Abstract
This study investigates the gut microbiota’s role in hibernation. It will involve euthermic 13-lined ground squirrels (Ictidomys tridecemlineatus) during summer when they are active and winter during hibernation season. We investigated this in aroused squirrels using cavity ring-down spectroscopy (CRDS), a technique that provides a real-time indication of fuel use by measuring the ratio of 13C to 12C in the exhaled CO2 to verify the role of the microbiota, all experiments will be performed on squirrels with antibodies and antibiotic-depleted gut microbiotas.
Intro
Many animals hibernate in order to survive extremely cold temperatures or in times where food is scarce; it is not fully known how hibernating animals survive months without food entering the body. Food deprivation challenges the gut microbiota, which relies heavily on host diet for metabolic substrates and the gastrointestinal tract, which is influenced by enteral nutrients and microbial activity. “Winter fasting in hibernators shifts the microbiota to favor taxa with the capacity to degrade and utilize host-derived substrates and disfavor taxa that prefer complex plant polysaccharides” (Carey, Hannah V.). Microbiota changes can aid with hibernation in the intestinal immune system. If we are able to understand the mechanisms by which the hibernating animal and its gut microbes and how they adapt during winter fasting will hopefully provide insight about the mechanisms in non-hibernating species like humans and how to be able to go through a long period of time without nutrients entering the body.
The recent explosion of work on the gut microbiota has revealed that microbes affect their animal hosts through the metabolites they produce. These metabolites, which vary depending on microbial species and the host’s nutritional state, enhance the host’s metabolic capabilities. (Wiebler JM, Kohl KD, Lee Jr) To prove these ideas, we will be comparing ABX treated squirrels and non-ABX treated squirrels. ABX is an antibiotic that is mixed in with the squirrel’s water, this wipes out the enzymes in the intestine. If there is a transporter that bring urea into the gut lumen and if there are enzymes that break urea, then the results will prove this.
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An article states “Amphibians exhibit a low-energy lifestyle, and thereby tolerate environmental circumstances that impose nutrient limitation. They would benefit from recouping the nitrogen in surplus urea” (Wiebler JM, Kohl KD). These animals lack internal urease which is an enzyme that is required to hydrolyze urea. However, many animals recycle nitrogen from urea through a symbiotic relationship with a specific gut bacteria that produces urease. (Malcolm F. Fuller, Peter J. Reeds) The idea of nitrogen recycling from urea would be helpful in amphibians that cumulate urea during times of activity and dormancy. If the microbiota can break urea then it could help hibernating animals, a study concluded that “approximately 80% of the hydrolyzed urea nitrogen is retained in amino acids within the body”. (Michael A. Singer)
Urea can be gathered by two main mechanisms: one is increasing urea synthesis (Balinsky, ’81) and the other is increasing urea retention. Urea retention is physically holding urea in your bladder, this is not as helpful because there is no chemical breakdown that could help in hibernation.
No animal can break down urea for energy but hibernating animals have this enzyme in their intestine. This allows us to hypothesize that the breakdown of urea is connected to hibernation in some form. The nitrogen comes from urea that enters the gut lumen by both secretion and diffusion and is broken down by urease, making extra nitrogen for microbial growth. Nitrogen can be used to make amino acids, into “microbial protein that can then be digested and absorbed and thus contribute to the amino acid supply of the organism” (Michael A. Singer). There has been increasing evidence that agrees with the idea of nitrogen metabolism being important for amino acid nutrition of non-ruminant species. (Michael A. Singer).
In UNS, host-generated urea enters the gut lumen by active or passive transport across the epithelial layer where urease, a gut bacteria, is hydrolyzed by bacteria ureases to CO2 and NH3 (Wiebler JM, Kohl KD, Lee Jr). Urea gets broken down to CO2 and NH3+ in the intestine. This product of CO2 portion of the CO2 produced is absorbed into the blood and expired in breath, released as gas, or go through fixation which can produce molecules such as acetate which is helpful to the body and can be taken up by the animal as energy. Not every molecule formed will become an energy source, other molecules can only be helpful to bacterial cells and other times the molecule will not be helpful at all to the animal.
Intestinal microbes degrade urea and uric acid to produce NH3+ which can either be utilized by gut microbes or it can be absorbed via MCT transporters. most of the absorbed NH3 is recycled back into urea in the liver, but If it absorbed my MCT transporters it may contribute to new protein synthesis (70, 102). The ability of gut microbiota to “recycle” urea nitrogen back into the host would represent a vital benefit of host-microbial co-metabolism to the hibernator. Ammonia (NH3) can be utilized as a nitrogen source to make proteins. These synthesized amino acids are then taken back to the host animal. Nitrogenous substance’s net movement is from the gut lumen and then into the body. (Andrew J., Rosendale, Jon P. Costanzo)
The rate of urea recycling depends on two factors: one is the rate of its diffusion and secretion of urea into the gut lumen, and the other is the rate of urea hydrolysis by enzymes. (Michael A. Singer) In a previous experiment, the lab found that summer concentrations of ambient nutrients were greatest. During the winter NO3− concentrations increased drastically, but all the “uptake rates fell to extremely low levels, and urea uptake rates were the lowest of those measured NH4+” (Layton, A.T. Bull. Math. Biol). A research article has found that hibernating/non-feeding frogs, whose gut lumen had around 33% less bacterial population, exceeded that of active/feeding frogs, this may be because of “reasoned effect of high urea on urease expression and/or remodelling of the microbial community” (Wiebler, Kohl, Lee Jr.) Urea hydrolysis in hibernating frogs gives evidence that we could expect the same to happen with our experiments with 13 lined ground squirrels.
13-lined ground squirrels scientifically known as Ictidomys Tridecemlineatus are placed in a sealed chamber connected to a cavity ring-down spectroscopy (CRDS) to sample breath every 20 min. The change in 13CO2/12CO2 ratio (δ13C) in exhaled breath reflects bacterial metabolism of the injected 13 Urea, 13C-labeled substrate if the compound is resistant to mammalian enzyme degradation but can be metabolized by bacteria. Comparison of breath δ13C responses to dietary 13C-labeled substrates with others that could be present in the gut lumen during winter fasting will provide insight into microbiome function over the seasons. To accomplish this, I will IP inject ground squirrels with 13C-labeled Urea and then quantify the microbiota’s degradation function in real time using cavity ring-down spectroscopy to monitor the 13CO2:12CO2 ratio (δ13C) of the squirrel’s exhaled CO2, where higher values indicate higher microbial function. Based on research from other labs and experiments, we assume that δ13C will be higher during summer compared to winter squirrels. Also, we assume that ABX treated animals would have a δ13C at around baseline and non-ABX will have a higher δ13C value because if there are no enzymes to break the urea, then no 13C will be released from the animal.
Methods
This study used 25 13-lined ground squirrels scientifically known as Ictidomys tridecemlineatus born in an enclosed laboratory research setting on the UW-Madison campus. The mothers of this sample were wild-caught around Madison, Wisconsin in May 2018. The members of the lab go out to fields when it is known that most female squirrels are pregnant. They catch female and male squirrels but later release the male squirrels back in the field and take the female squirrels back to lab assuming most of them will be pregnant. Pregnant squirrels were housed individually at Ta of 22°C room in 12 hours of light and 12 hours of darkness. “Water and rodent chow” (Harlan Tekland) were given with no restriction; diets were supplemented with apples and sunflower seeds once per week. All squirrels gave birth in late May. Pups stayed with their mothers for 5 weeks following delivery and were then moved to individual cages called hibernacula. Following 2 weeks of ad libitum chow and fruit, the pups were then restricted to 12 grams of chow per day and supplemented with 1 g of sunflower seeds per week to stop extreme increase in weight. The consistent diet among squirrels was important because diet can significantly impact the molar ratio of 13C to 12C in exhaled CO2 (32, 47). Squirrel pups were held under these conditions until the beginning of the hibernation season.
In mid-September, hibernacula were moved to a 4°C room in the UW-Madison Biotron, a building located adjacent to the research laboratory. The cold room was held in darkness except for about 5 min of dim light every day to be able to enter and do activity checks on the squirrels. Food and water were then removed after squirrels began regular torpor arousal cycles, which was usually 24 to 72 hours after being moved into the cold room.
We are supposed to perform CRDS breath analyses (explained in the methods) on individual ground squirrels during summer (mid-July to early-September 2018), winter (late-December 2018 to late-February 2019; n=21), and spring (early-March to early-April 2018; n=13) seasons. During my time in the lab I was only able to perform the CRDS breath samples for summer (August and September; 2018) and Early Winter (early to mid November; 2018).
The Cavity ring-down spectroscopy also known as the CRDS machine is an instrument that gives us reliable δ13C measurements in the squirrel’s breath. Air flow rate through the box was regulated by a vacuum pump to maintain a CO2 concentration between 0.5% and 1.2%, the necessary concentration to make reliable δ13C measurements. Every 15 to 20 minutes an air sample from the box, which included CO2 expelled from the squirrel’s breath and from the anus, was automatically inserted into the CRDS unit for isotopic analysis. The δ13C (13CO2:12CO2) was calculated using the equation pasted below. where δ13C is expressed as parts per mil (‰).
For summer and winter squirrels used in the seasonal measurements, δ13C readings commenced around 30 min after transfer to the Plexiglas box; for winter squirrels, readings commenced around 3.5 h after transfer to the box to allow arousal from torpor.
Arousal process
During the summer, squirrels are awake and active but during the “early winter” squirrels, they are in hibernation and therefore there is a slightly different procedure that is done. On the day of an experiment, a squirrel was aroused from torpor by moving it from the cold room (Ta=4°C) to the laboratory (Ta=22°C). Squirrels were only aroused for an experiment if they were more than 75% of the length of their current torpor bout, which we estimated based on the daily activity records.
This is to try to get the animal’s arousal as natural as possible. 75% allows us to make sure the animal is still in hibernation but is around the time it will be naturally arousing. The daily activity record is a record where one member in the lab checks of the squirrels every day. There are small circular physical markers that is kept on top of the ground squirrel. When it is under torpor, the squirrel does not move and therefore the markers are still on the squirrel. If the markers have fallen off the squirrel, we assume it has aroused from its torpor bout and therefore this is how we measure how long an animal has been in torpor and when it arouses. The University of Wisconsin-Madison Institutional Animal Care and Use Committee approved of all procedures.
Statistical Analysis:
I used a two sample ANOVA test to compare the mean δ 13C values between summer squirrels and early winter ground squirrels and the mean δ 13C value between ABX and non-ABX treated squirrels. We used a total of 25 squirrels in the data, few of these squirrels were complete outliers this could be due to health or going into hibernation too soon or too late, one also died during hibernation, all of these situations were removed from the overall mean data. We still managed to have between 4-6 squirrels in each category; for summer ABX we had 5 squirrels, winter ABX 4 squirrels, Summer Non-Abx 5 squirrels and winter non-Abx 4 squirrels. We are comparing the two seasons (summer and early winter) and comparing ABX and non-Abx for summer and early winter.
Results
The p-values both of these comparisons were < 0.05. This means that ABX and non-Abx were statistically different and therefore the treatment does make a difference in the δ 13C value. The results also concluded that summer non-Abx and early winter non-Abx is statistically significant, this means that there is a meaningful difference between δ 13C. There is no statistically significant conclusion we can make between the Labeled Urea and the un-labeled urea since the sample size for un-labeled urea was 2 to 3, this is not a large enough sample size to be able to make a strong conclusion. The mean δ13C value in the breath of early winter squirrels (aroused from torpor and at Tb around 37°C) was more negative than those for summer squirrels. Within seasons, δ13C values did change significantly.
Discussion
The purpose of the experiments was to determine if urea enters the intestinal lumen and if it is hydrolyzed by the urease enzyme. We assume that hydrolyzing urea by the gut microbes, generates ammonia as a nitrogen source which is helpful to hibernating animals since Ammonia can be used to make amino acid which is protein that could be helpful for the animals to survive long periods of not consuming foods. Based on the results of this experiment, we found significant results that supported our hypothesis. Our hypothesis was that during winter, 13C would decrease and ABX treated ground squirrels would decrease in 13C value. Our baseline measurements were around -22 δ 13C values. This means there is more C12 CO2 being exhaled or gassed out than 13C CO2. ABX treated squirrels were around baseline measurements because the antibiotic wipes out the bacteria and enzymes in the intestine, therefore there are no enzymes to break the urea that enters the gut intestine. This means the urea is not broken down, therefore the δ 13C values would be the baseline measurement (around -22 δ 13C).
When urea is broken down the CO2 can do potentially do three things. One in is diffused back into the body and exhaled, second is that the animal could release the CO2 through gas (flatulence), since the squirrel is in a box, the CRDS machine takes both ways into account. The third option the CO2 molecule can do is be used up by the squirrel itself. The CRDS machine cannot measure this value therefore there are limitations and the true value is not known unless we open the squirrel up and use a NMT machine to look at all the chemicals in a certain tissue like the liver, kidney. This will tell us how much 13C and N15 gets taken up into the tissues. The NMT machine tells us what chemicals and how much of each chemical is in a piece of tissue. This can help us for future research because if we notice an abundance of a certain enzyme in the winter compared to the summer this could aid the animal in hibernation. The product NH3+ can be absorbed via MCT transporters. There is a lot to learn about MCT transporters, this is assumed to aid hibernation.
A preliminary report examined the extent to which UNS contributes to protein synthesis during hibernation in Wyoming ground squirrels (Urocitellus elegans). Two weeks after injecting fed or fasted summer squirrels or hibernating squirrels with 15N-urea, tissues were harvested to determine the change in 15N enrichment in small intestine, liver, and skeletal muscle. Tissues from hibernating squirrels (which continued torpor-arousal cycles after 15N-urea injection) exhibited the greatest 15N enrichment of the three groups. In other ground squirrel species, expression of urea cycle enzymes is reduced during hibernation relative to summer, which supports routing of bacterially derived urea nitrogen to amino acid rather than urea synthesis. More research is needed to confirm these intriguing results and characterize the microbial contribution to UNS in hibernating animals.
Many articles claim that the UTB transporter that transports urea into the gum lumen, have different amounts during summer and winter. This could affect the amount of urea that enters the lumen and therefore giving us different C13 values. The exact causes of seasonal variation in urea transporter abundance is something we can base our future experiments on.
