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Vol 48 No 1 January 2009

Pages 23ndash27

Journal o the American Association or Laboratory Animal ScienceCopyright 2009

by the American Association or Laboratory Animal Science

23

Because laboratory rodents are the most common animalsused in research today optimizing the methods used or eutha-nasia o these animals is important to researchers veterinariansand care sta Unlike many other laboratory animals rodentsoten are bred in research acilities Breeding may require eu-thanasia o very young animals most requently because o undesired phenotype or genotype or the production o surplusanimals Although many studies have evaluated the ethical andphysiologic considerations o various euthanasia methods onadult laboratory rodents the literature is relatively sparse onthe euthanasia o neonatal rodents

Because most research institutions do not have to euthanize

large numbers o neonates at one time the limited research onthe euthanasia o neonatal rodents is unsurprising Howeverthis paucity leaves rodent producers and others who musteuthanize groups o neonatal rodents at a loss or best practiceguidelines The American Veterinary Medical Association(AVMA) Panel on Euthanasia denes ldquoacceptablerdquo methodso euthanasia or rodents and other small mammals as bar-

biturates inhalant anesthetics carbon monoxide potassiumchloride in conjunction with general anesthesia microwaveirradiation and CO

22 For some o these methods their use

to ensure the humane death o large numbers o neonatal ratswould present several challenges Ideally the method chosenwould minimize distress to the animals would have a minimal

health impact on personnel perorming the euthanasia be easyto administer lack complicated record-keeping or disposalrequirements not be distressing to personnel perorming orwitnessing euthanasia and be cost-eective to employ or largenumbers o animals In addition an ideal euthanasia methodwould leave the euthanized animals suitable or other useswhether or research or as ood or rodent-eating animals Noneo the methods approved by the AVMA meet all these criteria

but CO2

is the closest European Union recommendations orthe euthanasia o neonatal rodents include decapitation concus-sion rapid reezing and hypothermia7 o these methods onlyrapid reezing could meet the criteria just listed but hazards

are associated with the use o liquid nitrogen In additionimmersion in liquid nitrogen is not recommended as an eutha-nasia method in the United States unless the animals are rst

rendered insensible215 In many cases the method used priorto reezing or hypothermia is CO

2inhalation Carbon dioxide is

widely accepted as an euthanasia agent due to the quick onseto eects including unconsciousness451629 minimal tissueartiact production1215 and ease o use14

The use o CO2

or euthanasia o adult rodents is the subjecto much opinion and debate32022 The current study was notintended to evaluate the merits o using CO

2to euthanize labora-

tory animals but rather to determine the parameters o its usein the euthanasia o neonatal rats The aim was to determine thelength o exposure to 100 CO

2necessary to euthanize 100

o rats 0 to 10 d o age

Materials and MethodsAnimals Pregnant CrlCD(SD) and F344DuCrl emale rats

(Rattus norvegicus) were obtained rom Charles River Kingston(Kingston NY) and Charles River Raleigh (Raleigh NC) respec-tively The strain and stock chosen or testing were based on thenumbers sold by Charles River and was thought to representa reasonable proportion o animals in use in the United StatesAll experiments were conducted at Charles Riverrsquos Wilming-ton MA acility Animals arrived at the Wilmington acility atapproximately day 12 o gestation and were housed in 1 areauntil the termination o the experiment All experiments were

approved by Charles Riverrsquos Institutional Animal Care and UseCommittee All work was conducted according to standards setorth in the Guide for the Care and Use of Laboratory Animals17

Animals were housed in solid-bottomed plastic cages byusing paper-based contact bedding (CareFresh Midwest Fil-tration Cincinnati OH) Cages were sanitized once weeklyEnvironmental conditions were maintained at 21deg plusmn 2 degC (70deg plusmn 3 degF) with 50 plusmn 20 relative humidity and 15 air changeshourly Animals were kept on a 1212-h lightdark cycle andprovided ad libitum access to water and eed (Lab Diet 5K52 orLab Diet 5L79 Purina Mills Richmond IN) All animals wererom colonies that tested negative or the ollowing viral agentsSendai virus pneumonia virus o mice sialodacryoadenitis

virus Kilham rat virus H1 virus rat minute virus reovirusrat theilovirus lymphocytic choriomeningitis virus hantavirusmouse adenovirus rat respiratory virus and rat parvovirus Inaddition the colonies were ree o the ollowing bacterial andungal agents Bordetella bronchiseptica CAR bacillus Clostridium

piliforme Corynebacterium kutscheri Encephalitozoon cuniculi Heli-

Euthanasia of Neonatal Rats with Carbon Dioxide

Kathleen R Pritchett-Corning

Exposure to CO2

is a common method used to euthanize rodents in biomedical research and rodent production The pur-pose of this study was to determine the length of CO

2exposure required to euthanize neonatal rats (0 to 10 d old) Multiple

groups of rats were exposed to 100 CO2

for 5 to 60 min After CO2

exposure rats were placed in room air for 20 min to allowfor possible recovery No difference was found in comparing 1 inbred strain and 1 outbred stock of rats Time to death variedinversely with the age of the animals requiring as long as 35 min on the day of birth The time to death decreased steadilywith increasing age with 100 of the rats euthanized after 5 min of CO

2exposure at 10 d of age The time required for 100

mortality decreased by 3 min for every 1 d increase in age between days 0 and 10

Received 29 May 2008 Revision requested 24 Jun 2008 Accepted 21 Jul 2008Charles River Domaine Des Oncins LrsquoArbresle France

Email kathleenpritchettcrlcom



Vol 48 No 1 Journal o the American Association or Laboratory Animal Science January 2009

observation o cessation o respiration and palpation or a lacko heartbeat beore being placed in a holding reezer

Statistical evaluation This study was designed to makerecommendations or the euthanasia o neonatal rats withinhaled 100 CO

2 The study did not attempt to determine the

exact time required to kill 100 o the animals at each day o age A pilot study used a limited number o inbred animals to

determine approximate times around which to evaluate initialexposure Then 2 separate groups o 10 neonates were exposedto CO

2at each time point Recovery rom CO

2exposure was

an unacceptable result and indicated an inadequate exposuretime Because the recovery o any animal was considered anundesirable outcome that exposure time was discarded and alonger exposure time was tested Once the 100 mortality timepoint was reached another 2 groups o 10 animals was tested ata yet longer exposure to determine whether the 100 mortal-ity ound at the prior time point was an anomaly Regressionanalysis (Minitab State College PA) was used to relate the timeto reach 100 mortality to the age o the pups

ResultsTime to death did not dier between inbred and outbred

animals on day 0 (Table 1) Ater evaluation o these resultsI tested only outbred rats thereater (Table 1 Figure 1) Ratso both sexes were equally susceptible to CO

2and regardless

o age appeared to be unconscious within 60 s o exposure toCO

2 Animals diered in their susceptibility to CO

2by age with

younger animals being less susceptible than older The stronglinear negative relationship between the time to reach 100mortality and the age o the pups is shown in Figure 2 Theregression equation was time = 357 ndash (30 times age) The coeciento multiple correlation (R2) was 951 Although euthanasia o

day 0 animals required at least 35 min exposure to CO2 a 5-minCO

2exposure was sucient or euthanasia at 10 d o age When

animals recovered ater CO2

exposure the pups that recoveredappeared dead at the beginning o the recovery period with norespiration or heartbeat noted However some pups did recovereven ater CO

2exposures o 30 min or longer Recovery occurred

as long as 10 min ater removal rom CO2

exposure in 0- to4-d-old rats Whenever recovery occurred in a group multipleanimals (which were in dierent bags) recovered indicatingthat the exposure time was insucient and that the recoverywas not due to ailure o the euthanasia chamber

DiscussionThis work conrms that neonatal rats euthanized with CO2

take substantially longer to die than the euthanasia times re-ported and recommended in the literature or adult rats Thenecessity o a prolonged CO

2exposure time or neonatal rats

can be explained by the mechanisms o action o CO2

and theinnate resistance o the neonate to hypercarbia and hypoxiaBecause embryonic hemoglobin is replaced by adult orms o hemoglobin by day 18 o gestation18 the increased anity o etal hemoglobin or oxygen is not a mechanism o resistance toCO

2in the neonatal rat Blood has a buering capacity or CO

2

because CO2

is also a byproduct o normal cellular metabolicprocesses I the capacity o this buering system is exceeded

an excess o CO2 in the blood leads to acidosis or a loweringo the pH o the blood and associated fuids Mild respiratoryacidosis leads to a compensatory increase in depth and rate o respiration in an eort to lsquoblow orsquo the excess CO

2 Proound

respiratory acidosis quickly suppresses the respiratory centerso the brain leading to a slow gasping respiratory pattern1323

cobacter hepaticus Helicobacter bilis and other Helicobacter sppKlebsiella oxytoca Klebsiella pneumoniae Mycoplasma pulmonisPasteurella pneumotropica Pseudomonas aeruginosa Salmonella spp Streptobacillus moniliformis Streptococcus pneumoniae and

beta-hemolytic Streptococcus spp Animals were also ree o endoparasites and ectoparasites Pregnant emale rats were al-lowed to litter naturally and the day o birth was recorded as

day 0 o the puprsquos lie Pups used or experiments were selectedarbitrarily rom litters at the desired age or experimentation andcombined into experimental groups regardless o sex

Procedures The total number o animals tested was 791comprising 71 inbred animals at 0 to 10 d o age that were testedin a preliminary study and 720 outbred animals 0 to 10 d o agethat were tested in the main study Each experimental groupincluded 10 rats o mixed sex o the same age Groups wereplaced in a clear plastic bag [17 times 75 times 39 cm (6 times 3 times 15 in)low-density polyethylene Polymer Packaging North CantonOH] containing an unmeasured small quantity o room air thatentered the bag when it was opened to insert the animals The

bag was lled to 4 L in volume with 100 CO2

rom a com-

pressed gas cylinder delivered at a rate o 05 Ls and thenplaced upright in a bedded cage

A pilot exposure study at 0 d o age was perormed with lim-ited numbers (10 per time point) o inbred rat pups to determinethe maximal exposure time at that age that would be requiredForty minutes was chosen or the upper limit o CO

2exposure

Because all animals in the pilot experiment died within 35 mino exposure and because only 1 group o animals was testedin the pilot the next exposure time (40 min) was chosen as themaximal exposure time or the actual study to aord a margino saety Five minutes was chosen as the lower limit o exposure

based both on the policies at many acilities which require a

5-min exposure to CO2 or adult rodents and the AVMA Panelrsquosrecommendations For each group o animals except day 0 therst exposure time was based on the successul euthanasia time(that is the exposure leading to death o all animals in the group)o the animals a day younger CO

2exposure time was measured

starting rom the initial introduction o gas into the bag Duringthe exposure animals were observed or movement apparentconsciousness and respiratory pattern or the rst 30 s ater CO

2

exposure and then at 1-min intervals until 5 min Ater 5 mino exposure animals were monitored at 5-min intervals untilthe end o the exposure period At the end o the CO

2exposure

period animals were removed rom the plastic bag placed in a bedded cage and observed continuously or 20 min or recovery

Recovery was considered to have occurred i any CO2-exposedanimal took a single breath during the recovery period I anyanimal rom a group recovered the CO

2exposure period was

lengthened by 5 min and another group o that age was testedAnimals were not palpated or instrumented to detect cessationo heartbeat ailure to recover when exposed to room air wasconsidered death Neonatal rats that recovered were euthanized

by using a physical method (decapitation)Unconsciousness was determined by loss o righting refex

cessation o spontaneous respiration and ailure to respondto nocioceptive stimuli (or example a gentle toe or tail pinchapplied without opening the plastic bag) Animals were notinstrumented to determine conscious state All potentialstimulation o the rats was avoided during the recovery periodThereore recovering rats were not palpated or a heartbeat Ratswere kept at room temperature during the recovery period tosimulate conditions under which animals might recover rom aneuthanasia attempt All rats were conrmed to be dead through



Euthanasia o neonatal rats

The cerebrospinal fuid lacks the buering capacity o the blood such that during acute respiratory acidosis the pH o the cerebrospinal fuid will drop precipitously25 This lower-ing o the pH o the cerebrospinal fuid is directly associated

with anesthetic depth and insensibility to pain and results in aquick onset o stupor and coma in humans21 in addition to therespiratory eects noted above Acidosis leads to depressiono myocardial contractility in addition CO

2has a direct eect

on the myocardium inducing hyperkalemia precipitating ar-rhythmias and slowing the heart rate2426 Thereore hypoxiais not the primary means by which CO

2exposure causes death

but rather it is the direct action o CO2

on vital systems thatproduces unconsciousness and death

Although hypoxia is not the means by which CO2

producesdeath the resistance o neonates to both hypoxia and hypercar-

bia appears to explain why altricial neonates show prolonged

Table 1 Numbers o neonatal and adult rats tested

Age (d)CO

2exposure(min)

No o pupsexposed

No o pupseuthanized

Pilot study

0 20 11 1

0 25 10 1

0 30 10 8

0 35 10 10

0 40 10 10

0 50 10 10

0 60 10 10

Main study

0 25 20 11

0 30 20 18

0 35 20 20

0 40 20 20

1 25 20 6

1 30 20 18

1 35 20 20

2 25 20 11

2 30 20 20

2 35 20 20

3 20 20 11

3 25 20 20

3 30 20 20

4 20 20 194 25 20 20

4 30 20 20

5 15 20 20

5 20 20 20

5 25 20 20

6 5 20 0

6 10 20 16

6 15 20 18

6 20 20 20

7 10 20 11

7 15 20 20

7 20 20 20

8 5 20 15

8 10 20 20

8 15 20 20

8 20 20 20

9 5 20 15

9 10 20 20

9 15 20 20

10 5 20 20

10 10 20 20

10 15 20 20

Figure 1 Mortality as a unction o exposure time or rats o each agegroup Each point is the percentage mortality in a group o 20 ani-mals

Figure 2 Time to 100 mortality as a unction o age (days) Dashedlines represent the 95 prediction intervals The regression equationis time = 357 ndash (30 times age) R2 = 951 The regression coecient is 30indicating that the time required or 100 mortality decreased by 3min or every 1-d increase in age between days 0 and 10

The pilot study was perormed by using the inbred strain F344DuCrl and the main experiment was done by using the outbred stockCrlCD(SD) Pups were considered to be euthanized i they did notrecover within the allotted recovery time

Table 1 Continued




The majority o the current work was perormed in a singleSpraguendashDawley-derived stock o outbred rats due to theirpopularity in research and their prevalence in commercial breed-ing situations Unlike the apparent dierences between inbred

and outbred mice regardless o the strain o inbred mouse27 a pilot experiment with a single strain o inbred rats did notreveal a dierence in response rom outbred rats so I did notuse inbred rats or urther testing In contrast in mice markeddierences between inbred and outbred animals occurred withregard to euthanasia with CO

2during the rst ew days o lie

but the time to death or the inbred mice was equivalent tothat o outbred mice by 7 d o age27 No such dierence wasnoted in the current experiment which could be due to the actthat only 1 strain and 1 stock were tested or to some intrinsicdierence in the development o rats compared with mice Anintrinsic dierence seems more likely because the dierences

between inbred and outbred mice were readily apparent at day0 regardless o the strains and stocks compared27 Comparedwith mice27 neonatal rats appear to be more sensitive to theeects o 100 CO

2

The CO2

exposure time necessary or euthanasia declinedlinearly as the age o the neonatal rats increased Age andattendant development appear to be the critical actors in de-termining susceptibility to CO

2 thereore developmental cues

may be used to determine the age o the animal I the exactage o animals to be euthanized is unknown physical devel-opment rather than size should serve as an indicator o age I the age is questionable one should err on the side o assumingthe animalrsquos immaturity and use a longer CO

2exposure I an

error is made in aging pups animals might be underexposedto CO

2and inadvertently recover whereas overexposure to

CO2

will prevent recovery o the animal A sample exposurechart is given in Table 2 The divisions chosen were based oneasily recognizable physical signs (or example eyelid open-ing) associated with specic days o age in the rat In Table 2 asaety margin is added to each exposure time recommendationto minimize the chance o postexposure recovery This saetymargin is added to accommodate inevitable human error andpossible mechanical ailure in any recommendations and toaid in recognition o susceptibility to CO

2by physical signs o

maturity Recommendations on exposure times or animals 14to 21 d o age and adult rats are extrapolated rom the data orday 10 animals and common recommendations in place or theeuthanasia o adult rats

The recommendations made or euthanasia times or neona-tal rats in Table 2 all roughly into week-long divisions in thedevelopment o the rat Alternatively acilities may want tomake exposure time decisions less complicated and categorize

survival times when exposed to CO2 Acute protective responses

in the neonate to severe hypoxia and hypercarbia include hy-pothermia reduction o heart and respiration rates (the divingrefex) and reduction o blood pH30 All the mechanisms o ac-tion o CO

2on animals are compensated or by the newbornrsquos

normal responses to hypoxic or hypercarbic stress and thereoreare better tolerated by newborns than adults30 This hypoxic

hypometabolism seen in newborns also may explain why eu-thanasia agents that appear to act solely through hypoxia suchas N

2 have extended neonatal survival times when compared

with CO21101119

Although adult rats are oten the subjects o CO2

euthanasiastudies I ound only 2 published reerences to the euthanasiao young rats with CO

2 In 1 study rats at 1 2 3 4 5 10 and

12 d o age were exposed to 100 CO2 recorded survival times

were 23 28 13 13 7 and 5 min with survival times generallydecreasing as the animals aged11 The authors o that studylisted 7 ages but only 6 survival times and graphs were notprovided to aid in interpretation o the listed results11 In theother published study the responses o young Wistar rats with

an average weight o 88 g were compared with those o moremature Wistar rats with an average weight o 220 g4 The youngrats took longer to ldquocollapserdquo when exposed to 100 CO

2but

had a shorter time to death4 Although the stock and sourceo rats were not specied in the study i a Wistar stock wereselected rom a commercial breeder the age based on a typicalagendashweight correlation would be 29 to 34 d old or the 88-grats and 49 to 81 d old or the 220-g weight group6 Accordingto usual weaning practices these animals were well past thetypical weaning age o 21 d and thereore this study providesno insight into the immediate neonatal period

Several comments and recommendations may be made with

regard to euthanasia o neonatal rats with CO2 I used a simpleplastic bag as the euthanasia chamber or several reasons Aplastic bag allowed easy observation and manipulation o therats without changing the CO

2levels within the chamber Plastic

bags can be sealed completely and provide immediate evidenceo ailure i they defate Because the euthanasia o neonatesrequires extended CO

2exposure times the use o a plastic bag

allows the concurrent use o more sophisticated euthanasiaequipment or the euthanasia o adult animals Care should betaken to avoid overlling the bag with animals however be-cause doing so could aect time to euthanasia or cause distressIn this experiment relatively ew animals were placed in theplastic bag all animals could stand or move reely in the bag

In addition the bag was placed in a bedded cage to provide astable platorm or the animals

The decision to expose the animals to 100 CO2was based on

research showing that supplementation with oxygen or room airprolongs time to death in adult rats7829 The rate o delivery o CO

2in this experiment was chosen to minimize potential animal

distress due to hypothermia and noise associated with lling o the euthanasia chamber The small size o the chamber resultedin rapid lling and the animals may have been chilled by thegas Chilling neonatal rats lowers their metabolic rate28 per-haps prolonging the time to death in this experiment Animalsare more likely to remain normothermic when kept in groupsand allowed to huddle28 In this experiment animals were notprevented rom huddling together which may have mitigatedany chilling eects o the gas or being removed rom the damHeated gas delivery systems may be a way to decrease the timeto euthanasia o neonatal rats Heating the CO

2delivered rom

24 degC to 34 degC decreased the time to euthanasia in a previousstudy11

Table 2 Suggested CO2

exposure time or euthanasia o rats o vari-ous ages

Appearance o animalCO

2exposure

time (min)

Nonhaired pups (0 to 6 d o age) 40

Haired pups eyes closed (7 to 13 d o age) 20

Haired pups eyes open preweaning (14 to

20 d o age)

10

Weanlings and adults (21 d o age and older) 5

The sample exposure chart presented here applies to the exposure chamber and lling parameters used in this study The use o other chambertypes and ll rates will require separate calibration All euthanasia timesrecommended in this table should be validated at each acility usingcurrent equipment and animals




animals into 2 age groups to determine necessary times orCO

2exposure unweaned animals and adults Exposure times

should be validated at each acility using the intended equip-ment and gas delivery system Because the present study onlyaddresses exposure times or F344 and CD rats acilities maywish to validate exposure times or other stocks or strains in useAll equipment should be calibrated and revalidated regularly

to ensure eective euthanasia without the need or backupmethods to ensure death

AcknowledgmentsThe author would like to acknowledge B Hayes or animal care over

the course o this experiment M Ripperton or editorial assistance andMFW Festing or statistical consultation

References1 Adolph EF 1969 Regulation during survival without oxygen in

inant mammals Respir Physiol 7356ndash368 2 AVMA Panel on Euthanasia 2000 2000 report o the AVMA panel

on euthanasia J Am Vet Med Assoc 218669ndash696

3 Bennett T 2005 Letter to the editor Comp Med 55114 Blackshaw JK Fenwick DC Beattie AW Allan DJ 1988 The

behaviour o chickens mice and rats during euthanasia withchloroorm carbon dioxide and ether Lab Anim 2267ndash75

5 Cartner SC Barlow SC Ness TJ 2007 Loss o cortical unctionin mice ollowing decapitation cervical dislocation potassiumchloride injection and CO

2inhalation Comp Med 57570ndash573

6 Charles River [Internet] Research animal models pricing and lit-erature the Wistar rat [cited 5 January 2009] Available at httpwwwcrivercomSiteCollectionDocumentsrm_rm_c_wistar_ratspd

7 Close B Banister K Baumans V Bernoth EM Bromage N Bunyan J Erhardt W Flecknell P Gregory N Hackbarth H Morton D Warwick C 1997 Recommendations or euthanasia o

experimental animals part 2 DGXI o the European CommissionLab Anim 311ndash32

8 Coenen AM Drinkenburg WH Hoenderken R van Luijtelaar EL 1995 Carbon dioxide euthanasia in rats oxygen supple-mentation minimizes signs o agitation and asphyxia Lab Anim29262ndash268

9 Danneman PJ Stein S Walshaw SO 1997 Humane and practi-cal implications o using carbon dioxide mixed with oxygen oranesthesia or euthanasia o rats Lab Anim Sci 47376ndash385

10 Duffy TE Kohle SJ Vannucci RC 1975 Carbohydrate and energymetabolism in perinatal rat brain relation to survival in anoxia J Neurochem 24271ndash276

11 Fazekas JF Alexander FAD Himwich HE 1941 Tolerance o thenewborn to anoxia Am J Physiol 134281ndash287

12 Feldman DB Gupta BN 1976 Histopathologic changes in labora-tory animals resulting rom various methods o euthanasia Lab Anim Sci 26218ndash221

13 Forslid A Augustinsson O 1988 Acidosis hypoxia and stresshormone release in response to 1-minute inhalation o 80 CO

2

in swine Acta Physiol Scand 132223ndash231

14 Hackbarth H Kuppers N Bohnet W 2000 Euthanasia o rats withcarbon dioxidemdashanimal welare aspects Lab Anim 3491ndash9615 Hauser R Jankowski Z Gos T Krzyzanowski M 2001 Haemor-

rhages in head tissues during the asphyxiation process Forensic Sci Int 124235ndash236

16 Hewett TA Kovacs MS Artwohl JE Bennett BT 1993 A compari-son o euthanasia methods in rats using carbon dioxide in prelledand xed fow-ratendashlled chambers Lab Anim Sci 43579ndash582

17 Institute of Laboratory Animal Resources (ILAR) National Re-search Council 1996 Guide or the Care and Use o LaboratoryAnimals Washington DC National Academy Press

18 Iwahara SI Abe Y Okazaki T 1996 Identication o ve embry-onic hemoglobins o rat and ontogeny o their constituent globinsduring etal development J Biochem 119360ndash366

19 Kabat H 1940 The greater resistance o very young animals toarrest o the brain circulation Am J Physiol 130588ndash59920 Marris E 2006 Bioethics an easy way out Nature 441570ndash571 21 Meyer JS Gotoh F Tazaki Y 1961 CO

2narcosis An experimental

study Neurology 11524ndash53722 Morton DB 2005 Letter to the editor Comp Med 551123 Petty WC Sulkowski TS 1971 CO

2narcosis in the rat I Eects

on respiration and blood parameters Aerosp Med 42547ndash55224 Petty WC Sulkowski TS 1971 CO

2narcosis in the rat II Eects

on the ECG Aerosp Med 42553ndash55825 Ponteacuten U 1966 Consecutive acidndashbase changes in blood brain

tissue and cerebrospinal fuid during respiratory acidosis and baseosis Acta Neurol Scand 42455ndash471

26 Price HL 1960 Eects o carbon dioxide on the cardiovascularsystem Anesthesiology 21652ndash663

27 Pritchett K Corrow D Stockwell J Smith A 2005 Euthanasia o neonatal mice with carbon dioxide Comp Med 55275ndash281

28 Saiki C Mortola JP 1996 Eect o CO2

on the metabolic andventilatory responses to ambient temperature in conscious adultand newborn rats J Physiol 491261ndash269

29 Sharp J Azar T Lawson D 2006 Comparison o carbon dioxideargon and nitrogen or inducing unconsciousness or euthanasiao rats J Am Assoc Lab Anim Sci 4521ndash25

30 Singer D 2004 Metabolic adaptation to hypoxia cost and beneto being small Respir Physiol Neurobiol 141215ndash228




observation o cessation o respiration and palpation or a lacko heartbeat beore being placed in a holding reezer

Statistical evaluation This study was designed to makerecommendations or the euthanasia o neonatal rats withinhaled 100 CO

2 The study did not attempt to determine the

exact time required to kill 100 o the animals at each day o age A pilot study used a limited number o inbred animals to

determine approximate times around which to evaluate initialexposure Then 2 separate groups o 10 neonates were exposedto CO

2at each time point Recovery rom CO

2exposure was

an unacceptable result and indicated an inadequate exposuretime Because the recovery o any animal was considered anundesirable outcome that exposure time was discarded and alonger exposure time was tested Once the 100 mortality timepoint was reached another 2 groups o 10 animals was tested ata yet longer exposure to determine whether the 100 mortal-ity ound at the prior time point was an anomaly Regressionanalysis (Minitab State College PA) was used to relate the timeto reach 100 mortality to the age o the pups

ResultsTime to death did not dier between inbred and outbred

animals on day 0 (Table 1) Ater evaluation o these resultsI tested only outbred rats thereater (Table 1 Figure 1) Ratso both sexes were equally susceptible to CO

2and regardless

o age appeared to be unconscious within 60 s o exposure toCO

2 Animals diered in their susceptibility to CO

2by age with

younger animals being less susceptible than older The stronglinear negative relationship between the time to reach 100mortality and the age o the pups is shown in Figure 2 Theregression equation was time = 357 ndash (30 times age) The coeciento multiple correlation (R2) was 951 Although euthanasia o

day 0 animals required at least 35 min exposure to CO2 a 5-minCO

2exposure was sucient or euthanasia at 10 d o age When

animals recovered ater CO2

exposure the pups that recoveredappeared dead at the beginning o the recovery period with norespiration or heartbeat noted However some pups did recovereven ater CO

2exposures o 30 min or longer Recovery occurred

as long as 10 min ater removal rom CO2

exposure in 0- to4-d-old rats Whenever recovery occurred in a group multipleanimals (which were in dierent bags) recovered indicatingthat the exposure time was insucient and that the recoverywas not due to ailure o the euthanasia chamber

DiscussionThis work conrms that neonatal rats euthanized with CO2

take substantially longer to die than the euthanasia times re-ported and recommended in the literature or adult rats Thenecessity o a prolonged CO

2exposure time or neonatal rats

can be explained by the mechanisms o action o CO2

and theinnate resistance o the neonate to hypercarbia and hypoxiaBecause embryonic hemoglobin is replaced by adult orms o hemoglobin by day 18 o gestation18 the increased anity o etal hemoglobin or oxygen is not a mechanism o resistance toCO

2in the neonatal rat Blood has a buering capacity or CO

2

because CO2

is also a byproduct o normal cellular metabolicprocesses I the capacity o this buering system is exceeded

an excess o CO2 in the blood leads to acidosis or a loweringo the pH o the blood and associated fuids Mild respiratoryacidosis leads to a compensatory increase in depth and rate o respiration in an eort to lsquoblow orsquo the excess CO

2 Proound

respiratory acidosis quickly suppresses the respiratory centerso the brain leading to a slow gasping respiratory pattern1323

cobacter hepaticus Helicobacter bilis and other Helicobacter sppKlebsiella oxytoca Klebsiella pneumoniae Mycoplasma pulmonisPasteurella pneumotropica Pseudomonas aeruginosa Salmonella spp Streptobacillus moniliformis Streptococcus pneumoniae and

beta-hemolytic Streptococcus spp Animals were also ree o endoparasites and ectoparasites Pregnant emale rats were al-lowed to litter naturally and the day o birth was recorded as

day 0 o the puprsquos lie Pups used or experiments were selectedarbitrarily rom litters at the desired age or experimentation andcombined into experimental groups regardless o sex

Procedures The total number o animals tested was 791comprising 71 inbred animals at 0 to 10 d o age that were testedin a preliminary study and 720 outbred animals 0 to 10 d o agethat were tested in the main study Each experimental groupincluded 10 rats o mixed sex o the same age Groups wereplaced in a clear plastic bag [17 times 75 times 39 cm (6 times 3 times 15 in)low-density polyethylene Polymer Packaging North CantonOH] containing an unmeasured small quantity o room air thatentered the bag when it was opened to insert the animals The

bag was lled to 4 L in volume with 100 CO2

rom a com-

pressed gas cylinder delivered at a rate o 05 Ls and thenplaced upright in a bedded cage

A pilot exposure study at 0 d o age was perormed with lim-ited numbers (10 per time point) o inbred rat pups to determinethe maximal exposure time at that age that would be requiredForty minutes was chosen or the upper limit o CO

2exposure

Because all animals in the pilot experiment died within 35 mino exposure and because only 1 group o animals was testedin the pilot the next exposure time (40 min) was chosen as themaximal exposure time or the actual study to aord a margino saety Five minutes was chosen as the lower limit o exposure

based both on the policies at many acilities which require a

5-min exposure to CO2 or adult rodents and the AVMA Panelrsquosrecommendations For each group o animals except day 0 therst exposure time was based on the successul euthanasia time(that is the exposure leading to death o all animals in the group)o the animals a day younger CO

2exposure time was measured

starting rom the initial introduction o gas into the bag Duringthe exposure animals were observed or movement apparentconsciousness and respiratory pattern or the rst 30 s ater CO

2

exposure and then at 1-min intervals until 5 min Ater 5 mino exposure animals were monitored at 5-min intervals untilthe end o the exposure period At the end o the CO

2exposure

period animals were removed rom the plastic bag placed in a bedded cage and observed continuously or 20 min or recovery

Recovery was considered to have occurred i any CO2-exposedanimal took a single breath during the recovery period I anyanimal rom a group recovered the CO

2exposure period was

lengthened by 5 min and another group o that age was testedAnimals were not palpated or instrumented to detect cessationo heartbeat ailure to recover when exposed to room air wasconsidered death Neonatal rats that recovered were euthanized

by using a physical method (decapitation)Unconsciousness was determined by loss o righting refex

cessation o spontaneous respiration and ailure to respondto nocioceptive stimuli (or example a gentle toe or tail pinchapplied without opening the plastic bag) Animals were notinstrumented to determine conscious state All potentialstimulation o the rats was avoided during the recovery periodThereore recovering rats were not palpated or a heartbeat Ratswere kept at room temperature during the recovery period tosimulate conditions under which animals might recover rom aneuthanasia attempt All rats were conrmed to be dead through






2has a direct eect









Age (d)CO

2exposure(min)

No o pupsexposed

No o pupseuthanized

Pilot study

0 20 11 1

0 25 10 1

0 30 10 8

0 35 10 10

0 40 10 10

0 50 10 10

0 60 10 10

Main study

0 25 20 11

0 30 20 18

0 35 20 20

0 40 20 20

1 25 20 6

1 30 20 18

1 35 20 20

2 25 20 11

2 30 20 20

2 35 20 20

3 20 20 11

3 25 20 20

3 30 20 20

4 20 20 194 25 20 20

4 30 20 20

5 15 20 20

5 20 20 20

5 25 20 20

6 5 20 0

6 10 20 16

6 15 20 18

6 20 20 20

7 10 20 11

7 15 20 20

7 20 20 20

8 5 20 15

8 10 20 20

8 15 20 20

8 20 20 20

9 5 20 15

9 10 20 20

9 15 20 20

10 5 20 20

10 10 20 20

10 15 20 20




Table 1 Continued









2

The CO2




2exposure I an



CO2











with CO21101119







2but













2delivered rom





2exposure

time (min)




20 d o age)

10




























2


























2has a direct eect









Age (d)CO

2exposure(min)

No o pupsexposed

No o pupseuthanized

Pilot study

0 20 11 1

0 25 10 1

0 30 10 8

0 35 10 10

0 40 10 10

0 50 10 10

0 60 10 10

Main study

0 25 20 11

0 30 20 18

0 35 20 20

0 40 20 20

1 25 20 6

1 30 20 18

1 35 20 20

2 25 20 11

2 30 20 20

2 35 20 20

3 20 20 11

3 25 20 20

3 30 20 20

4 20 20 194 25 20 20

4 30 20 20

5 15 20 20

5 20 20 20

5 25 20 20

6 5 20 0

6 10 20 16

6 15 20 18

6 20 20 20

7 10 20 11

7 15 20 20

7 20 20 20

8 5 20 15

8 10 20 20

8 15 20 20

8 20 20 20

9 5 20 15

9 10 20 20

9 15 20 20

10 5 20 20

10 10 20 20

10 15 20 20




Table 1 Continued









2

The CO2




2exposure I an



CO2











with CO21101119







2but













2delivered rom





2exposure

time (min)




20 d o age)

10




























2





























2

The CO2




2exposure I an



CO2











with CO21101119







2but













2delivered rom





2exposure

time (min)




20 d o age)

10




























2














































2



















