, NIH.R1 and NIH.R2 were model cooperative space biology efforts that fostered novel cross-disciplinary interactions among scientists from diverse disciplines and led to numerous published reports describing reproductive, neural, vestibular, locomotor, immune, musculoskeletal, and circadian factors in dams and offspring (Ronca, 2003).
In 1996 and 1998, the NIH.R3 (STS-72) and Neurolab (STS-90) carried nursing rat dams and their litters into space for the first time. Sensorimotor, neural, muscular, vestibular, cardiovascular, and cognitive processes were studied in the offspring (Ronca, 2003; Buckey and Homick, 2003). These missions also yielded important “lessons learned” (Ronca, 2003; Maese and Ostrach, 2002). The maternal-offspring system in mammals is exquisitely sensitive to changes in gravity, particularly during the early postnatal period when infants are dependent upon maternal care for their survival. Flight conditions and hardware, including caging, food delivery, and waste removal, therefore exert extraordinary influences on the animals’ health and development. Significant mortality and feeding difficulties were observed in young infant rats flown on NIH.R3 and Neurolab missions, requiring intervention from the astronauts (Maese and Ostrach, 2002). Similarly, quail fledglings hatched on the Russian Space Station Mir were unable to regulate their body position in space, requiring cosmonaut assistance to accomplish feeding (Jones, 1992). These experiments identified a clear need for specialized habitats for flying young postnatal animals in the weightless space environment. The highlights of these efforts are:
Animal Enclosure Modules (AEMs) onboard the shuttle adequately supported late-pregnant rats. Pregnant rats that experienced spaceflight and were subsequently returned to Earth within 48-72 hrs of normal birth underwent delivery at the expected time. The duration of the birth process was similar in spaceflight-exposed and ground control rats although spaceflight dams exhibited two times more labor contractions.
Pups suckled on anesthetized dams during 25 seconds of weightlessness during parabolic flight on NASA’s KC-135 airplane and stayed on the nipple during parabolic maneuvers, obtaining milk. Dams were injected with oxytocin that successfully caused milk letdowns during the parabolic flight. Pups showed milk-letdown reflexes, stretching and extending their hindlimbs, but they remained attached – with their bodies “out in space” around the mother’s ventrum during periods of microgravity and hypergravity that occur in the parabolic flight. Suckling also was demonstrated during the Neurolab mission, STS-90, in 1998.
STS-72 (NIH.R3) 5-day-olds (housed in the Nursing Facility AEM-NF) and STS-90 (Neurolab) 8-day-olds held in the Research Animal Holding Facility (RAHF) within the Shuttle/Spacelab had high mortality rates and low body weights. Habitat design played a crucial role in neonatal survival in microgravity, particularly in the youngest neonates (Maese and Ostrach, 2002; Ronca, 2003).
Males mated within 5 days of landing with new females produced offspring, but pups were developmentally delayed: (evidence of epigenetic influence on males?). Males mated 2 months post-flight produced normal pregnancies and offspring (no developmental anomalies support epigenetic effect on sperm). No data are available from follow-on assessments of females. There was no video monitoring during this flight. Therefore, it was not possible to distinguish failed fertilization from failure to mate. Ground controls may have also failed to become pregnant (Keefe, 1985). There was no assessment of hardware design to ascertain whether mating could be accomplished in the habitat/caging provided.
The three experiments used female C57BL/6 or BalbC mice. Microgravity negatively impacted ovarian histology in mice as evidenced by lower numbers of corpora lutea, and unhealthy oocytes. Estrogen receptor levels were lower in flight mice, while gene expression of the HSPH-1 stress marker was down regulated. Oocyte maturation and production were blocked or terminated after 12-15 days (3 estrous cycles) of spaceflight (post-flight recovery has not been ascertained). These findings raise the possibility that COSMOS 1129 rats had no eggs to fertilize and/or refused to mate in absence of estrogen-dependent mating behavior, though the sensitivity of female mouse and rat reproductive factors to spaceflight is unknown.
Since its inception at the NASA Ames Research Center in the mid-1970s, many laboratories around the world have used the rat hindlimb unloading (HLU) model to mimic the effects of weightlessness and to study various aspects of musculoskeletal loading (Morey-Holton et al., 2005). In this model, the hindlimbs of rodents are elevated to produce a 30 degrees head-down tilt resulting in a cephalad fluid shift and to avoid weight-bearing by the hindquarters. Long-term HLU inhibits spermatogenesis in adult male rats in the absence of cryptorchidism, changes in testicular function due to hyperthermic testes (Tash et al., 2002). All HLU animals were sterile in 2 mating attempts whereas controls were 100% fertile. Chronic testicular hyperthermia was observed with temperature elevated above normal by 2.2°C, P<.00001). Other findings included: (1) Invasion of inflammatory cells (≥3 weeks), (2) Catastrophic apoptosis in the testes, (3) These factors cause aspermatogenic dysfunction.
Less information is known for mice – much more is known for rats.
Rats live in colonies with multiple males and females. They are characterized by a promiscuous mating system – males mate with multiple females; females mate with multiple males. Mice live in demes with a single territorial male maintaining a range that encompasses homes of multiple females with which it mates. Females may mate with multiple, territorial males. Both species rely on
Colonial life of rats is associated with high levels of tolerance in close proximity (a “contact species”), seen in both females and males, although males are larger and tend to be more aggressive. Social organization of mice is associated with lower levels of tolerance among unrelated, unfamiliar adult males. Amicable contact behaviors are frequently seen among female mice but are not well understood.
Females exhibit an estrous cycle, 4–5 days continuously. Some seasonality is suspected but cycling occurs throughout the year in the lab. Females are sexually “receptive” during the estrus phase of the cycle, but they are more accurately defined as proceptive—because the estrus female actively solicits the male’s sexual attention. Female proceptive behavior includes: Moving into vicinity of male, making available their arousing, and estrus odors, which attract male approach behavior and sniffing. The female then “darts” away and stops, the male approaches again, the female darts and “ear wiggles.” The “dance” continues and escalates until copulation occurs.
During copulation: The male mounts and the female exhibits
Biomechanics of copulatory sequences are worth examining in relation to habitat configuration and implications for performance in microgravity
Some strain differences exist in the form of copulatory moves, such as mounts, clasps, dismounts, and grooming sequences (known especially for mice)
Surprising lack of information on interruptions of estrous cycle by general “stress” factors; this would be worth investigating
Some data exist on habitat configuration (at 1g) and patterned mating in rats, but comparable information has not been not collected for mice
The STS-90 Neurolab Mammalian Development Team consisted of six principal investigators. Collectively, the experiments had 12 cross-fostered litters of PN8 and PN14 Sprague Dawley rats in each of the flight, asynchronous ground control, and vivarium control conditions. The initial assignment of litters as primary for specific investigators was discarded due to the high in-flight mortality of the PN8 pups, causes unknown, that necessitated reapportioning to maximize science return.
The effects of spaceflight on development ranged from unimpaired to permanently altered. Interpretation of these results as to whether gravity is required for normal development must be tempered by the facts that all of the pregnant flight animals experienced at least one week of gravity just prior to launch, and the flight duration of 16-day gravity deprivation was likely too brief to have a major impact on some systems.
Effects of spaceflight on development and growth—the space-flown rodents were pre-exposed to gravity, and the exposures to the spaceflight environment were too short to complete development, growth, maturation, and aging such that deficiencies would manifest. Some findings indicate existence of critical periods requiring gravity environment stimulation for normal (1g) development and growth of systems. Studies of rodents generated in space and never exposed to one-gravity are necessary to elucidate the full impact of the spaceflight environment on development, maturation, and aging (i.e., studies across the lifespan and multiple generations in space). Return of live space-reared organisms is necessary for assessment of exposure to 1g stress to ascertain system weaknesses and repair capacities and the existence of irrevocable structure and function. An on-orbit centrifuge is needed to determine the level of gravity exposure (0-1g) sufficient for 1g comparable development. Science return is enhanced by forming integrated-discipline teams and by judicious sharing of litters. On-orbit animal microsurgery, complex tissue processing, and stable storage of specimens is feasible. Sample return is required for full analysis requiring techniques unavailable in space. Rodent habitat redesign is essential to maintain the neonate and dam interactions that facilitate adequate nutrition, sleep, body temperature control, and other factors of viability provided in a nest environment on Earth (Ronca, 2003).
Life on Earth evolved with a single environmental constant, gravity (1g). Chronic acceleration or hypergravity is the resultant of 1g Earth gravity combined with centrifugal force. The UC Davis Chronic Acceleration Research Unit (CARU) provides an experimental facility for exposing animals to short or long duration hypergravity.
Hypergravity has been used to establish the “Principle of Continuity” (Wade, 2005), the idea that gravitational fields are continuous above and below the gravitational field on Earth, and that biological responses to changes across the spectrum of gravity exhibit a similar continuity. On Earth (1g), fractional increments in g-load exceeding 1g can be continuously applied for extended periods, and dose-response relationship established. While the principle of continuity has not been rigorously tested and validated across the gravity continuum, there is a sizeable corpus of data suggesting that the principle is valid across multiple systems. Reproduction and development, size/growth, energy and metabolism, musculoskeletal, cardiovascular, immune, and CNS sensory/vestibular responses are among those that have been studied in space.
The lack of definitive data on exposure to centrifugation in space for rats and mice makes the extrapolation of the continuum to levels below Earth-gravity problematic. However, in systems where responses are detected for both spaceflight and acceleration by centrifugation, a gravitational continuum is present, supporting the Principle of Continuity or a systematic dose-response relationship between gravity load and the magnitude of physiological response. Accordingly, the use of hypergravity holds significant potential for predicting responses to spaceflight.
This model simulates certain features of microgravity exposure by elevating the hindquarters 30 to produce head-down tilt. Support structures are unloaded (bone, muscle), and cephalic fluid shifts are induced. Thus HLU mimics spaceflight effects for certain key systems. Limitations of the technique include immobility of the animals, no limitations or alterations of CNS sensory input such as that which would typically occur in spaceflight.
Will females hormonally cycle in the novel space environment? If females cycle, will they solicit and will males respond? In microgravity, can pairs manage mechanics of repeated intromissions needed for ejaculation?
Use proven breeders. Cage design should incorporate the biomechanics of mating. Test specific strains of each species in a flight-like habitat. Include some relevant environmental stressors (noise, thermal spikes, lighting) and evaluate breeding success.
Detrimental stress effects on the gestating offspring seem likely for both rats and mice. Disrupted onset and initiation of maternal behavior patterns is a major risk (unaddressed by Neurolab experiences), although maternal behavior was intact in late-pregnant dams exposed to spaceflight, then returned to Earth just prior to birth. If there is an absence of nesting material, then we must understand implications of depriving dam of nest building experience as part of maternal behavior and, importantly, the thermal consequences of no nest insulation!
It is vital to collect data on thermal requirements of both rats and mice in absence of nesting material. Collect quantitative and qualitative data on mouse parturition in selected strains.
For the dam—the energetic demands of lactation are considerable, so there must be an adequate balance of energy intake and output (including that for milk production). Adequate diet (enriched in most labs) is essential, as well as feeders capable of delivering more food than is reasonable to expect. For the first 12-14 days postpartum, the dam approaches a unified group of pups. This is important because all nipples acquire milk at once per letdown and thus all pups should be attached to get their fair share. Neurolab revealed the difficulties of maintaining litter integrity. For pups—ready access to dam from Day 14 to 28, as well as appropriate presentation of food for weaning.
Collect data on energetics of lactation cycle and pup growth under flight-like thermal conditions. Anticipate food presentation and quantities appropriate for dams and for weanlings.
Two additional presentations were made. Mike Hines presented the current rodent habitat designs for transport to the ISS on the Space X Dragon, the Animal Enclosure Module–Transfer (AEM-T) and housing on the ISS (AEM-X). Cecelia Wigley presented ISS Mission Characteristics including launch, concept of operations, and Recovery. These presentations can be viewed at URL (
The workshop concluded with a plan for achieving lifespan and multigenerational rodent studies on the ISS. Spaceflight research offers unique insights into the role(s) of gravitational forces omnipresent on Earth, but absent in orbital flight. These forces may actively shape genomes in ways that are heritable. The 2011 NRC Report determined that studies of structural and functional changes induced by exposure to space during development and the transmission across generations of such effects are a major priority for Reproduction and Developmental Biology research in space promising fundamental new knowledge about how genetic and epigenetic factors interact with the environment to shape gravity-dependent processes, other changes induced by the space environment, and their penetrating influence(s) across generations.
The Roadmap to Multiple Generations in Space calls for a staged approach with defined milestones involving ground-based and spaceflight efforts to address habitat development and enabling science gaps in the specific areas of: (1) Breeding, (2) Birth through Weaning, then (3) Multiple Generations (Figure 2). Ideally, singular flights will verify that breeding, birthing, and weaning occur successfully before multiple generations are attempted. At each step along the roadmap, cross-disciplinary translational science will be possible and encouraged.
The Mark III Rodent Habitat Workshop assembled a diverse range of science, engineering, and program experts to identify necessary requirements and specifications for a flight habitat to increase the probability of successful mating, birth, and development of rodents in space. Detailed discussions of existing knowledge, research gaps, risks, and risk mitigation at key reproductive and developmental phases, and comparison of rats versus mice enabled the group to reach consensus on the development of top-level science requirements and an expansion of the ISS Rodent Research Science Requirements Envelope Document (SRED) (URL: Pending) to include detailed requirements for Reproductive and Developmental Biology.
The recommendations set forth in the 2011 National Academy of Sciences Report,
Concerns, Risks, and Knowledge Gaps from the Workshop Discussion on Breeding. Birthing, and Postnatal Development in Space
Subcategory | Basic Science (S) / Hardware (H) / Enabling Science (ES) | Risk (R) / Concern (C) / Gap (G) | Concerns, Risks, & Knowledge Gaps | Risk Mitigation Approaches |
---|---|---|---|---|
Cross-cutting issues | S | G, R | What are the changes in endocrine status, including HPG axis and prolactin during spaceflight? | |
S | G, R | Are there fertilization issues during spaceflight? | Artificial Insemination | |
S | G | Does the elevated radiation level affect reproduction or fetal/neonatal development? | Localized dosimetry at or near (or that closely resembles the shielding characteristics of) the habitat and any exposure from DXA machine. | |
H, ES | C, G | Information is needed to enable the understanding of behavior and of experiment failures. | Video and monitoring of key parameters | |
H, ES | C, G | Does the environment (i.e., air circulation, noise) within the habitat impede acoustic and olfactory communication between neonates and dam? | Define and measure indices of survivability and thrivability | |
H | C, G | Required crew access to animals should inform cage design and procedures (science, safety, etc), see SRED. | ||
H | C | Cage design will play an important role in promoting or inhibiting success of all categories of reproduction and development. | Wall surface texture and grip; dimensions of birthing space; space for the young to huddle and suckle; temperature control | |
ES | C, G | Does the current diet present any nutritional deficiencies impeding aspects of reproduction? | Pair feeding. Measure food and water intake. | |
ES | C, G | What are the enrichment requirements for Breeders? | ||
ES | G | What is the acclimation period before harems are ready to mate? | Generate data from a Mark I experiment. | |
ES | C, G | How long have prior foodbar tests/studies been performed? Are there any foodbar inadequacy or stability issues with long-term use of the foodbar? | ||
ES | C | Most of the knowledge is from studies using rats. Issues can emerge from extrapolation to mice. | Continue using rats to plan validation flight study and to test new hypothesis. Replicate some of the earlier rat-studies with mice in a stepwise fashion. | |
Mating Behavior | S | G, R | Is copulation affected by space flight? | Selection of proven breeders or breeding pairs |
S | G, R | Is estrous cycling altered by space flight? | ||
H, ES | G, R | Are pre-mating behaviors and courtship affected by space flight or the habitat design? | Selection of proven breeders or breeding pairs | |
Lactation | S | G, R | What are the changes in endocrine status during spaceflight, including HPG axis and prolactin? | Observing through skin: milk bands (acquiring milk); tooth eruption. Gauging milk let-downs through video |
S | G, R | Is the quality and quantity of milk altered during space flight? | Pre- vs. during- flight comparison | |
S | G, R | Do changes in immune function (antibody status in milk; absorption) affect lactation? | ||
H, S | G, R | Are nursing behaviors of the dam and pup, including suckling and retrieving, altered during space flight? | Consider cage design to promote these behaviors. Use video to analyze behaviors. Also, apply a pre-flight candidate selection filter of the dam (define whether she is a gatherer, or not, and define the quality of her milk). | |
ES | C, G | Is the foodbar adequate for lactation? | ||
Pre-Fertilization | S | G, R | Are gonad and gametogenesis and their functions compromised during space flight? | |
S | G, R | Is the endocrine status, including HPG axis, altered by spaceflight environment? | ||
S | G, R | Are post-gonad gamete formation, function, and maturation compromised? | ||
Fertilization | S, ES | G | Does the current diet present any nutritional deficiencies impeding aspects of fertilization? | Pair feeding. Measure food and water intake. |
S | G, R | Are sperm-egg signaling and interactions compromised? | ||
S | G, R | Is pre-implantation development compromised in the dam? | Consider results of STS-131, -133, -135 for underlying mechanisms in mouse. | |
S | G, R | Is signaling in the fertilized egg compromised by mechanisms including altered gene regulation and DNA damage? | ||
Implantation | S | G, R | Does mating trigger proper signaling to prepare uterine epithelium for implantation? | |
S | G, R | Are adhesion and implantation gravity dependent? | ||
S | G, R | Are uterine epithelial health and stem cells compromised? | ||
Placentation | S | G, R | Does decreased connexin-43 levels affect placentation during spaceflight? | |
S | G, R | During spaceflight, does placentation depend on changes in vascular tone? | ||
S | G, R | Are the signals for placental formation intact? | ||
S | G, R | During spaceflight, does placentation depend on changes in the immune system? | ||
S | G, R | What are the statuses of prolactins and other pro-placentation endocrine signals during spaceflight? | ||
Organogenesis | S | G, R | Are organ formation, maturation, and function compromised during spaceflight? | |
S | G, R | Are the endocrine-driven fetal-development phases that define the sex of offspring altered during spaceflight? | ||
Birth (also see cross-cutting) | S | R | Will reduced strength in abdominal musculature affect birth? | Caesarean section |
S | C, R | Will altered uterine contraction strength affect birth? | Cage design - wall surface texture and grip; dimensions of birthing space | |
S | G, R | Does the likelihood of a successful birth depend on genetic strain of rat? | ||
H, S | G, R | Is the maternal care pattern at birth intact? | 1 hour of pup cooling - temperature regulation; Video and temperature monitoring; space for the young | |
ES | C | With regards to testosterone formation, the temperature exposure of male pups should be closely monitored and potentially regulated. | Include capability for thermography of the pups within the habitat. | |
Perinatal Development (PND 0-8) | H, S | G, R | Are the pups receiving milk in similar manner as ground controls? | Observing through skin: milk bands (acquiring milk); tooth eruption. Gauging milk-let downs through video |
H, ES | G | Is the amount or quality of sleep that the neonates receive affected by spaceflight or cage design? | Video | |
H, ES | C | In case of cannibalism, how to quantify the number of pups that were initially born? | Video. Count # that are born. Higher frequency of monitoring around key milestones. | |
ES | C | How will individual pups be identified for further monitoring? | Ink in footpad or tail tatoo/marking, toe-clipping. Crew access. ANG; coat color. | |
ES | C | Body mass shall be recorded 2x/week. | Repeated measurement of body mass is important during the first few weeks post-natal to assess growth. | |
Infant Development (PND 8-14) | S | G | Do changes in the development of ovaries and testes occur during spaceflight? | |
H, S | G, C | Is the amount or quality of sleep that the neonates receive affected by spaceflight or cage design? | Video | |
H, ES | C | Huddle formation needs to be considered during cage design, as it is an integral aspect of neonatal development. | Cage design (artificial nest) | |
H, ES | G | Does the relative humidity need to be regulated within a specific range for neonates? | ||
H, ES | G | Are there thermal-regulation requirement differences between pups and mother? | Cage design (compartments with unique thermal regulation). Thermography capability needed. | |
Pre-Weaning (PND 15-21) | S | G | Do changes in the development of ovaries and testes occur during spaceflight? | |
S | C | Milestone: Independent ingestion should occur around PND15 (Neurolab). | ||
Adolescence (PND 28-35) | ES | C, R | Consider the risk of impregnation of siblings at sexual maturity when establishing age to separate the litter. | Male-female separation Mice: approx. 21-35 days Rats: approx. 45-80 days |