Comparison of Environment and Mice in Static and Mechanically Ventilated Isolator Cages with Different Air Velocities and Ventilation Designs

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1 Comprison of Environment nd Mice in Sttic nd Mechniclly Ventilted Isoltor Cges with Different Air Velocities nd Ventiltion Designs FARHAD MEMARZADEH, PHD, PE, 1 PAUL C. HARRISON, PHD, 2* GERALD L. RISKOWSKI, PHD, PE, 3 AND TONJA HENZE, MS, LATG 4 The purpose of this study ws to compre environmentl conditions nd mice in cges with four different mechnicl ventiltion designs nd sttic isoltor cge. Environmentl conditions (ir velocity, temperture, reltive humidity, bedding weight chnge, irborne dust, NH 3, ) were compred for ech cge type (n = 5 per cge). Bedding type ws chipped hrdwood. Mouse response in ech cge type ws evluted by body weight, feed consumption, wter intke, loction of specific behviors, nd building of bedding mounds. Commercil polycrbonte mouse cging units ( cm shoebox style, stinless-steel round wire br lids, nd 7-cm-deep isoltor cge filter tops) were modified to fit the mechnicl ventiltion cge types nd were used for the sttic isoltor cges. Mechniclly ventilted cges were fitted with forced ir inlets centered 5 cm bove the cge floor on the 19.1 cm-side of the cge. Inlet ir velocity ws either 40 or 200 feet/min (n = 10 cges ech), nd the ir volume exchnge rte ws 9.3 L/min. In hlf of the mechniclly ventilted cges, the exhust ir ws forced through filter in the isoltor cge top, wheres in the remining mechniclly ventilted cges, the ir ws forced through single exhust port fixed in the nrrow side of the cge top directly bove the ir inlet. Inlet ir velocity but not exhust design ffected intrcge ir velocity distribution. Other environmentl conditions were similr between the four mechnicl ventiltion designs. Reltive to the mechniclly ventilted cges, the sttic isoltor cges hd lower ir velocities, higher reltive humidities, higher NH 3 levels, higher CO 2 levels, lower body weight gin, nd lower wter consumption; tempertures, prticulte levels, nd feed consumption rtes did not differ significntly between cge types. Loctions of bedding mounds nd behviors were similr in ll cge tretments. The gol to improve environmentl conditions in niml housing fcilities nd the microenvironment to which nimls re exposed hs given rise to n evolution of ventiltion designs for rodent isoltor cging systems (ICS). In comprehensive review of sttic nd mechniclly ventilted ICS, Lipmn (1) indicted tht the lck of common fetures mkes comprtive opertionl evlutions difficult. Physicl nd biologicl ICS dt from the literture hve been used to develop computtionl fluid dynmic (CFD) models tht cn be used to predict ICS ventiltion response (2). Becuse CFD model verifiction experiments were conducted with sttic mouse cges, min objective of our study ws to evlute the intrcge environments of ICS with two different mechnicl ventiltion supply ir velocities nd two different ir exhust systems. The four mechnicl ventiltion cge tretments were compred to sttic isoltor cges. In order to fcilitte comprtive opertionl evlutions, ll physicl nd niml vribles were kept s uniform s possible. Another specific objective of our reserch ws to define how live mice would ffect the intrcge environment nd to evlute how the environments from the vrious cge tretments would ffect the mice. Prior to the niml experiment, initil environmentl mesurements were tken with Simulted Mouse Objects (SMO) in the cges to ensure tht environmentl conditions in the cges were cceptble. Dt collected under SMO conditions cn be integrted into previously developed computtionl fluid dynmic (CFD) Division of Policy nd Progrm Assessment, Office of Reserch Fcilities, Ntionl Institutes of Helth 1 ; Animl Science Deprtment 2 ; Texs A&M University, Biologicl & Agriculturl Engineering, Formerly University of Illinois 3 ; College of Agriculturl, Consumer nd Environmentl Sciences, University of Illinois 4 * Corresponding uthor models to ensure incresed ccurcy in predictions of ICS ventiltion. The individully ventilted ICS used in this study were designed, fbricted, nd their physicl ttributes mesured in order to reduce the possible vribles tht would be ssocited with different commercilly vilble ICS. We used our experimentl ICS to better define nd compre experimentl prmeters; they were not intended to replicte ny commercilly vilble ICS. Mterils nd Methods Generl protocol nd mcroenvironment. Approvl of ll fcilities nd procedures for the use of lbortory mice ws obtined from the University of Illinois Institutionl Animl Cre nd Use Committee prior to the initition of this reserch. Prior to the conduct of this study, fcilities, recording equipment, nd environmentl conditions within ll cges were evluted using bedded, fully ssembled cges contining n SMO. The SMO pproximted the re displcement nd therml emissions (2.6 W) of group of five mture mice (3). Individul cge nd SMO dimensions were the sme s defined by Memrzdeh (2). After the SMO stge of the experiment, the SMO ws replced with five femle mice (Mus musculus) per cge. The mice (Hsd:ICR, CD-1; men body weight, 26 g) were trnsported in dedicted vehicle operted by the vendor (Hrln Sprgue Dwley, Indinpolis, Ind.). The mice were housed in polycrbonte shoebox-style cges (pproximtely 29.2 cm long 19.1 cm wide 12.7 cm deep) with stinless-steel round wire br lids for 1-week period prior to experimentl dt collection. The number of mice per cge ws the mximum llowble for the mouse weight nd cge re (4). During both SMO nd live niml dt collection periods, cges were rndomly ssigned to one of two levels of stinless-steel cge rck tht 14

2 ws locted inside m controlled environment chmber (Hotpck, Phildelphi, P.). Air ventiltion into the controlled environment chmber supplied pproximtely 18 ir chnges per hour, ws HEPA-filtered, nd served s the inlet ir source for ll cges. Air from the environmentl chmber ws exhusted directly outside the niml fcility. The environmentl chmber hd 12:12-h photoperiod (lights on t 1:00 pm). Photophse illumintion ws from four cler (60-W) incndescent light sources (Generl Electric Co., Clevelnd, Ohio) tht were eqully spced round the stinless-steel cge rck (intensity, 20 to 40 foot-cndles, s mesured beside the cges t rck level). Becuse the lest mount of photoctive responses pper to occur in the blue light spectr (5), scotoperiod illumintion ws from four blue (25-W) incndescent light sources (Generl Electric Co., Clevelnd, Ohio; intensity, 0.2 to 0.4 foot-cndles) tht were locted djcent to the cler lights. The temperture nd reltive humidity were continuously monitored with hygrothermogrphs (model WD , Okton Instruments, Vernon Hills, Ill.). Temperture in the environmentl chmber ws 23.7 ± 0.1 C, nd reltive humidity ws 43% ± 1.2%. Animl cre records nd mouse conditions were checked dily by Office of Lbortory Animl Resources personnel. Cge ventiltion design. Fifteen new commercil polycrbonte mouse isoltion cging units (pproximtely 29.2 cm long 19.1 cm wide 12.7 cm deep shoebox-style cges with stinless steel round wire br lids nd 7-cm-deep isoltor cge filter tops; N10 series, ctlog no. N10MBTC, Ancre, Bellmore, N.Y.) were used for ll cges in this study. There were five ir ventiltion tretments four of the ventiltion tretments used mechnicl ventiltion systems tht provided 60 ir chnges per hour (ACH), nd one tretment ws sttic isoltor cge with no mechnicl ventiltion. The five ventiltion tretments were designted s: high-velocity ir supply with the ir exhusted through single-point exhust (HS), highvelocity ir supply with the ir exhusted through porous cge top (HP), low-velocity ir supply with the ir exhusted through singlepoint exhust (LS), low-velocity ir supply with the ir exhusted through porous cge top (LP), nd sttic isoltor cge with porous filter top but no mechnicl ventiltion (SM). Refer to Fig. 1 for schemtic digrm of the mechniclly ventilted cges. The configurtions of the mechnicl ventiltion systems consisted of combintions of two types of ir inlets nd two types of ir exhusts. High-velocity ir inlet nozzles were cylinders (dimeter, 3.8 cm) with six openings (inner dimeter, 0.56 cm) on ech. Air supply through the high-velocity nozzle openings results in velocities of pproximtely 200 ft/min t the nozzle. The inlet ir nozzle for the low-velocity configurtion consisted of cylinder (dimeter, 7.0 cm) with 162 (inner dimeter, 0.28 cm) openings on ech cylinder. The low-velocity ir supply ws designed to provide velocities of pproximtely 40 ft/min t the nozzle. Air-supply nozzles were instlled centrlly on one of the short (19.1-cm) sides of the cgebottom, nd the center of the ir supply cylinder ws pproximtely 5 cm bove the cge floor. Supply irflow rte in ll mechniclly ventilted cges ws 9.3 L/min, which provided 60 ACH. The singlepoint ir exhust ws mnufctured from stndrd copper pipe cp (dimeter, 6.3 cm) with hose connector (outer dimeter, 1.0 cm) ttched through the solid flt side, nd the open side ws covered with filter medi (no. 2024, Reemy, Old Hickory, Tenn.). The open (filtered) end ws inside the cge-top, directly bove the ir supply, nd the hose connector extended through the cge wll. The porous (diffuse) type of exhust ws through filter medi (no. 2024, Reemy) sndwiched between the porous plstic ssembly screens in the top of the cge top (the sme setup nd mteril s for stndrd isoltor cge filter tops). A solid plexigls sheet ws seled over the porous plstic ssembly in the tops of the single-point exhust Figure 1. Schemtic digrm of mechniclly ventilted mouse cges, with porous nd single point exhust tops longitudinl cross-sections re shown. High- nd low-velocity mechnicl ventiltion cges hd ir inlets tht were centered in the cge bottom. Sttic isoltor cges hd porous exhust cge tops but no mechnicl ventiltion ir inlets. cges. Cge tops of the mechnicl ventiltion cges were seled to the cge bottom section for the entire period of ech experiment so tht ll inlet ir ws exhusted from the ventilted cges through either the filtered single-point outlet or the porous tops under positive pressure. The seling mteril ws replceble wether-stripping (M-D Building Products, Inc., Oklhom City, Okl.). SM cge filtered cge tops were not seled to the cge bottoms, nd the tops remined in plce for the entire period of ech experiment. Fresh ir ws supplied eqully into ech forced ventilted cge t 9.3 L/min. An ir pump (4F7 4EA, Gst Mnufcturing, Inc., Benton Hrbor, Mich.) ws used to supply ir to common (multiport) sttic pressure tnk, which distributed the ir mong the cges through flexible hoses. The irflow rte supplied by the pump to the sttic pressure tnk ws djusted using bypss vlve. Airflow from the sttic pressure tnk to ech cge ws mesured with flow meters (model U , Cole-Prmer Instrument Co., Vernon Hills, Ill.) nd controlled using flow meter vlves. Flow meters were clibrted using Mini-Buck Clibrtor (model M-30, A.P. Buck, Inc., Orlndo, Fl.). Correction fctors were formulted by regression of cge flow meter vlues to the Mini-Buck Clibrtor. Experimentl design nd nlysis. Seventy-five mice were llocted rndomly into 15 designted cge groups tht remined together throughout this study s five-mouse experimentl units (MU). Three MU then were rndomly ssigned to ech cge unit (A through E), nd ech cge unit (CU) ws initilly rndomly ssigned to prticulr cge ventiltion tretment. Cge units were ressigned (sequentilly rotted) to different cge ventiltion tretment on weekly bsis over the 5-week study until ll CU (three MU ech) hd experienced ech of the five cge ventiltion tretments. This procedure llowed us to evlute whether mesurement of niml 15

3 responses by prticulr CU (repliction unit) ws independent of cge ventiltion tretment. During ech week, the following mesurements were recorded: temperture, reltive humidity, dust (mss nd prticle count), NH 3, (cge environment prmeters); body weight nd food nd wter utiliztion (niml response prmeters); nd ctivity nd mound building t mid-photophse nd mid-scotophse (niml behvior prmeters). Environmentl conditions were smpled from one cge in ech of the cge ventiltion tretments by fitting it with n instrumented cge top which remined in plce for the entire week of ech experiment. Animl responses were recorded weekly for ll 15 cges nd 75 mice. Significnt differences between vribles were ssessed using nlysis of vrince nd Fischer s lest significnt difference tests t n inference level of P < 0.05 (SAS Institute, Inc., Cry, N.C.). Dt collection protocol nd methods. The following dt collection protocol ws followed on weekly bsis: Fridy Mice, feed, wter, nd bedding were weighed-out of one cge, nd the mice were plced in clen experimentl cge of different cge ventiltion tretment. Sturdy nd Sundy Animl sttus ws visully checked, but no mesurements were collected. Mondy Temperture, reltive humidity, nd dust were recorded from the five cges fitted with instrumented cge-tops (one cge for ech cge ventiltion tretment). Tuesdy NH 3 gs levels were smpled from the five cges with instrumented tops. NH 3 mesurements were tken 5 dys fter the mice were plced on new bedding, becuse it is common prctice in mny lbortories to chnge bedding t lest every 5 dys. Wednesdy Loction nd ctivity of ll 75 mice nd the loctions of bedding mounds were recorded for ll 15 cges t mid-photophse nd mid-scotophse. After the weigh-bck mesurements were obtined on Fridy, the mice were mintined in their seprte mouse experimentl units (MU) nd housed in individul open top rodent cges for pproximtely 2 to 4 h. During this time period, ll of the experimentl cges (15) were clened, snitized, nd dried. All cges were given fresh (weighed) supply of feed (Tekld 22/5 Rodent Diet [W] 8640; Hrln Tekld, Indinpolis, Ind.), bottled tp wter, nd n pproximtely 1.3-cm depth of evenly spred bedding (Course Grde Bet Chips, 100% Hrdwood; Northestern Products Corp., Wrrensburg, N.Y.). After this clening period, the mice were gin weighed s MU nd ssigned s CU to different cge ventiltion tretment for the next week. Weekly chnges in body weight, feed nd wter use, nd bedding weight were bsed on the weigh-in vlues from the previous Fridy. This procedure ws repeted five times so tht ll mice hd been recorded in ech of the experimentl cge ventiltion tretments (HS, HP, LS, LP, nd SM). The procedure lso llowed the evlution of responses for the week fter the chnge to different experimentl cge ventiltion tretment (HS to HP, HP to LS, LS to LP, LP to SM, nd SM to HS; this sequence ws selected t rndom). This sequence of chnge in cge ventiltion tretment ws rbitrrily ssigned; however, time nd fcilities would not llow for ll possible combintions. T-type thermocouples were used to mesure nd record tempertures outside nd inside the cge. The thermocouples were clibrted using wter bth. Wter bth tempertures were determined using totl immersion mercury thermometer (model , vendor unknown, Tiwn). Correction fctors were formulted by regression of thermocouple redings on thermometer redings. Thermocouples were plced in the room, in supply ir (inside the tube connected to the ir inlet nozzle in cges with mechnicl ventiltion), nd in ech of the five instrumented cge tops. Six thermocouples were inside the cges t heights of 12.5 cm nd 19 cm from the cge bottom (three thermocouples t ech height, spced t 6.5, 0, nd 6.5 cm from the cge center long the medin xis). Mesurements t lower positions were not possible becuse the mice would interfere with the sensors. Thermocouples were connected to dt cquisition system (Model Personl DAQ 56 + PDQ2, Iotech, Inc., Clevelnd, Ohio). In ech cge loction nd for ech cge ventiltion tretment, temperture ws determined from nine seprte mesurements of 20 smples ech ( smple ws mesured every 10 sec). Air velocities were mesured using n omni-directionl probe (model 8455, TSI, Inc., Shoreview, Minn.) clibrted in benchtop wind tunnel (model 8390, TSI, Inc.). During velocity mesurements, the cges contined bedding, wter bottle, feed, nd n SMO but no mice. Mesurements were tken t three points 4 cm bove the bottom of the cge ( 6.5, 0, nd 6.5 cm from the cge center long the medin xis). In ech cge loction nd for ech cge ventiltion tretment, velocity ws determined from nine seprte mesurements of 20 smples ech ( smple ws mesured every 10 sec). Air velocity redings below 10 ft/min re not s ccurte s higher ones, but the redings were consistent cross mesurements. Reltive humidity ws evluted electroniclly using thermohygrometer (model 900, Generl Electric, Woburn, Mss.). The reltive humidity sensor probe ws inserted through port into the center of ech instrumented cge top, nd redings were tken fter 10-min stbiliztion period. The reltive humidity sensors were clibrted with psychrometer prior to recording the room nd cge. The signl from the sensor ws collected on dt cquisition system (Model Personl DAQ 56 +PDQ2, Iotech, Inc., Clevelnd, Ohio) connected to n IBM comptible PC. A lser prticle counter (Aerodynmic Prticle Sizer TSI 3320, TSI, Inc., Shoreview, Minn.) ws used to evlute the irborne prticle size distribution t 52 subrnges within the overll rnge of 0.5 to 19.8 µm, nd dust mss ws determined ssuming stndrd prticle density (density of wter, 1.0 g/cm 3 ). A clibrted volume of ir ws vcuumed into the nlyzer through smpling hose (inner dimeter, 1.3 cm) tht ws connected to plstic tube seled into the middle of one side of the instrumented cge-top. The plstic tube hd removble cp, which remined closed except during ir smpling, ws locted 2.5 cm down from the top horizontl surfce, nd extended 3.8 cm inside the cge. Air smples for NH 3 nlysis were obtined through the sme cge-top port tht ws used for dust smpling. Air from the smpling port ws pumped to n infrred CO 2 nlyzer (model 880A, Rosemount, Inc., Chnhssen, Minn.) tht ws connected to Kipp- Zonen strip chrt recorder (Cole Prmer). Vlues recorded for CO 2 were clibrted ginst four certified CO 2 stndrds (1.49%, 0.998%, 0.506%, nd %). NH 3 gs smples were tken with Mtheson Kitgw pump (model A, certified model 42 CFR84, Montgomeryville, P.) connected with either model 105 SD (0.2 to 20 ppm) or model 105 SC (5 to 20 ppm) Kitgw Precision Gs Detector Tubes (Mtheson Sfety Products, Est Rutherford, N.J.). Vlues recorded for NH 3 were clibrted ginst two certified NH 3 gs stndrds (52.5 nd 74.8 ppm). All wire nd tube connections tht coupled the mouse cges to physicl environment sensors ttched through the polycrbonte isoltor cge top nd were seled in plce with plible, replceble wether-stripping mteril (M-D Building Products, Inc., Oklhom City, Okl.). The totl volume of intrcge ir extrcted during ech prticulte, CO 2, nd NH 3 smpling ws 2.5, 3.4, nd 0.2 L, respectively. The totl internl volume of ech cge ws just under 11 L nd time per smple ws under 2 min, so there should hve been little long-term distur- 16

4 wter supplies dictted tht the et behvior could only occur in res A, B, D, H, nd G; likewise, the drink behvior could only occur in res C nd F. Bedding mounds were recorded in res tht ppered to be elevted 2 or more cm bove the surrounding level of bedding nd were drwn directly on the recording chrts. Video recordings were not used in this study, becuse only generl ctivity nd loctions cn be observed. Becuse records were obtined during the middle of the 12-h photophse nd 12-h scotophse, cler video records were not ccurte for precise counts of mice or mound loctions. Figure 2. Mp of mouse cge floor re showing the nine potentil res (A through I) used for the behviorl nd mound-building recordings. Becuse of the loction of feed nd wter in the wire lid bove the mouse re, recorded eting behvior could only occur in res A, B, D, H, nd G, nd drinking behvior could only occur in res C nd F. bnce of cge conditions from these smples. Cge loctions of behviors nd bedding mounds were recorded on stndrdized chrts ssigned to ech cge, by n observer tht quietly entered nd remined in the mouse room until cge-floor re nd one of six designted behviors were recorded for ech mouse. Designted ctegories of behvior were sleep, groom, move, drink, dig, et, nd climb. The number of mice involved in designted behvior nd their loctions during the time of observtion were recorded. The recording chrts schemticlly mpped the floor of the mouse cge into nine seprte eqully sized res in reltion to the sides nd centers of the long nd short xes of the cge (Fig. 2). Ech re ws designted by n lphbetic chrcter; res A, B, nd C represented the one-third of the cge t the ir inlet end (front); D, E, nd F represented the trnsverse third (mid); nd G, H, nd I represented the remining third (rer). Ares B, E, nd H were center (medin) long the nrrow xis. The loctions of the feed nd Results Physicl results. (i) Air velocities. Air velocities (ft/min) mesured t 4 cm bove the cge floor, long the medin xis of empty cges tht were fully ssembled nd bedded, t three evenly spced cge loctions (front, center, nd rer) for ll cge ventiltion tretments re shown in Tble 1. Men ir velocities for the high- nd low-velocity cges were 65.3 nd 42.3 ft/min (P < 0.05), respectively. Exhust design did not ffect ir velocity, but intrcge pressures were ± nd ± cm of wter column (P < 0.05) for the single nd porous exhust designs, respectively. (ii) Air tempertures. Air tempertures ( C) mesured t 12.5 nd 19 cm bove the cge floor, long the medin xis of cges contining five mice ech, t three evenly spced cge loctions (front, center, nd rer) for the five different ventiltion tretments re shown in Tble 2. Points of mesurement within the cges showed significntly different ir tempertures, nd there ws no min effect of cge ventiltion tretment or interction. The top nd middle mesurement res of the cges differed (P < 0.05) cross ll ventiltion designs, nd overll mens for ech were ± 0.05 nd ± 0.06 C, respectively. (iii) Reltive humidity. Men reltive humidity (%) inside the different cge ventiltion tretments is shown in Tble 3. When the Tble 1. Effect of cge ventiltion tretment on cge ir velocity Points of mesurement Air velocity (ft/min, men ± stndrd error of the men) ccording to cge ventiltion tretment Loction h (cm) L (cm) HS HP LS LP SM Bottom front ± ± ± ± ± 0.78 Bottom center ± ± ± ± ± 0.25 Bottom rer ± ± ± ± ± 0.0 h, height bove bottom of cge; HP, high-velocity ir supply with ir exhust through filter top; HS, high-velocity ir supply with single-point ir exhust through filter; L, distnce from cge center long medin xis; LP, low-velocity ir supply with ir exhust through filter top; LS, low-velocity ir supply with single-point ir exhust through filter; SM, no mechnicl ir supply. Anlysis of vrince for significnce of min effects: Ventiltion Design = P < 0.001, Points of Mesurement = P < 0.001, Interction = P < Ech vlue obtined from nine seprte mesurements of 20 smples ech with no mice in the cges. Tble 2. Effect of cge ventiltion tretment on ir temperture ( C) nd distribution in cge 6 Points of mesurement Temperture ( C, men ± stndrd error of the men) ccording to cge ventiltion tretment Overll men Loction h (cm) L (cm) HS HP LS LP SM Room 23.7 ± ± ± ± ± ± 0.1 Middle front ± ± ± ± ± ± 0.1 Middle center ± ± ± ± ± ± 0.1 Middle rer ± ± ± ± ± ± 0.1 Top front ± ± ± ± ± ± 0.1 Top center ± ± ± ± ± ± 0.1 Top rer ± ± ± ± ± ± 0.1 Overll men n n 24.8 ± ± ± ± ± 0.1 n h, height bove bottom of cge; HP, high-velocity ir supply with ir exhust through filter top; HS, high-velocity ir supply with single-point ir exhust through filter; L, distnce from cge center long medin xis; LP, low-velocity ir supply with ir exhust through filter top; LS, low-velocity ir supply with single-point ir exhust through filter; n, not pplicble; SM, no mechnicl ir supply. Anlysis of vrince for significnce of min effects: Ventiltion Design = P < 0.117, Points of Mesurement = P < 0.001, Interctions = P < Ech vlue obtined from nine seprte mesurements of 20 smples ech, with five mice per pge. 17

5 Tble 3. Effect of cge ventiltion tretment on ir environment inside cge HS HP LS LP SM Reltive humidity (%) ± ± ± ± ± 2.39 b Prticultes (mg/m 3 ) ± ± ± ± ± NH 3 (ppm) ± ± ± ±28.5 b CO 2 (%) 0.11 ± ± ± ± ± 0.07 b HP, high-velocity ir supply with ir exhust through filter top; HS, high-velocity ir supply with single-point ir exhust through filter; LP, low-velocity ir supply with ir exhust through filter top; LS, low-velocity ir supply with single-point ir exhust through filter; SM, no mechnicl ir supply. Ech vlue (men ± stndrd error of the men) represents replicte mesurements tken weekly over 5-week period. There were five mice per cge nd different mice ech week. b Significntly different (P < 0.05) from vlues for other ventiltion designs. Tble 4. Effect of photoperiod on ctivity nd floor re 2 use 3 for ll cge ventiltion tretments (HS, HP, LS, LP nd SM) 1 Percents of Overll Totl for Observed Behvior nd Loction Photoperiod = Light: Loction: A-front B-front C-front D-mid E-mid F-mid G-rer H-rer I-rer Totl Sleep Groom Move Drink Dig Et Climb Totl Percents of Overll Totl for Observed Behvior nd Loction Photoperiod = Drk: Loction: A-front B-front C-front D-mid E-mid F-mid G-rer H-rer I-rer Totl Sleep Groom Move Drink Dig Et Climb Totl (HS = High velocity ir supply - single point ir exhust through filter, HP = High velocity ir supply - ir exhust through porous filter top, LS = Low velocity ir supply single point ir exhust through filter, LP Low velocity ir supply - ir exhust through porous filter top nd SM = Cge without mechnicl ir supply). 2 Ares A, B, nd C represent the one-third of the cge re t the ir inlet end. Ares B, E, nd H represent the medin third of the cge. Feed nd wter re centered over res D, E, nd F. (see Fig. 2). 3 Vlues represent percent of 375 observed behviors recorded during five replicte evlutions of 15 cges ech (recorded t mid-photophse nd midscotophse). reltive humidity inside ech cge ws subtrcted from the reltive humidity of the room (inlet) ir, the men difference in percent reltive humidity ws 4.5%, 5.0%, 5.6%, 4.6%, nd 34.6% in the HS, HP, LS, LP, nd SM tretments, respectively. When the differences in reltive humidity between inside the cge nd room were compred to vlues when no mice were present in the cges, the men differences were 0.04% nd 10.9% for cges with no mice nd mice (five per cge), respectively. When mice were not present, reltive humidity ws the sme cross ll cge ventiltion designs, s expected. (iv) Prticultes. Airborne prticle mss (mg/m 3 ), smpled on the sme dy s temperture nd humidity, is shown in Tble 3. Prticle counts per cm 3 in the 0.5- to m rnge were skewed to the smller end of the size rnge in ll cge ventiltion tretments. Prticles in the 0.5-to-2- nd 2-to-4- m rnges represented 65% nd 24% of the totl count, respectively. There were no sttisticl differences in prticle counts cross the cge ventiltion tretments. (v) NH 3. NH 3 levels were consistently higher in the SM cges (P < 0.05) thn ny of the other cge tretments (Tble 3). Response of mice to cge environment. (i) Behvior nd bedding mound loctions. From the dt summrized in Tble 4, it is pprent tht more mice were observed t both ends of the cges thn in the center. The stinless-steel cge lid extends to within 4.5 cm of the cge floor in the cge center, in the mid-cge re (floor res D, E, nd F in Fig. 2). Sleeping in group ws the most frequent observtion t both mid-photophse nd scotophse (84% nd 66%, respectively). The percentges of the totl bedding mounds (196) recorded in the front, middle, nd rer res of the cges were 3.0%, 84.2%, nd 12.8%, respectively. Ventiltion tretment did not influence the number or distribution of bedding mounds. When bedding mounds were observed, they generlly extended into two or three res cross the nrrow xis of the cge. Mice generlly slept in hollowed re (nest), nd this nest nd rim ws not recorded s mound. Mounds in the middle of the cge would often extend up to the bottom of the feed nd wter res of the stinless-steel cge lids. (ii) Animl mngement conditions mouse, feed, wter, nd bedding weights. Body weight gin, body weight gin fter the sequentil chnge in cge design, nd wter consumption of mice were lower (P < 0.05) for the week of SM cge ssignment (Tble 5). Weekly increse in bedding weight ws 65 ± 2, 68 ± 4, 68 ± 2, 18

6 Tble 5. Effect of cge ventiltion tretment on mice HS HP LS LP SM Body weight gin 1.59 ± ± ± ± ± 0.32 b Gin for week fter chnge 1.57 ± 0.38 SM to HS 1.76 ± 0.32HS to HP 1.50 ± 0.27HP to LS 1.41 ± 0.29LS to LP 0.54 ± 0.37 b LP to SM Feed consumed 25.8 ± ± ± ± ± 0.61 Wter consumed ± ± ± ± ± 1.08 b HP, high-velocity ir supply with ir exhust through filter top; HS, high-velocity ir supply with single-point ir exhust through filter; LP, low-velocity ir supply with ir exhust through filter top; LS, low-velocity ir supply with single-point ir exhust through filter; SM, no mechnicl ir supply All vlues (g/mouse/week) represent the men ± stndrd error (n = 15) of the men of weekly mesurements from three cges of five mice ech during the week of cge design exposure. Mesurements were replicted over 5-week period. b Significntly different (P < 0.05) from vlues for other ventiltion designs. 71 ± 4, nd 106 ± 4 g/cge in the HS, HP, LS, LP, nd SM cge designs, respectively. All mechniclly ventilted cge designs showed lower bedding weight increse (P < 0.05) thn did the sttic isoltor (SM) cges. When evluted over ll tretments nd replictions, body weight differences did not vry significntly (P < 0.05) between MU nd CU, nd they were independent of cge ventiltion tretment. Men MU (five mice) body weight incresed (P < 0.05) from ± 0.92 g to ± 1.24 g t the beginning of the first nd fifth week of the experimentl period, respectively. Discussion Cge ventiltion designs, which cused different ir velocities within the cge nd different venues of ir exhust, did not influence the men cge ir temperture, nd our finding is similr to results previously reported for sttic nd ventilted cges (6-8). Forced convection het exchnge my hve vried between mice nd their environment t different ir velocity tretments; however, this study ws not designed to evlute different perceived tempertures t different ir velocities. Prticulte levels were not significntly different between the vrious ventiltion configurtions. In generl, the prticulte level of our HEPA-filtered room ir ws low (0.04 mg/m 3 ), nd the men intrcge level ws mg/m 3 higher. In previous study (9) in which room ir hd higher men prticulte level (0.98 mg/m 3 ) thn in our study, the men intrcge level ws 1.03 mg/m 3, which is smller increse thn we mesured. Sttic isoltor cges hd higher men levels of reltive humidity nd NH 3 concentrtions thn did mechniclly ventilted cges, regrdless of ventiltion inlet velocity nd exhust design. Intrcge humidity control ppers more relted to ir exchnge rte thn to other ventiltion prmeters. The ir exchnge rte used in our mechniclly ventilted cges ws round 60 ACH, nd the men intrcge reltive humidity ws 4.95 ± 0.71% higher thn inlet (room) ir. In studies in which vented cges received round 23 ACH, reltive humidity ws 18% higher thn in the room (6), nd significnt decrese in intrcge humidity hs been reported to occur between 40 nd 60 ACH (8). When ir exchnge rte ws in the rnge of 70 to 196 ACH, the cge bedding moisture content ws only 2% while sttic cge bedding moisture content ws 16.4% (10). In our study, the men bedding weight gin ws 1.9 g/mouse/ dy with 60 ACH nd 3.0 g/mouse/dy in the sttic isoltor cge. The most likely explntion for this trend is tht incresed moisture evportion from the bedding occurs in the mechniclly ventilted cges. Where comprisons were mde, intrcge NH 3 levels, like humidity, generlly relted more to ir exchnge rte thn ventiltion design or velocity (1, 6, 8, 9). In our study, with the exception of high NH 3 mesurements from two SM cges (men, 115 nd 120 ppm), NH 3 levels both were consistent within given ventiltion design. Even if these two excessively high NH 3 smples were dropped from the dt nlysis, the NH 3 in the sttic isoltor cges continued to be significntly higher thn in other cge designs, nd the mens were 0.0, 0.18, 0.16, 0.12, nd 1.3 ppm for the HS, HP, LS, LP, nd SM cge ventiltion tretments, respectively. In generl we hve noted in our reserch nd s reported by others tht when men intrcge NH 3 levels re high (> 25 to 50 ppm), there is low ir exchnge, high ir reltive humidity, nd high bedding moisture content. There is lso lrge rnge of NH 3 levels. Another fctor tht should be ssocited with NH 3 levels in mouse cges is bedding ph. In reserch ddressing the mss genertion rte of mmoni from poultry mnure, the ph of the mnure is more closely relted to gseous NH 3 genertion thn to ny other mnure or bedding chrcteristic (11). In tht study, very little NH 3 genertion occurred t phs lower thn 6.5 to 7.0, nd this ssocition is relted to the pk (9.3) of NH 4+. Another importnt fctor tht my hve cused the higher vribility in NH 3 mesured in the SM tretment ws the type of bedding used. Although chipped hrdwood bedding is commonly used in mouse cges, it is not s effective s ground corncob bedding for controlling NH 3 emissions (12). The loctions of certin designted behviors nd bedding mounds we reported were intended to be used for more relistic ppliction of ctul cge conditions in future CFD modeling of mouse cge ventiltion. For exmple, in light of the behvior loction results, n estimted het production from mice sleeping in group likely would be representtive bout 75% of the time. However, becuse mice occupied both ends (especilly the corners) of the cge, locting the mouse group in the center of the rer re of the cge would not lwys be n ccurte representtion. The presence of bedding mounds cross the center width of the cge tht we noted in 30% of observtions should lso to be considered for ventiltion modeling. This fctor would pper to be especilly importnt for modeling mechniclly ventilted cges tht force ir long the long xis ner the floor (bedding) level. Another considertion is tht during the 150 recordings of mound observtions, 61 cges hd no distinct bedding mounds. During the light phse of the dily photoperiod, 55% of the cges hd mounds nd 45% hd no mounds, nd in the drk, 64% hd mounds wheres 36% hd no bedding mounds. These mound loction dt indicte tht the mounds re rerrnged or modified dily nd my be result of the digging behvior tht ws noted for 3.5% of the mice during mid-scotophse. When compred with those of mice in mechniclly ventilted cges, the wter consumption nd body weight gin of our mice were lower when they were housed in the sttic isoltor cges. Men body weight gin during the week tht mice were in the SM cges ws less thn hlf tht of the week before or fter being housed in the SM cges. Body weight gin ws consistently lower in SM cges during ech week of the experiment. During Weeks 4 nd 5, the mice tht were trnsferred into nd housed in the SM cges showed men loss in weight ( 0.09 nd 0.4 g/mouse/week, respectively). Men weekly feed consumed ws not significntly different nd ws 132 nd 127 g/cge of five mice in the mechniclly ventilted cges (HS, HP, LS, nd LP) nd SM cges, respectively. It is possible tht 19

7 more feed ws spilled into nd left in the wetter bedding of the SM cges thn in the ventilted cges, but tht cnnot be confirmed from the dt collected in this study. The results of our study support the need for dditionl reserch tht tests CFD models of ventiltion ptterns in mouse cges nd tht tkes into ccount intrcge therml distribution, humidity, nd bedding mound loction. In ddition, studies ddressing the reltionships between bedding moisture, ge, nd ph nd the mss genertion rte of gseous NH 3 my be beneficil for explining the wide rnge of intrcge NH 3 levels (2). References 1. Lipmn, N. S Isoltor rodent cging systems (stte of the rt): criticl view. Contemp. Top. Lb. Anim. Sci. 38(5): Memrzdeh, F Ventiltion design hndbook on niml reserch fcilities using sttic microisoltors. Ntionl Institutes of Helth, Bethesd, Md. 3. Gordon, C. J Temperture regultion in lbortory rodents. Cmbridge University Press. 4. Ntionl Reserch Council Guide for the cre nd use of lbortory nimls. Ntionl Acdemy Press, Wshington, D. C. 5. Hrrison, P. C., J. D. Ltshw, J. M. Csey, et l Influence of decresed length of different spectrl photoperiods on testis development of domestic fowl. J. Reprod. Fert. 22: Lipmn, N. S., B. F. Corning, nd M. A. Coiro, Sr The effects of intrcge ventiltion on microenvironmentl conditions in filter-top cges. Lb. Anim. 26: Simmons, M. L., D. M. Robie, J. B. Jones, et l Effect of filter cover on temperture nd humidity in mouse cge. Lb Anim. 2: Reeb, C. K., R. B. Jones, D.W. Berg, et l Microenvironment in ventilted niml cges with differing ventiltion rtes, mice popultions nd frequency of bedding chnges. Contemp. Top. Lb. Anim. Sci. 37(2): Murkmi, H Differences between internl nd externl environments of the mouse cge. Lb. Anim. Sci. 21: Wu, D., G. N. Joiner, nd A. R. McFrlnd A forced-ir ventiltion system for rodent cges. Lb. Anim. Sci. 35: Koelkebeck, K. W., P. C. Hrrison, nd G. L. Riskowski Effect of feed dditive or topiclly pplied tretments on the mss genertion rte of mmoni from lying hen mnure. Poultry Sci. 80(Suppl.): Perkins, S. nd N. S. Lipmn Chrcteriztion nd quntifiction of microenviromentl contminnts in isoltor cges with vriety of contct bedding. Contemp. Top. Lb. Anim. Sci. 34(3):