Introduction
From an evolutionary point of view, the endothermic homeothermic thermoregulation in small altricial mammals, like rodents, develops at around one third to a half of the animal's adult size 8. By having a small hairless body, pups are constantly losing heat, having to manage a higher and unbalanced energetic challenge, relying on adult parents or nests to keep a high and stable body temperature. To overcome this challenge, they usually resort to heterothermic physiology. This way, during their development, pups can have a controlled reduction of their metabolism and body temperature, saving energy that is crucial for them to grow 8.
Although this heterothermic physiology is very likely to be employed by many species, until the present date, it has only been reported in 4 bird orders and 1 marsupial order. In these two cases, the hatchlings are poikilothermic at birth following a heterothermic thermoregulation afterwards, during cold exposure. By this, it is possible to infer that daily heterothermy is phylogenetically old and likely plesiomorphic. Besides birds and marsupials, daily heterothermy in placental mammals has only been described in two orders, Insectivora and Rodentia. However, juveniles from other orders, like bats, are known to enter a torpor state 9. Yet, no studies were published on detailed developmental sequences, identifying the transition between poikilothermy and heterothermy or endothermy. The main difference between the studied marsupials and the placentals is that, in the latter, poikilothermy at birth is followed by a homeothermic phase after which the endothermic thermoregulation is established, meaning that the ability to employ daily heterothermy appears later in life (i.e. poikilothermy–endothermy–heterothermy) 8 This suggests that in placentals, daily heterothermy is a trait that evolved secondarily after a homeothermic phase, in response to energetic challenges, probably due to climate change 5. As mammals and birds arose from different reptilian lineages, it is likely that endothermy emerged from two separate classes, and daily heterothermy seems to have evolved at least three times, given the differences in developmental sequence between marsupials and placentals 8.
On an ontogenetic point of view, a recent study compared pups (0-4 days) from four different rodent species, ranging in different heterothermy levels as adults 10. They tested the adaptation to cold during hypoxia conditions, concluding that heterotherms retain traits that are common to all newborn mammals 11, including high tolerance to body temperature decreases. Even as newborns, the heterotherms species were more tolerant of cold temperatures and O2 limitation than homeotherms12,13. Additionally, from the tested species, the rat was the one showing the greatest thermogenic response, followed by the facultative heterotherm species, and lastly by the hibernator (squirrel) with almost no response. These differences were coherent with the ones seen on the same adult species14, indicating that the adult phenotype characteristics are already present at birth. This was the first comparative study that attempted to test a link between pup and adult stages in different species10, concerning the heterothermy and endothermy physiologies, studying some environmental parameters (temperature and O2 levels). Nonetheless, the question of when (ontogenetically) is the homeothermic stage achieved and how (which genes trigger it), still remains unanswered. For this project we will test whether the endothermic regulation starts around weaning age. Specifically, we aim to answer the following questions: 1) is endothermy developed gradually, rather than abruptly?; 2) is endothermy a pleiotropic trait, where previously studied metabolic control and temperature-related genes take part?; and 3) is a high level of gene expression after weaning indicative of emergence of endothermy?. We will use an inbred Mus musculus strain (C57BL/6J) in a laboratory setting as a model for a facultative heterotherm species15 as adults. To test our main hypothesis and answer our additional experimental questions, we will reproduce the laboratory mice in-house. Firstly, we will measure and study the relation between acute and short-term surface (Ts) and internal (Tb) body temperature loss in hairless pups temporarily within the litter (0 and 3min – pup mass) and separated (6min) from and, during their active (night) and inactive (day) periods; and monitor Ts and Tb body temperature in juveniles. To account for the effect of nesting material in insulation and thermoregulation, we will remove it for 60 min prior to measurements, in half of the cages (assigned randomly), from P15 to P40. To identify the ontogenic onset of endothermy, two tissue samples will be taken before weaning and two afterwards, in spaced time-points (at P4, P15, P23 and P40), namely brain (hippocampus and amygdala), and liver, which are markedly related with thermoregulation control and thermogenesis 16–19, that will be posteriorly sequenced (RNAseq) in order to assess their gene expression.
Type of research
Confirmatory
Hypothesis of your study
Main hypothesis being tested: Endothermic regulation starts around weaning age Secondary hypotheses: 1. Endothermy develops gradually, rather than abruptly 2. Endothermy is a pleiotropic trait, where previously studied metabolic control and temperature-related genes take part. 3. High level of gene expression after weaning indicates the emergence of endothermy
Study design
Experimental unit: litter
Timeline:
P0 - pups birth
from P0 to P40 - Daily TS and TB measurements of the pups (0, 3 and 6 min)
P4 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling; P15 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling; P21 - separation of the males and females in two cages by sex; pups weaning
P21-P40 (every 2 days: P22, P24, P28, P30, P32, P34, P36, P38) - random assign of "control" and " experimental" groups to either the male or female cage for the nesting inhibition experiment (30min-1h)
P23 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling;
P40 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling;
Method of blinding
The researcher analyzing the data will not be aware of the experimental groups. Allocation concealment for data collection will not be possible, but data extraction will rely on technology (pit tag readers and thermal cameras) which output that cannot be interfered with by the researcher. Performance bias will be addressed by randomization of order by which each litter and each pup within each litter is assessed.
Method of randomization
Software assisted (Excel RAND() function) randomization of treatments to each litter (the order to perform each method/procedure); to each pup of each litter - each pup will have an assigned number that will be randomized; to the cage position in the rack - the cages are also going to be identified with labels and randomized each time.
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