| Cross-cutting issues | S | G, R | What are the changes in endocrine status, including HPG axis and prolactin during spaceflight? |
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| S | G, R | Are there fertilization issues during spaceflight? | Artificial Insemination |
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| 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. |
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| H, ES | C, G | Information is needed to enable the understanding of behavior and of experiment failures. | Video and monitoring of key parameters |
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| 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 |
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| H | C, G | Required crew access to animals should inform cage design and procedures (science, safety, etc), see SRED. |
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| 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 |
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| ES | C, G | Does the current diet present any nutritional deficiencies impeding aspects of reproduction? | Pair feeding. Measure food and water intake. |
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| ES | C, G | What are the enrichment requirements for Breeders? |
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| ES | G | What is the acclimation period before harems are ready to mate? | Generate data from a Mark I experiment. |
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| 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? |
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| 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 |
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| S | G, R | Is estrous cycling altered by space flight? |
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| 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 |
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| S | G, R | Is the quality and quantity of milk altered during space flight? | Pre- vs. during- flight comparison |
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| S | G, R | Do changes in immune function (antibody status in milk; absorption) affect lactation? |
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| 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). |
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| ES | C, G | Is the foodbar adequate for lactation? |
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| Pre-Fertilization | S | G, R | Are gonad and gametogenesis and their functions compromised during space flight? |
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| S | G, R | Is the endocrine status, including HPG axis, altered by spaceflight environment? |
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| S | G, R | Are post-gonad gamete formation, function, and maturation compromised? |
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| Fertilization | S, ES | G | Does the current diet present any nutritional deficiencies impeding aspects of fertilization? | Pair feeding. Measure food and water intake. |
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| S | G, R | Are sperm-egg signaling and interactions compromised? |
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| S | G, R | Is pre-implantation development compromised in the dam? | Consider results of STS-131, -133, -135 for underlying mechanisms in mouse. |
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| S | G, R | Is signaling in the fertilized egg compromised by mechanisms including altered gene regulation and DNA damage? |
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| Implantation | S | G, R | Does mating trigger proper signaling to prepare uterine epithelium for implantation? |
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| S | G, R | Are adhesion and implantation gravity dependent? |
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| S | G, R | Are uterine epithelial health and stem cells compromised? |
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| Placentation | S | G, R | Does decreased connexin-43 levels affect placentation during spaceflight? |
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| S | G, R | During spaceflight, does placentation depend on changes in vascular tone? |
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| S | G, R | Are the signals for placental formation intact? |
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| S | G, R | During spaceflight, does placentation depend on changes in the immune system? |
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| S | G, R | What are the statuses of prolactins and other pro-placentation endocrine signals during spaceflight? |
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| Organogenesis | S | G, R | Are organ formation, maturation, and function compromised during spaceflight? |
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| S | G, R | Are the endocrine-driven fetal-development phases that define the sex of offspring altered during spaceflight? |
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| Birth (also see cross-cutting) | S | R | Will reduced strength in abdominal musculature affect birth? | Caesarean section |
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| S | C, R | Will altered uterine contraction strength affect birth? | Cage design - wall surface texture and grip; dimensions of birthing space |
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| S | G, R | Does the likelihood of a successful birth depend on genetic strain of rat? |
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| 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 |
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| 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 |
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| H, ES | G | Is the amount or quality of sleep that the neonates receive affected by spaceflight or cage design? | Video |
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| 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. |
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| 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. |
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| 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? |
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| H, S | G, C | Is the amount or quality of sleep that the neonates receive affected by spaceflight or cage design? | Video |
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| 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) |
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| H, ES | G | Does the relative humidity need to be regulated within a specific range for neonates? |
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| 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? |
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| S | C | Milestone: Independent ingestion should occur around PND15 (Neurolab). |
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| 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 |
