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Assessing the Upper Critical Limit of theThermoneutral Zone in Laboratory MiceTeumbo Ngunte

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ASSESSING THE UPPER CRITICAL LIMIT OF THE THERMONEUTRAL ZONE IN LABORATORY MICE

By

Teumbo Ngunte

A THESIS Submitted in Partial Fulfilment of the

Requirements for the Degree of Master of Sciences

(in Zoology)

The Graduate School

The University of Maine

December 2018

Advisory committee

Danielle Levesque, Assistant Professor of Mammalogy and Mammalian Health, Advisor

Kristy Townsend, Assistant Professor of Neurobiology

Leonard J. Kass, Associate Professor of Biological sciences

ii

Copyright 2018 Teumbo Ngunte

ASSESSING THE UPPER CRITICAL LIMIT OF THE THERMONEUTRAL ZONE IN LABORATORY MICE

By Teumbo Ngunte

Thesis Advisor: Dr. Danielle Levesque

An Abstract of the Thesis Presented

In Partial Fulfillment of the Requirement for the

Degree of Master of Science

(in Zoology)

December 2018

Endothermic organisms such as mammals and birds rely on high energy budgets to regulate body

temperature. Many studies have previously investigated the thermo-regulation of mammals under the

lower critical temperature of the thermoneutral zone, yet our knowledge in determining the upper

critical limits is still scarce. As an endotherm, lab mice (Mus musculus) are perfect models to determine

the cost to maintain constant body temperature as ambient temperature increases. The upper critical

temperature of the thermoneutral zone of this species has been estimated to be above 32°C and below

34°C. By utilizing different genetic backgrounds of lab mice in this study will help us to understand how

genetic variation may impact thermoregulation. My research, questions were: 1) Is the upper critical

limit similar for all mice, regardless of genetic background? And 2) Do resting metabolic rate (RMR),

body temperature (Tb) and evaporative water loss (EWL) increase similarly all mice as ambient

temperature increases? To investigate the upper critical temperature between the standard inbred lab

strain C57BL6/JUM (N =12) and mice with a mixed genetic background (N=8), I used flow through

respirometry which measured VCO2, evaporative water loss and body temperature over a range of

ambient temperatures from 25-38°C. Body mass was measured on a 0.1g scale and there was a

significant difference in body mass between both strains of mice, which is well documented. Break

points in resting metabolic rate and body temperature were higher C57BL6/JUM mice compared to the

mixed strain. On the other hand, evaporative water loss was higher in the mixed strain. The results were

not statistically significant since I had a low degree of freedom due to exclusion of data points based on

activity of the mice, which confounded data collection in those samples and the goal was to determine

the resting metabolic rate, not active metabolic rate. However, my results do indicate differences in

RMR, Tb and EWL due to genetic differences. This information could be important for understanding the

most efficient way mammals spend their metabolic energy to maintain a tightly balanced energy

budget. Future work needs to be done to understand what causes these variations in upper critical

limits.

iii

ACKNOWLEDGEMENT

This study was financed by USDA National institute of food and Agriculture. Hatch project number 21623

through the Maine Agricultural & Forest Experiment Station and teaching assistantships from the school

of Biology and Ecology at the University of Maine.

Many Thanks and Gratitude to Dr. Danielle Levesque for her supervision over my course of study and

concern about my wellbeing throughout this study. Gratitude to Vanessa Hensley for being an

awesome lab colleague helping me whenever I had some trouble-shooting with my research. The

Townsend’s lab are thanked for providing the mice that was used in this study and also teaching me how

handle laboratory lab mice.

Big Thank you to J.D. Janna Gau, Dr. Faharad Dastoor, Dr. Danielle Levesque, Dr. Mary Tyler, Dr

Michelle Goody, Robert Kirk, M.s Beverly Antonitis, Mr.&Mrs. Jim and Carol Toner, Robert Kirk, Graham

Morehead, Mrs. Sarah Joughin and Orlina Boteve. I thank them for their Help and support to reinstate

me back into the University after when I was suspended due to Racial profiling and discrimination by the

Orono Police department assisted by Mr. David Fiacco, Director of Community Standard, Rights and

Responsibilities. Without their help, I could never finished this thesis.

Thanks to my Big brother and Mentor Dr. Gaston Tolo’o for his support and day to day advice he gave

me. A big thanks to my father Mr. Ngunte for all his support, sacrifices and love.

Without forgetting God and my ancestors a big thanks for their protection and guidance throughout my

life.

iv

TABLE OF CONTENTS

ACKNOWLEDGEMENT …………………………………………………………………………………………………………(iii)

TABLE OF CONTENT……………………………………………………………………………………………………………..(iv)

LIST OF TABLES…………………………………………………………………………………………………………………….(v)

LIST OF FIGURES ………………………………………………………………………………………………………………….(vi)

LIST OF ABBREVIATIONS………………………………………………………………………………………………………(viii)

CHAPTER 1

INTRODUCTION ……………………………………………………………………………………………………………………1

CHAPTER 2

METHODS………………………………………………………………………………………………………………………………7

ANIMAL OF STUDY AND SURGICAL PROCEDURES…………………………………………………………………..7

AMBIENT TEMPERATURE AND BODY TEMPERATURE MEASUREMENT …………………………………..8

GAS EXCHANGE MEASUREMENT…………………………………………………………………………………………..10

CHAPTER 3

DATA ANALYSIS.……………………………………………………………………………………………………………………12

CHAPTER 4

RESULTS ………………………………………………………………………………………………………………….……………16

CHAPTER 5

DISCUSSIONS………………………………………………………………………………………………………………………...20

FUTURE DIRECTIONS AND CONCLUSIONS………………………………………………………………………………23

REFERENCES ………………………………………………………………………………………………………………………….24

APPENDIX ……………………………………………………………………………………………………………………………..26

AUTHOR’S BIOGRAPHY………………………………………………………………………………………………………….29

v

List of Tables

Table 1. Data for Transgenic B6;129 Mixed genetic background and C57BL6 mice at different Ta Break

points Temperatures are in parenthesis indicates break points temperatures for Resting metabolic rate,

body temperature and evaporative water loss ……………………………………………………………………………………..18

Table 2. Data for C57BL6 and Transgenic B6;129 mixed genetic background mice at high ambient

temperatures (37 – 38°C). Mean and standard deviation for resting metabolic rate, body temperature

and Evaporative water loss………..………………………………………………………………………………………………………….19

Appendix 1. All data point for both active and non-active mice…………………………………………………………….26

vi

LIST OF FIGURES

Fig. 1. Heat balance diagram showing the schematic relationship between metabolic heat production

(MHP), evaporative heat loss (EHL), and body temperature (Tb) for an endothermic mammal. TNZ:

thermoneutral zone; Tlc: lower critical temperature; Tuc: upper critical temperature …………………………… 3

Figure. 2. Flow diagram of the experimental protocol. Starting at the far left, representing surgically

implant of pit tags to experiments conducted under respirometry to obtain data under different ambient

temperatures and finally to the far right at the end of each experiment mouse was euthanized ………… 9

Figure. 3. Representative sample trace used to determine the activity level of a mouse 6077069

(Appendix A) in the respirometry system used in these experiments. The flat baselines where when the

composition of the air flowing into the chamber where measured as a control. The higher values were

measurements of flowing out of the animal’s chamber. Note that the first trace that shows the highest

peaks show high CO2 levels indicating relatively higher activity level for the mouse. The second segment

shows fairly flat and low CO2 levels indicating a relatively non-active mouse ……………………………………… 13

Figure. 4A. A scatter plot of the relationship between CO2 standard deviation and ambient temperature

(°C) of all mice for both strains studied: the 129mix strain (N= 31: red open circles) and the BL6 (N=55:

black open circles) are shown. B. A scatter plot of the relationship between CO2 standard deviation and

ambient temperature (°C) of the mice considered at relative rest for both strains studied: the 129mix

strain (N= 13: red open circles) and the BL6 (N=36: black open circles) are shown………………………………14

Figure. 5a. Resting metabolic rate (RMR) in two different strain of mice over a range ambient

temperatures (Ta). 129 mix strain (N=13: red circles) and BL6 (N=36: black circles). Piecewise linear

regressions were used to estimate the break points in the relationship between RMR and Ta for both

strains. Break points for 129mix strain (solid vertical red line) and BL6 (black vertical line) are shown. B.

vii

Body temperature in (Tb) in two different strains of mice over a range of ambient temperature (Ta).

129mix strain (N=13: red circles) and BL6(N=36: black circles). The black diagonal line represents the

point where Tb = Ta . C. Evaporative water loss (EWL) in two different strains of mice over a range of

ambient temperature (Ta) 129mix strain and BL6…………………………………………………………………………………17

viii

LIST OF ABBREVATIONS

BMR BASAL METABOLIC RATE

EWL EVAPORATIVE WATER LOSS

RMR RESTING METABOLIC RATE

RQ RESPIRATORYQUOTIENT

Ta AMBIENT TEMPERATURE OF THE CHAMBER

Tb BODY TEMPERATURE

�̇�CO2 RATE OF CARBON DIOXIDE PRODUCED

�̇�O2 RATE OF OXYGEN CONSUMED

Mix 129 TRANSGENIC 129-MIXED GENETIC BACK GROUND MICE SRAIN

B6 C57BL6

1

CHAPTER 1

INTRODUCTION

Unlike ectotherms, mammals are endothermic and can regulate their body temperature physiologically

to maintain constant body temperature as environmental temperature changes (Angellitta et al. 2010).

This allows them to perform optimally over a range of environmental temperatures. Energetic cost to

maintain constant body temperature varies over the range of different environmental temperatures

experienced by these species over their lifetime (Hafez, 2017). The relation between metabolic heat

production in a resting animal and ambient temperatures in respirometry provides us a broader

understanding of energy requirements of a species and their ability to live and survive in different

climates (Gordon, 1993).

As climate changes, vulnerability of small mammals will vary across the globe. Mammals found in tropic

and artic regions will tolerate an increase in environmental temperatures differently. At 0°C, mammals

such as polar bears will be experiencing thermal stress since 0°C is consider high for these species

(Derocher, 1993). The relationship between metabolic heat production and environmental temperature

can give us a better understanding to predict the survival of mammals in different environments and

climates(Gordon, 2012). The thermo neutral zone (TNZ) a range of ambient temperatures within which

an endotherm is capable of maintaining its body temperature without increase in metabolic rate

(McNab 2012). TNZ is also defined as the range of ambient temperature at which temperature

regulation is achieved only by controlling sensible heat loss (IUPS, 2001). These definitions of (TNZ) can

occur without conflict only if metabolic rate increases above basal metabolic rate at the same

temperature that water loss increases. (Mitchell et al. 2018). Basal metabolic rate of an endotherm is an

important aspect in thermo-physiology which is known as the minimum sustained rate of energy

turnover of an endotherm, when the animal is tested under a range of ambient temperature

2

maintaining standardized laboratory conditions. This standardized state of an endotherm at basal

metabolic rate (BMR) includes: non-active, post absorptive, adult, non- reproductive and in a resting

phase of their circadian rhythms (White and Seymour 2003). It is very challenging to create a state

where an animal is in an absolute resting phase but not sleeping (Gordon, 1993). It is unlikely for any

mammal in its natural condition to be within these precise set of conditions necessary for measuring

BMR (McNab, 2015) For this reason, the resting metabolic rate is more commonly measured as

conditions when an animal is resting within its thermo neutral zone and allows for conditions to violate

other criteria for BMR (Speakman et al. 2003).

Below the TNZ, mammals have a lower critical temperature (LCT) which is the temperature at which

metabolic rate starts increasing above the BMR as ambient temperature decreases to bring back the

body temperature to normal. On the other hand, upper critical temperature of the TNZ, is the

temperature at which as ambient temperature increases, the rate of evaporative cooling or metabolic

rate increases to bring back body temperatures to normal (Gordon, 2012). The metabolic elevation at

ambient temperatures can easily be determined at the LCT (Gordo 1993), yet there are still some gaps in

determining the exact break point at which the resting metabolic rate elevate as ambient temperature

increases. As temperature increases, above normal ambient temperatures, small mammals use both

behavioral and physiological responses to regulate their body temperature and maintain homeostasis

(Gordon, 2010). Behavioral response under higher temperatures could be panting, increase in blood

flow through capillaries in the tail and grooming of saliva on the fur to be able dissipate heat easily to

the environment (Gordon, 2010). A newly developed technique has been used to access the upper

critical limit to determine the sensitivity to heat in a number of birds globally (Whitfield et al. 2015). This

technique includes measuring the amount of CO2 and EWL produced and measuring body temperature

over a range of ambient temperature. Birds are endothermic and regulate their body temperature

3

behaviorally and physiologically over a range of ambient temperatures. A similar technique was used in

my research to determine the upper limits in different strains of mice. Laboratory mice are

known to have small thermoneutral zones compared to species living in the artic with ambient

temperatures averaging to -25°C (Derocher, 1993). Species such as polar bears are very insulated to

withstand cold environmental conditions leaving them with a very low critical limits compared to species

in the tropics with higher Ta (Scholander et al., 1950). The estimated upper limit temperature for

increase in metabolic rate is 34°C for albino, male mice (Herrington, 1940).

Fig. 1. Heat balance diagram showing the schematic relationship between metabolic heat production

(MHP), evaporative heat loss (EHL), and body temperature (Tb) for an endothermic mammal. TNZ:

thermoneutral zone; Tlc: lower critical temperature; Tuc: upper critical temperature.

4

Currently, the core temperature measurements for metabolic phenotyping for a mouse can be done

three different ways(Meyer, Ootsuka, & Romanovsky, 2017). Firstly, Tb can be obtained by inserting a

probe into the mouse’s rectum, secondly by pre-implanting a probe in the abdominal cavity or

subcutaneous part and finally, by measuring surfaces by infrared thermography (Meyer et al., 2017). In

this research, I used a passive integrative transponder tag (Biothermo 13, Biomark , Biose ID) implanted

into the peritoneal cavity which enabled me to monitor the core temperature of an unstressed and

undisturbed animal (Gordon, 2012). Telemetry can help develop a better understanding of body

temperature measurement because lifting a mouse which has been telemetered by its tail and holding it

above the floor for about 5 seconds leads to an increase in heartrate to about 200 beats/min and may

last upwards of 10 minutes leading to a significant rise in core temperature that lasts close to an hour

(Meijer et al.2006). Radio-telemetric monitoring of mice when maintained at a fixed environmental

temperature for 48 hours revealed that ambient temperature for the thermoneutral zone is between 32

and 34°C (Gordon 2012). Mean core temperature was essentially the same at ambient temperatures of

25, 28 and 34°C. There is an abrupt rise in core temperature of about 1°C when ambient temperature is

maintained at 34°C (Gordon 2012). It is therefore reasonable to assume that the break point for upper

limit of thermoneutrality is above 32 and below 34°C.

Mammals and birds use evaporative water loss as an important mechanism for thermal balance

(Withers et al. 2016). This process is distributed into passive and active mechanisms (Gordon, 2012).

Passive evaporative water lost occurs through respiratory surfaces and diffusion across the skin. This

process increases in drier environments and at warmer ambient temperatures (Edward and Haines,

1978). Reducing evaporative water loss (EWL) has an important adaptive role for animals living in

extremely hot environments (Williams, 1996; Tieleman et al. 2003).

The ability to regulate gene expression in mice has turned mice, into a good model for biomedical

research (Karp, 2012). Despite millions of years of evolutionary divergence mice have a close related

5

genome and physiology with human and other endotherms (Speakman, 2013). The objective of my

study is to determine the upper critical temperature of the thermoneutral in two strains of laboratory

mice and determine if there are variations in upper critical limits between both strains. Comparing two

distinct strains of mice will provide an indication of the level of variability within a species. Body

temperature can impact other physiological functioning such as development, reproduction etc. Other

researchers that are not specialists on thermal physiology should be aware of the impact of

thermoregulation on their end results of interest (Gordon, 2012). C5BL/6JUM inbreed strain of mice are

most widely used in biomedical research and the first strain of mouse to have their entire genome

sequenced (Smith et al. 2018). Knowing the entire genome of B6 mice makes it easier to understand the

physiology of mice and relate it to other species with a similar genetic background. These mice are more

likely to be influenced by diet-induced obesity and also used to research many neurological disorders

which also deals with thermo physiology. Transgenic mice 129 mice (B6;129 mixed genetic background)

come from the combination of C5bL/6J, Balb/C and 129 genetic background. Mice with 129 strains used

in the ES cell line from which mutants are derived are known as the best control mice for targeted

mutation (Simpson et al. 1997; Threadgill et al 1997). B6;129 mix genetic F1 generations these mice are

less suitable for biological research than B6;129 mix genetic F2 generations due to different segregation

of the parental F1 hybrids (Simpson et al. 1997).

Based on Gordon’s findings (1985), the upper critical temperature for evaporative water loss for Balb/c

mice(albino laboratory mice) is between 32-34.5°C (Gordon 1985) Since BMR of laboratory mice is

estimated to be between 32-34°C, mouse such as Balb/C show significant rise in EWL ambient

temperature of 34.5°C in Balb/C mice(Gordon, 2012).

6

For this thesis, my question is, will the upper critical be the same for both mice? What mechanism will

both strains use more efficiently to regulate their body temperature back to normal? Will metabolic

rate, body temperature and evaporative water loss increases at the same rate in both strain of mice? I

measured resting metabolic rate, evaporative water loss and body temperatures to determine break

points in this parameter in 2 different strains of mice as ambient temperature increases. A similar

schematic approach as Withers et al (2016, Fig. 1) was used attain this objective.

I hypothesized that there will be variability in resting metabolic rate, evaporative water loss and body

temperature response as ambient temperature increase between C57Bl6/JUM and transgenic 129-

mixed genetic background. Energy cost of self-maintenance varies within species. Difference

morphology such as body mass can play a role on the different way heat gets dissipate as ambient

temperature increase and species uses the less costly and more favorable form of heat dissipation to

allocate more energy for other physiological response such as growth, strengthening the immune

system or reproduction.

7

CHAPTER 2

METHODS

ANIMAL OF STUDY AND SURGICAL PROCEDURE

Male C57BL6 mice ( 23.41 ± 1.64g, n= 7), male Transgenic B6; 129 mixed genic background (28.38 ±

2.02g, n=3), female C57BL6 mice (21.68±0.70g, n=5) and female 129 mix (26.68 ± 4.05g, n=5) were all

bred and genotyped by the Townsend lab in the Small Animal Research Facility at the University of

Maine. C57BL6/JUM (BL6) mice were mated at the University of Maine and transgenic 12mixed strain

mouse (129mix strain) were bred from both parent Jax # 004781 and Jax # 004339 by the Townsend lab.

Mice were place in cages which contained a maximum of 5 mice per cage. They were fed with mouse

diet 9F (mouse diet 9F, St. Louis, MO, USA) which is a complete life-cycle diet containing 9% of fat and

provided with water daily. Daily maintenance of cages where performed and housing temperatures

ranged from 20°C-26°C.

Before any surgery, surgical equipment was autoclaved for 45 minutes at a temperature of about 120°C.

The temperature sensitive PIT-tags (Biothermo 13, Biomark , Biose ID) were placed in 70% ethanol

solution for sterilization. I introduced each mouse into a surgical chamber containing 5% of Isoflurane in

0xygen. Once anesthesia was attained, and the mouse did not respond to stimuli, I transferred the

mouse to a mask from which 1.5% of isoflurane with oxygen was introduced throughout the rest of the

procedure. Surgical area was prepared by soaking a razor blade into betadine scrub. Cotton with

betadine was used to wet the surgical area on the abdomen of the mouse and with a razor I shaved the

surgical site of the mouse. Alcohol and betadine solutions were used to clean the stomach site post

shaving. Surgical drapes were placed over the animal with an opening over the shaved region of the

stomach. A small incision was made in the skin, and forceps were used to hold some muscle tissue

8

beneath the skin and with the help of a 12mm syringe containing a sterilized pit-tag, I could introduce

the pit-tag successfully into the peritoneal cavity of the mouse. Surgical glue was used to close the

incision. Post-surgery, the animal was placed on their back in clean cages lining on clean towels to

prevent the surgical glue from sticking to the towel. Each mouse was kept under normal housing

temperature and transferred into clean cages lining with Beta Hardwood chip (Northeastern Products

Corp, New York, NY) Once the mice completely arose from anesthesia, food and water was provided.

The animal where given at least a week to recover before the experimental trail began under

respirometry (Leon et al. 2004).

AMBIENT AND BODY TEMPERATURE MEASUREMENT

Within the respirometry chamber, Ta was set to experimental temperature using a PELT-5 temperature

controller (Sable Systems, Las Vegas, NV). 30°C,34°C,36°C and 38°C. These Ta could be measured in the

respirometer using a thermocouple couple probe (TC-2000 Thermocouple Meter) which was inserted on

one side of the plastic chamber through a hole. I recorded body temperature each second from the pit

tags throughout the experiments using a Biomark HPRreader (Biomark, Biose ,ID).

Experimental trials were done during the day, the mouse’s rest phase. Each mouse body mass was

recorded before placing the mouse into the gaseous exchange chamber. Experiments started at lower

temperatures, then I proceeded with higher Ta the following day. Each mouse was exposed at list to two

lower Ta (25°C, 30°C and 34°C) and two higher temperatures (36°C-38°C) Ta over a two days period. Mice

were placed in respirometry chamber for two hours per Ta except at 38°C where the mouse was left at

that Ta for an hour. This was because at Ta 38°C mouse showed behavioral signs of heat stress. I was able

to monitor the activity and behavior of mice with the aid of the video camera placed inside the cooler

box. In case of heat stress indicators such as agitation, loss of coordination, body temperature greater

9

than 41°C, or mouse lying on their backs, the experiment was terminated and the mouse is removed

from the cage and placed in a cooler cage.

Fig. 2. Flow diagram of the experimental protocol. Starting at the far left, representing surgically implant of pit tags to experiments conducted under respirometry to obtain data under different ambient temperatures and finally to the far right at the end of each experiment mouse was euthanized.

Surgical procedure For P-tag

implantation in mouse 607701

Was performed on 05/01/2018

Body mass recorded on

o5/08/2018 before starting

respirometry measurements

Respirometry recordings starts

under lower ambient temperatures of 25

and 30°C on the 05/08/2018for 2

hours per Ta. Temp set at 25 °C baseline 10mins then switch

to chamber recording for 50mins. This is

done twice. Temp set to 30°C recordings

are done the same as at 25 °C

Recordings under high Ta of 34,36 and 38°C on 05/09/2018

were done similarly as

lower temperature

except the duration under 38°C lasted for

an hour

Body mass recorded

and mouse euthanized

under excess CO2

10

GAS EXCHANGE MEASUREMENTS

Using an open flow respirometry system, oxygen consumption (VO2), carbon dioxide production (VCO2)

and evaporative water loss (EWL) were measured over a range of Ta. mouse was placed in a plastic

airtight container which had a volume of 2.04 liters. The plastic chamber(respirometer) was placed

inside an insulated foam box containing a temperature generator at the top. The respirometer had a

metal grate at the bottom which elevated the mouse and separated the mouse from approximately 1

cm of mineral oil that was placed at the bottom of the chamber to prevent evaporation of any urine or

feces produced by the mouse during measurement periods. Prior to every experiment, the mouse was

fasted for an hour before any respiratory measurement was made.

I recorded the chamber on empty for an hour to provide baseline values for CO2 and H2O. Ta setpoint

was controlled using the PELT-5 temperature controller to achieve the appropriate temp set for each

measurement.

Atmospheric air was obtained by a pump (KNF type MPU3381-NMP830) and dried in a column of

Drierite (WH Hammond Drierite Co.). A flow measurement system (FB-8 Sable Systems, Las Vegas, NV)

determined the amount of air going through the respirometry system (incurrent air). Air streams were

divided by a mass/flow control system to divide incurrent air streams into the baseline and the chamber.

Baseline carbon dioxide measurement was less than 500ppm. Flow rate (400ml/min) was chosen to

maintain chamber humidity at low levels. Subsample excurrent air from the chamber and baseline was

measured every 50 mins. Meaning after every 50mins chamber measurements, the multiplexer (RM8

sable systems, Las Vegas, NV, USA) switches to baseline for 10mins this is a control measure to make

sure air is flowing through the chamber and this was done using a RM-8 flow multiplexer. Air leaving the

chamber was pulled into a CO2 and water analyzer (LI-COR model LI-840A CO2/H2O analyzer Lincoln, NE,

USA). Excurrent air had to pass through columns of drierite connected in series to absorb any excess

11

water or moisture before entering the Licor. Thermocouple data and voltage output from the analyzer

were digitized by using an analog-digital converter (UI3- universal interference, Sable, System, Las

Vegas, NV) and recording was done every second using Expedata software (Sable System, Las Vegas, NV,

USA). The RMR for each ambient temperature consist of the lowest 10minutes section of the 2 hours

experiment. This was determined by averaging the lowest 10mins for VCO2 and EWL in a 50 minutes

sequential recording.

12

CHAPTER 3

DATA ANALYSIS

To determine resting data for analysis, I obtained the lowest 10mins value in a 50 minutes recording

interval at each ambient temperature. Using equations from Withers (2001) to calculate �̇�CO2 I used

�̇�CO2 = (FeCO2 – FiCO2) * FR/(1-FeCO2 * (1-(1/RQ))) from �̇�CO2 I obtained �̇�O2 by using RQ=VCO2/VO2

considering RQ to be 0.8. I used the oxy caloric equivalent (6.0913*RQ+15.439J.molO2) to convert �̇�O2

from ml/min to J/s(W). To obtain values for EWL, I used VH2O= FRi(FeH2O-FiH2O)/(1-FeH2O) then I

converted water vapor pressure(mmol/l) into water vapor density (g/l) and , using the chamber flow

rates(in ml/hr) further converted water vapor density to EWL (g/hr)

To select RMR for each animal, I selected the lowest 10mins section for CO2 from each 50 minutes

measurement period as the RMR. �̇�CO2, �̇�O2 and EWL were calculated using equations modified from

Withers (2001). �̇�O2 was converted from ml/min into MHP in Watt (J/s) I used the oxycaloric

equivalence of oxygen calculated from RQ as in Withers (1992), (6.0913*RQ+15.439J.molO2). To

determine whether a mouse was active, I used the average of standard deviation of CO2 at 10minute

intervals over the 50 minutes recording under each ambient temperature from which respirometry

measurement was obtained and also based on observation made during the experiment with the

camera inside the respirometry chamber. Any movements such as the animal walking in the cage or

grooming of saliva on fur was considered active. Data points above 200 ml/mins CO2 standard deviation

were considered active and were excluded from the data analysis (Fig 3).

13

Fig. 3. Representative sample trace us ed to determine the activity level of a mouse 6077069 (Appendix A) in the respirometry system used in these experiments. The flat baselines where when the composition of the air flowing into the chamber where measured as a control. The higher values were measurements of flowing out of the animal’s chamber. Note that the first trace that shows the highest peaks show high CO2 levels indicating relatively higher activity level for the mouse. The second segment shows fairly flat and low CO2 levels indicating a relatively non-active mouse

0

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38 Ta°C

CO

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pm

)

Time(hh:mm:ss)

sample trace showing Activity and non active mouse

14

Fig. 4A. A scatter plot of the relationship between CO2 standard deviation and ambient temperatures

(°C) of all mice for both strains studied: the 129mix strain (N=31: red open circles) and the BL6 (N=55:

black open circles) are shown. B. A scatterplot of the relationship between CO2 standard deviation and

ambient temperature (°C) of the mice considered at relative rest for both strains studied the 129mix

strain (N= 13: red open circles) and the BL6 (N=36: black open circles) are shown.

To determine the breakpoint in the relationship between RMR and Ta for both strains of mice, I used

piecewise Linear regression with mass as a covariate to determine break point in the slope of RMR

against Ta which will identify the upper limit of the thermoneutral zone (p.425 Crawley 2007). For the

15

relationship between Tb and EWL against Ta, I used break point analysis implemented using the R

package ‘segmented’ (Muggeo 2008) with mass as covariate. I compared the means of RMR, EWL and

body temperature between the transgenic 129mix strain and BL6 mice. Using a 0.1g scale, Body mass

was also compared between both strains using two tail t-test in strain and sexes.

16

CHAPTER 4

RESULTS

I found that mice made up of different genetic backgrounds showed differences in break points between

RMR, Tb, and EWL against Ta. The break point for RMR against Ta for BL6 mice at 35.8°C, which was higher

than break point for 129 mix mice at 34.6 (Fig. 5A). This difference of about a 1°C may indicate a higher

physiological cost for the 129 mix strain mice to thermoregulate under higher ambient temperatures

relative to the BL6 mice. The break point for Tb against Ta for BL6 mice was at 33.8°C, which was higher

than break point for 129 mix mice that was indicated at 32.5°C (Fig. 5b). This shows BL6 save more

energy by increasing their body temperature. This difference of about a 1°C may indicate that the 129

mix strain mice are less able to maintain a constant temperature and to thermoregulate under higher

ambient temperatures relative to the BL6 mice. The break point for EWL against Ta for BL6 mice was at

36.0°C, which was lower than break point for 129 mix mice that was at 37.4°C (Fig. 5C). This difference

of about a 1°C may indicate that the 129 mix strain mice are less efficient in cooling themselves through

evaporative water loss relative to the BL6 mice. When considering all 3 of these parameters (RMR, Tb

and EWL) against Ta suggests that the 129 mix strain mice are less efficient thermoregulating at those

higher temperatures.

17

Fig. 5A. Resting metabolic rate (RMR) in two different strain of mice over a range ambient temperatures (Ta). 129 mix strain (N=13: red circles) and BL6 (N=36: black circles). Piecewise linear regressions were used to estimate the break points in the relationship between RMR and Ta for both strains. Break points for 129mix strain (solid vertical red line) and BL6 (black vertical line) are shown. B. Body temperature in (Tb) in two different strains of mice over a range of ambient temperature (Ta). 129mix strain (N=13: red circles) and BL6(N=36: black circles). The black diagonal line represents the point where Tb = Ta . C. Evaporative water loss (EWL) in two different strains of mice over a range of ambient temperature (Ta) 129mix strain and BL6.

A

B

C

18

Table 1. Data for Transgenic B6;129 Mixed genetic background and C57BL6 mice at different Ta Break

points (indicative of the TNZ)

Transgenic B6; 129 Mixed genetic Background

C57BL6

RMR (W) 0.20 (34.57°C) 0.27(35.8°C)

Tb (°C) 38.13 (32.5°C) 36.92(33.8°C)

EWL (mg-1h-1) 185.47 (37.4°C) 90.17(36.5°C) RMR, resting metabolic rate; Tb, Body Temperature; EWL, evaporative water loss; EHL/MPH ratio of evaporative

heat loss to evaporative heat production all data at various ambient temperature breakpoints. Breaks points are

the temperatures in parenthesis

They may be other differences between these mice strains. The mean and standard deviation of body

mass between the mice of both strain and sex were calculated. Male BL6 mice had an average body

mass of 23.41g (± 1.64g, n= 7), whereas male 129 mix strain had an average body mass of 28.38g (±

2.02g, n=3). That is a significant difference in body mass between both groups of males (t2,3=3.89,

p=0.030). Female BL6 mice had an average body mass of 21.68g (±0.70g, n=5), whereas female 129 mix

mice had an average body mass of 26.68g (± 4.05g, n=5). However, there is no significant difference in

body mass between these female groups (t2,8= -2.19, p=0.06). Comparing differences in body mases

between sexes revealed a significant difference between BL6 males and females (t2,10 = -2.44, p=0.035).

No significant differences in body mass was found between 129mix strain males and females (t2,6=0.84,

p=0.43).

I used piecewise linear regression with mass as a covariate to determine the break point where RMR

starts increasing as Ta increases. I also measured RMR, body temperature and evaporative water loss

under a higher ambient temperature range (37- 38°C). Both body temperature and EWL means where

higher in 129-mix mice compared to B6 mice but RMR was higher in B6 mice under higher ambient

temperatures.

19

Table 2. Data for C57BL6 and Transgenic B6;129 mixed genetic background mice at the highest

ambient temperatures (37 – 38°C) at which the two strains of mice were tested.

C57BL6 Mice Transgenic B6; 129 Mixed genetic background

N 3 3

Mass (g) 22.41±1.23 26.915±1.63

Tb (°C) 38.65±1.16 39.0±0.51

EWL (mg-1 h-1) 340.11±108.04 364.88±340

RMR (W) 0.229±0.13 0.226±0.12

Tb , Body Temperature; EWL, evaporative water loss; RMR, resting metabolic rate; mass in grams. All data are

mean±s.d at Ta from 35 to 36°C.

20

CHAPTER 5

DISCUSSION

My study focused on analyzing upper critical limits of the thermoneutral in two different strains of mice

(B6 and 129-mix mice). I found a difference in the upper limits between both strains in RMR, body

temperature and evaporative water loss. It is assumed that the thermoneutral zone of lab mice is above

32°C and below 34°C (Gordon, 2012). At temperatures above 34°C, both BL6 and mix-129 mixed mice

showed some elevation in RMR, Tb and EWL (Fig. 5.), which indicates signs of physiological feedbacks to

facilitate and maintain constant body temperature (Withers & Cooper, 2009). At Ta > 36°C, both strains

showed behavioral characteristics such as grooming of saliva on their fur and feet and elongating the

tail. At the end of the experiment, proliferation of blood in their capillaries in feet and tail of each strains

exposed to higher ambient temperatures was observed. This indicates other means for dissipating heat

(Gordon, 2012). At higher Ta around 38°C, increase in motor activity could be observed and Tb reached

39°C. Not once was Tb observed at 40°C or above. One BL6 female mouse was resting at a Ta set at 37.8°C

and displayed a body temperature of 39.6°C. However, it showed no sign of escape nor stress. Tb varies

between mammals of different taxa, from about 31 to 39°C (Lovegrove, 2012 a.b). Above the

thermoneutral zone, it is energetically costly for evaporative cooling to occur through panting and

sweating (Withers 2012). The interaction of different strains with RMR, body temperature, EWL and the

ratio of evaporative heat loss (EHL) to metabolic heat production (MHP) shapes their thermoneutral

zone. Above the upper limits, EWL loss increases to bring back the body temperature to normothermic

temperature. This process occurs when the species is found in the heat challenge region within their

thermoprofile. The break points of 129-mix where higher than for BL6 mice. The break points were

higher in BL6 compared to 129mix strain which indicates a higher TNZ in Tb and RMR in BL6 mice. On the

other hand, break point for EWL against Ta was higher in 129mix strain compared to BL6 mice. These

differences show a higher physiological cost for 129mix mouse to perform under higher Ta since they

21

have a smaller thermoneutral zone. Above the TNZ, it is energetically costly means for evaporative

cooling to occur which implies sweating or panting (Withers et al. 2016). This shows a BL6 mice uses a

more efficient and less expensive way to maintain body temperature low as ambient temperature

increases compared to 129 mix strain. Physiological response such as sweeting is less expensive

compared to panting which uses more energy. BL6 EWL starts at 36.0°C which shows how these mice

start sweeting at a 1°C earlier than 129mix strain 37.4°C using a more energy efficient way of dissipating

heat and reserving more energy for other physiological demand. BL6 mice under higher temperature of

between 37-38°C had a higher RMR but body temperature and evaporative water loss was lower

compared to 129-mixed mice. I could not test how significant the difference in means were based on a

small degree of freedom. The study was conducted on both BL6 and 129 mix mice with both sexes

analyzed together. Analysis of both sexes were not done separately due to a low sample size between

sexes and I recorded 76 data point over the course of this research (see appendix 1). Within these 76

data points, 31 set of data were considered active and 45 nonactive. Only the nonactive mice data were

analyzed. Male and female mice possess morphological and physiological differences. It is common to

find some differences when it comes to gender specific thermoregulations (Gordon, 2010).

Thermoregulatory processes can be influenced by secretions of sex hormones, such as androgens and

estrogens. Females undergoes noticeable changes in thermoregulatory sensitivity during pregnancy and

lactation (Gordon, 2010). Rodent body temperature is about 0.5°C higher during estrous cycle at night,

but it appears to be unchanged during the day and the rest of the reproductive cycle (Yochim and

Spencer, 1976; Yanasa, Tanaka and Nakayama, 1989). Correlations exist between behavior of individual

mice in respirometry and their personality (Careau et al., 2008). Mice are known as very active species.

It is very difficult to keep these species in a resting state for a long time, especially when using

respirometry that has a pump that generate noise. Some mice might have some restless personality and

never settle in the chamber. I identified that noise produced by the respirometer pump and other

22

equipment can be source of noise that keeps the mouse active during recordings. A better sound proof

apparatus can be helpful. Whereas, other species of mice may calm down relatively quickly. Body mass

accounts for about 95.9% of variance in RMR between species (McNab 2008) smaller mammals have

higher mass specific thermal conductance and heat loss as ambient temperature increases. Difference in

body mass accounts for the difference in RMR, Tb and EWL. Difference in genetic sub-strains might

contribute and expression in heat shock proteins might account on how mice response to heat stress or

determine their thermoneutral zone.

23

FUTURE DIRECTIONS AND CONCLUSIONS

My research showed, under elevated temperature, mice use both behavioral and physiology means to

regulate their body temperature and as ambient temperature increase beyond the Ta break points we

observe elevation in RMR, body Temperature and EWL. From the differences obtained when

comparing both mice, I cannot really tell where the exact break point in the TNZ since some data were

excluded from data analysis based on activity. Also, there was cofounding effect of sex in data analysis.

Indeed, there were differences in means on RMR, Tb and EWL and that shows how genetic background

can influence the development and physiology of species. Behavioral personality should be considered

in future before conducting RMR on any mice. Comparisons should be done between same sex to avoid

risk of cofounding effects. Molecular work could be done on the transgenic 129 mice and BL6 to

understand what gene or protein (such as heat shock protein) expression causes this Difference in break

points in RMR, Tb and EWL between both strains.

We should also understand thermo physiology of mice as a model organism broadly used in biomedical

research such as in obesity, diabetes, CNS diseases and variety of other pathologies. Body temperature

can have a great impact when it comes to understand species physiology. Energy expenditure is

essential for respond to various physiological response. Variations in break points showed 129 mix strain

had a smaller TNZ compared to BL6. This show BL6 uses less energy in thermoregulate and potentially

allocates more of its energy to other physiological respond such as growth, repair and reproduction.

24

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APPENDIX 1: ALL DATA POINT FOR BOTH ACTIVE AND NON-ACTIVE MICE.

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29

AUTHOR’S BIOGRAPHY

My name Teumbo Ngunte I was born and raised in Cameroon, a country located between Central and

Western Africa. Cameroon is bordered by Nigeria to the west and North; Chad to the northeast; the

Central African Republic to the east; and Equatorial Guinea, Gabon and the Republic of Congo to the

south. Cameroon's coastline lies on the Bight of Biafra, part of the Gulf of Guinea. I left my country on a

student visa and moved to the United States during the summer of 2011. I was admitted at the Intensive

English Institute of the University of Maine to improve my English for 3 months. After my training at

the Intensive English Institute, I was then a polygot or quadrilingual, able to speak 4 languages fluently

(two African native languages, French and English). In the Fall of 2011, I became a UMaine Bridge

student. I was admitted into a degree program and took my first Biology course, but also attended

English class to continue to improve my academic English. I went on to complete my Bachelor’s degree

in Biology with a pre-med concentration in 2016. Toward the end of his undergraduate studies, I met

Dr. Danielle Levesque, my graduate academic advisor and mentor as I pursued my Master’s degree in

Biology. I intend to go to medical school after obtaining my Master's degree. I have a lot of passion and

love for medicine and I plans on going back to my home country with a doctorate in medicine. He is a

candidate for the Master of Science degree in Zoology from the University of Maine in December 2019.