EFFECTS OF HYPOXIA AND HYPEROXIA ON OXYGEN-TRANSFER PROPERTIES OF THE BLOOD OF A VIVIPAROUS SNAKE

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1 The Journl of Experimentl Biology 199, (1996) Printed in Gret Britin The Compny of Biologists Limited 1996 JEB EFFECTS OF HYPOXIA AND HYPEROXIA ON OXYGEN-TRANSFER PROPERTIES OF THE BLOOD OF A VIVIPAROUS SNAKE JAY K. HERMAN* AND ROLF L. INGERMANN Deprtment of Biologicl Sciences, University of Idho, Moscow, ID 83843, USA Accepted 1 My 1996 Red cell oxygen ffinity, red cell nucleoside triphosphte (NTP) levels nd lood oxygen-crrying cpcity were determined for mle, nonpregnnt nd pregnnt femle, nd fetl grter snkes Thmnophis elegns exposed to hypoxi (5 % oxygen) nd hyperoxi (1 % oxygen). Mle nd nonpregnnt femle snkes were mintined under these conditions for up to 3 weeks nd exhiited n pprent mximl chnge in oxygen ffinity fter 14 dys of hypoxi nd hyperoxi. Red cell NTP levels decresed nd oxygen ffinity incresed with exposure to hypoxi, while exposure to hyperoxi promoted n increse in red cell NTP concentrtions nd decrese in red cell oxygen ffinity in the mles. Hyperoxi-exposed nonpregnnt femles did not show significnt chnge in oxygen ffinity. After 14 dys of hypoxi, the pregnnt femles showed n increse in red cell oxygen ffinity which ws Summry ssocited with decrese in red cell NTP concentrtion nd in the molr rtio of NTP/hemogloin reltive to normoxic controls. Fourteen dys of hyperoxi did not result in chnge in oxygen ffinity of red cells from the pregnnt femle, ut did promote slight increse red cell NTP concentrtions. The lood prmeters of fetuses from femles exposed to hypoxi or hyperoxi did not differ from those of normoxic control fetuses. The fetuses of femles exposed to hypoxi suffered greter mortlity, ppered less developed nd hd lower verge wet mss thn the fetuses of normoxic- nd hyperoxic-exposed femles. Neither hypoxi nor hyperoxi ltered the oxygencrrying cpcity of the lood in ny group of snke. Key words: reptile, Thmnophis elegns, fetus, oxygen ffinity, grter snke, viviprity. Introduction Oxygen trnsfer from mother to fetus in the grter snke Thmnophis elegns ppers to e fcilitted y the lood of the fetus hving reltively high ffinity for oxygen nd y pregnncy-ssocited reduction in the oxygen ffinity of mternl lood (Berner nd Ingermnn, 1988; Ingermnn et l. 1991; Rgsdle et l. 1993). It is possile tht the high oxygen ffinity of fetl lood is consequence of fetl hypoxi nd tht the pregnncy-ssocited chnge in mternl lood is consequence of incresed oxygen vilility to the nucleted dult red cell. However, the influence of mient oxygen levels on the lood oxygen ffinity of snke is not known. The reltively high oxygen ffinity of the fetl red cell in T. elegns is not due to unique fetl hemogloin; rther it ppers to e due to reltively low red cell levels of nucleoside triphosphte (NTP, primrily ATP) (Berner nd Ingermnn, 1988). Levels of NTP rise nd oxygen ffinity decreses rpidly in neontl red cells following irth (Ingermnn et l. 1991). Consistent with these oservtions is the possiility tht the fetus is in reltively hypoxic environment which limits red cell oxidtive phosphoryltion, therey keeping NTP levels low nd oxygen ffinity high. A high ffinity would fcilitte the oxygen loding of fetl lood. Conceivly, upon irth, oxygen vilility to these cells increses, oxidtive phosphoryltion nd NTP levels increse, nd oxygen ffinity decreses. This decrese would enhnce oxygen unloding to the tissues. Tht red cell NTP levels nd oxygen ffinities my e ssocited with oxygen vilility in the snke is supported y the findings of Ogo et l. (1993), which show tht the red cells of the viper Bothrops lterntus contin functionl mitochondri nd tht uncoupling of oxidtive phosphoryltion in these cells results in lower ATP levels nd higher ffinity for oxygen. An ssocition etween oxygen vilility nd red cell oxygen ffinity is lso supported y the findings tht mient oxygen levels influence red cell orgnic phosphte levels in the chick emryo (Ingermnn et l. 1983) nd in numerous fishes (e.g. Greney nd Powers, 1978; Nikinm nd Soivio, 1982; Weer nd Lykkeoe, 1978; Wood nd Johnsen, 1972; Wood et l. 1975). One test of the puttive correltion etween the oxygen ffinity of fetl lood nd oxygen vilility vi oxidtive phosphoryltion is to expose the pregnnt femle to ltered levels of mient oxygen. In terms of the fetus, mient *Present ddress: Deprtment of Biology, University of Texs t Arlington, Arlington, TX 7619, USA. Author for correspondence.

2 262 J. K. HERMAN AND R. L. INGERMANN hyperoxi might e expected to rise red cell NTP levels nd decrese red cell oxygen ffinity; hypoxi might produce opposite results, ssuming tht the fetus cn survive sunorml oxygen levels. These expected responses re sed, however, on the ssumption tht oxygen vilility to the fetus is influenced y the gs tht the mother rethes. At lest in mmmls, this hs een shown in severl cute studies. In humns, fetl hert rte slows nd the fetus develops cidosis when the mother is cutely exposed to 1 % oxygen (Wood et l. 1971). In sheep, fetl rteril P O decreses within few minutes of exposure of the pregnnt ewe to % oxygen (Kitnk et l. 1989). The pregnncy-ssocited pproximtely 5 % rise in NTP concentrtions nd the concomitnt decrese in oxygen ffinity of red cells of the dult grter snke pper to e due, t lest in prt, to progesterone (Rgsdle et l. 1993). In mmmls, pregnncy is ssocited with mternl hyperventiltion due, in prt, to chnges in estrogen nd progesterone levels (Byliss et l. 1987; Hnnhrt et l. 1989; Regensteiner et l. 1989). Therefore, could the pregnncy-ssocited rise in red cell NTP levels in T. elegns e ssocited with incresed oxygen vilility to the mternl red cell? If so, hypoxi would e expected to cuse reduction in red cell NTP levels, perhps to levels noted in nonpregnnt nimls or elow these levels. Conversely, hyperoxi might prompt rise in NTP levels in red cells of nonpregnnt dults. We hve tested these possiilities in the current study. This project exmined the reltionship etween mient oxygen nd red cell orgnic phosphte levels nd oxygen ffinity y determining the effects of hypoxi nd hyperoxi on the lood chrcteristics of pregnnt nd nonpregnnt T. elegns. To ssess the time course of responses in red cell NTP levels nd oxygen ffinity to hypoxic nd hyperoxic exposures, mles nd nonpregnnt femles were exposed to 5, 21 or 1 % oxygen for periods of up to 3 weeks. Further, since chnges in mient oxygen levels re correlted with chnges in lood oxygen-crrying cpcity in numerous nimls (e.g. Pinder nd Burggren, 1983; Soivio et l. 198; Wells et l. 1989; Wood nd Johnsen, 1972), this study lso exmined the oxygencrrying cpcities of lood from dult snkes nd fetuses exposed to hypoxi nd hyperoxi for 2 weeks. Mterils nd methods Adult grter snkes, Thmnophis elegns (Bird nd Girrd), were collected in Lth County, ID, USA. Sex ws determined y proing the hemipenes; the mles were housed seprtely from the femles. Femles were determined to e pregnnt y dominl plption. The study used totl of 3 mles (pproximte mss 3 5 g), 28 nonpregnnt femles (5 85 g) nd 17 pregnnt femles (8 13 g). The snkes were kept in 4 l terrri with ccess to n electric light/het source on 12 h:12 h L:D photoperiod. Food ws given in the form of diced mice every 2 weeks (except during experiments) nd wter ws given d liitum. Snkes were mintined prior to nd during experiments t 2 C. Snkes were housed for t lest 2 weeks in the lortory prior to testing to ensure tht they were feeding. Mle snkes were tested throughout the yer nd nonpregnnt femles were studied fter Ferury to ensure tht they were in nonreproductive condition. Pregnnt snkes were ll exposed to the different oxygen tensions over the sme period in July; fetuses were therefore ssumed to e t similr stges of development t the onset of the experiment. Ech dult snke ws tested only once, nd snkes were divided into three experimentl groups: hypoxic (5 % oxygen), normoxic (21 % oxygen) nd hyperoxic (1 % oxygen). The hypoxic condition of 5 % ws chosen on the sis of the findings of Pörtner et l. (1991) in which the tod Bufo mrinus did not pper to e oxygen-stressed t levels ove 2.6 % oxygen. Consequently, the present study focused on the effects of 5 % oxygen; n oxygen concentrtion presumly within the snke s cpcity for eroic respirtion. The snkes were exposed to the differing oxygen tensions for vrious durtions to chrcterize the length of time involved in cclimtion to the mient oxygen level, except for the pregnnt femles which were only exposed to the differing oxygen tensions for 14 dys. Initil hemtologicl vlues were determined from pproximtely.4 ml of lood drwn from the herts of ethernesthetized mle nd nonpregnnt femle snkes, nd hemtocrit, hemogloin (H) concentrtion nd men corpusculr hemogloin concentrtion (MCHC) were mesured. For the determintion of hemogloin concentrtion,.25 ml of the lood ws mixed with 1.2 ml of Drkins solution, frozen nd thwed, then centrifuged t 1 g for 2 min to pellet the red cell ghosts. The hemogloin concentrtion of the superntnt ws determined spectrophotometriclly (Ingermnn et l. 1991). All hemogloin dt refer to tetrmeric hemogloin. MCHC vlues were determined using the hemtocrit vlues of the lood. For NTP nd oxygen ffinity determintions, red cells were wshed three times y centrifugtion (1 g, 5 min, 4 C) in sline uffered to ph 7.4 (in mmol l 1 : 143 NCl, 3 KCl, 1.5 MgCl 2, 1.5 CCl 2, 2 Tris, ph djusted with HCl). After the finl wsh, the cells were resuspended to give hemtocrit of pproximtely 12 %. Throughout the procedure, ll cell suspensions were kept on ice. NTP (=GTP+ATP) concentrtions were determined using Sigm Dignostic Procedure no. 366-UV (Sigm Chemicl Co., St Louis, MO, USA). Red cell NTP concentrtion (in mmol l 1 ) ws clculted s suspension NTP concentrtion divided y suspension hemtocrit. P 5 vlues of the wshed red cell suspension t ph 7.4 were determined using the method of Tucker (1967), with TC5 Tucker cell nd model 781 oxygen meter from Strthkelvin Instruments (Glsgow, Scotlnd). Cells were incuted for 1 min in wterth set t 2 C with prtil pressures of oxygen estlished y mixing nitrogen nd compressed ir. P 5 vlues were determined from Hill plots, ech using 6 1 dt points corresponding to cells tht rnged from 2 to 8 % sturted with oxygen (Hill plots ppered liner over this rnge).

3 Effects of mient O 2 on snke lood O 2 ffinity 263 Animls were plced in the testing chmers 24 h fter the initil lood smpling. The snkes were housed two per chmer; four snkes per experiment. These chmers consisted of 4 l glss continers contining wter owls. Two chmers were ligned in series nd the gs mixture flowed through the chmers t rte of pproximtely 8 ml min 1. Three-wy vlves were plced prior to the first chmer nd etween the two chmers, llowing the gs mixture to e nlyzed oth efore nd etween the chmers. The gs mixture ws nlyzed periodiclly with Servomex 57A Oxygen Anlyser (Syron, Anlyticl Products Division, Boston, MA, USA) to ensure tht the oxygen content of the gs mixture remined constnt throughout the procedure. For normoxic nd hypoxic runs, nitrogen nd compressed ir were mixed to the desired percentge oxygen with FLO-BOX gs mixer (MG Industries, Vlley Forge, PA, USA). For the hyperoxic nimls, 1 % oxygen ws directed first through floting ll flowmeter set t 8 ml min 1. After the test period, lood ws gin drwn s previously descried. Hemtocrit, lood hemogloin, MCHC nd NTP concentrtions, NTP/H rtios nd P 5 were determined for ech niml. Initil hemtologicl vlues were not collected from pregnnt snkes. The pregnnt femles snkes were plced in their respective oxygen environments for 14 dys, fter which they were stunned y low to the hed, decpitted nd lood collected into microcentrifuge tues. The femle s ody ws pcked under ice slush until fetl removl pproximtely 2 3 min lter. Whole-lood hemtocrit nd hemogloin concentrtion were determined. The remining mternl lood ws centrifuged t 1 g for 1 min nd plsm ws collected nd stored t 8 C for lctte concentrtion nlyses. The red cells were resuspended, wshed nd nlyzed for red cell NTP concentrtion, NTP/H rtio nd P 5. Lctte concentrtions were determined using Sigm Dignostics Procedure no. 735 (Sigm Chemicl Co.). Fetl snkes t Zehr stges (Zehr, 1962) were surgiclly removed from the pregnnt femles used ove. (Zehr stge 37 is the lst prentl stge.) The fetus nd its memrnes were trnsferred to n ice-cold sline th to remove the mternl lood. The fetus ws then removed from the fetl memrnes nd trnsferred to second ice-cold sline th. To determine whole-lood vlues, the fetus ws removed from the second sline th, lotted dry, smll portion of the til ws cut off, nd drop of lood collected onto sheet of Prfilm dusted with heprin powder. Smples of whole lood were then immeditely nlyzed s descried previously. The lood from the remining fetuses ws pooled y sectioning the fetuses into heprinized sline nd the crude cell suspension filtered through glss wool to remove tissue frgments. The suspension ws wshed three times nd the red cell prmeters determined s previously descried. A representtive fetus from ech pregnnt femle ws preserved to determine the Zehr stge nd fetl wet mss. Where pproprite, dt re presented s mens ± 1 S.D. For the tulr dt, pired t-test ws used to determine significnt differences etween initil nd finl vlues within group, while Student s t-test ws used to compre finl vlues etween tretment groups. For dt expressed s percentges of initil vlues, n nlysis of vrince (ANOVA) ws used to determine significnt differences etween tretment groups nd the Student Newmn Keuls method ws used when indicted y ANOVA. Since mximl responses ppered to occur t 14 dys, these dt were used for sttisticl nlyses. Results Oxygen ffinity To otin nonpregnnt dult seline dt nd to chrcterize cclimtion time, 14 mles snkes were exposed Percentge of initil P A B Time (dys) Fig. 1. (A) Percentge of initil red cell P 5 for oth mle ( ) nd nonpregnnt femle ( ) grter snkes exposed to hypoxi for differing periods. Initil vlues for mles nd nonpregnnt femles were 3.99±.67 nd 5.4±1.31 kp, respectively. (B) Percentge of initil red cell P 5 for oth mle nd nonpregnnt femle grter snkes exposed to hyperoxi for differing periods. Initil vlues for mles nd nonpregnnt femles were 4.22±.53 nd 4.51±.49 kp, respectively. N=3 for ech group, except t dy 14 where N=5. Vlues re mens ± 1 S.D.

4 264 J. K. HERMAN AND R. L. INGERMANN P5 (kp) 4 3 P5 (kp) Hypoxi Normoxi Hyperoxi Fig. 2. Red cell P 5 (in kp) for oth mle (open columns) nd nonpregnnt femle (crosshtched columns) grter snkes. Hypoxic, normoxic nd hyperoxic groups re compred fter 14 dys of exposure (N=5 for ech group). Like letters indicte significnt difference (P<.5) etween groups (e.g. red cell P 5 vlues differ etween mles exposed for 14 dys to hypoxi nd normoxi). Vlues re mens + 1 S.D. to 5 % oxygen nd 11 mles were exposed to 1 % oxygen for differing periods (Fig. 1A,B). Although no time point hd finl P 5 tht differed significntly from tht of the respective other non-initil time points, the pprent mximl chnge in P 5 ppered to e t dy 14 for oth groups of snkes. In the 14 dy hypoxic group, the P 5 hd decresed significntly from the initil vlues (Fig. 1A). Although there ws no sttisticlly significnt chnge in red cell oxygen ffinity for the hyperoxic mles when expressed reltive to their initil vlues, there did pper to e n upwrd trend in red cell P 5 with such exposure (Fig. 1B). Reltive to normoxic controls t dy 14, mle snkes exposed to hypoxi hd significntly lower P 5 vlue nd those exposed to hyperoxi hd significntly incresed P 5 vlue (Fig. 2). Twenty-two nonpregnnt femle snkes were lso exposed to hypoxi or hyperoxi for vrious times (11 in ech group) (Fig. 1A,B). There ws no sttisticlly significnt chnge in the red cell P 5 for ny of the groups t ny time point. Red cell P 5 t 14 dys for hypoxic femles ws not different from tht of the normoxic femles nor ws the percentge chnge from the initil P 5 vlue different (Figs 1A, 2). Nonetheless, trends in these dt do suggest response similr to tht seen in the mles. Mles nd femles exposed to hypoxi did not differ in their P 5 vlues t dy 14 (Fig. 2). For hyperoxic nonpregnnt femles t dy 14, the red cell P 5 vlue did not differ from tht of normoxic femles nor did the percentge chnge from the initil P 5 vlue differ etween the two groups (Figs 1B, 2). The P 5 vlues for mles nd nonpregnnt femles did not differ fter 14 dys of hyperoxic exposure (Fig. 2). Red cells of pregnnt snkes exposed to 5 % oxygen for 14 dys hd lower P 5 thn did those of pregnnt controls for this time; however, red cell P 5 of pregnnt femles exposed Hypoxi Normoxi Hyperoxi Fig. 3. Red cell P 5 (in kp) for oth pregnnt femles (verticl stripes) nd fetl (horizontl stripes) grter snkes following 14 dys of exposure to hypoxi, normoxi nd hyperoxi. Like letters indicte significnt difference (P<.5) etween groups. N=5 for normoxic nd N=6 for hypoxic nd hyperoxic groups. Vlues re mens + 1 S.D. to 1 % oxygen for 14 dys did not differ from these controls (Fig. 3). Hypoxic exposure of pregnnt femles did not result in chnge in fetl red cell P 5 reltive to normoxic controls (Fig. 3). Although there ws lso no sttisticlly signficnt difference in the red cell P 5 vlue of fetuses in the hyperoxic versus normoxic groups, P vlue of.68 suggests trend towrds significnce (Fig. 3). There were no pprent correltions etween Hill coefficient nd mient oxygen level. Vlues of Hill coefficients for 14 dys of hypoxi, normoxi nd hyperoxi were s follows: mle, 1.99±.24, 2.1±.23 nd 2.6±.41 (N=5,5,5); nonpregnnt femle, 3.17±1.15, 2.49±.52 nd 2.41±.35 (N=5,6,5); pregnnt femle, 2.69±.27, 2.53±.21 nd 2.32±.47 (N=6,5,6); fetus, 1.96±.78, 2.44±.64 nd 2.61±1.18 (N=5,5,6), respectively. Red cell NTP concentrtion nd NTP/H rtio The lower red cell P 5 of mles exposed to 14 dys of hypoxi reltive to normoxic controls (Fig. 2) ws ssocited with significntly lower red cell NTP concentrtion (in mmol l 1 ) nd nonsignificnt downwrd trend in the chnge from initil concentrtion vlues (Fig. 4A,B). In contrst, there were no hypoxi-ssocited chnges in the NTP/H rtio or in the chnge from initil molr rtios in these mles (Fig. 4C,D). The significntly higher red cell P 5 for mles incuted for 14 dys under hyperoxi reltive to normoxic controls (Fig. 2) ws ssocited with significnt increses in red cell NTP concentrtion, percentge chnge from initil concentrtion vlues, finl NTP/H vlues nd percentge chnge from initil NTP/H vlues (Fig. 4A D). There were no sttisticlly significnt differences in red cell P 5 etween nonpregnnt femles exposed to hypoxi versus normoxi nd hyperoxi versus normoxi for 14 dys of

5 Effects of mient O 2 on snke lood O 2 ffinity 265 [NTP] (mmol l 1 ) A d c c d Percentge of initil [NTP] B c c 6 NTP/H rtio C c c Percentge of initil NTP/H rtio D 1. Hypoxi Normoxi Hyperoxi Hypoxi Normoxi Hyperoxi Fig. 4. NTP vlues fter 14 dys of exposure to hypoxi, hyperoxi or normoxi (N=5 for ech group). (A) Red cell NTP concentrtions (mmol l 1 ) for oth mle (open columns) nd nonpregnnt femle (crosshtched columns) snkes. (B) NTP concentrtion s percentge of initil red cell NTP concentrtion for oth mle nd nonpregnnt femle grter snkes. Initil vlues (mmol l 1 ) for mles exposed to hypoxi, normoxi nd hyperoxi were 1.2±1., 1.2±1. nd 1.8±.6, respectively; initil vlues for the femles were 12.8±.8, 1.6±.6 nd 11.5±1.6 mmol l 1, respectively. (C) Molr rtio of NTP/H for oth mle nd nonpregnnt femle grter snkes. H represents tetrmeric hemogloin. (D) NTP/H rtio s percentge of the initil vlue for mle nd nonpregnnt femle grter snkes. Initil molr rtio vlues for mles exposed to hypoxi, normoxi nd hyperoxi were 2.2±.15, 2.27±.16 nd 2.35±.16, respectively; initil vlues for the femles were 2.68±.17, 2.82±.33 nd 3.47±.99, respectively. Like letters indicte significnt difference (P<.5) etween groups. Vlues re mens + 1 S.D. exposure. Nonetheless, there did pper to e trend (similr to the significnt chnge seen in the mles) of incresing P 5 with incresing mient oxygen level (Fig. 2). This trend ws ssocited with significnt differences in the percentge chnge from initil NTP concentrtion in the hypoxi- nd hyperoxiexposed femles reltive to normoxic controls nd significnt difference in NTP concentrtion nd NTP/H rtio etween normoxic nd hyperoxic nonpregnnt femles (Fig. 4A C). Red cells of mle snkes exposed to hyperoxi showed greter chnge in red cell NTP/H thn did those of the femles. Nonetheless, in oth the mles nd nonpregnnt femles, the generl trend ppered to e rise in red cell NTP levels ssocited with rise in P 5 s mient oxygen level incresed. Hypoxi resulted in lower red cell P 5 in pregnnt femles (Fig. 3). Associted with this chnge, hypoxi-exposed pregnnt femles showed lower red cell NTP levels nd NTP/H rtios thn did the normoxic pregnnt femles (Fig. 5A,B). Plsm lctte concentrtions did not differ etween these two groups of pregnnt femles (1.8±.42 mmol l 1, N=6, nd 1.24±1.21 mmol l 1, N=5, respectively). Hyperoxi did not pper to ffect red cell P 5 nor NTP/H rtio in the pregnnt femles (Figs 3, 5B). However, such exposure ws ssocited with greter red cell NTP concentrtion reltive to the normoxic pregnnt femles (Fig. 5A). Plsm lctte concentrtions did not differ etween hyperoxic pregnnt femles (2.4±1.21 mmol l 1, N=6) nd normoxic controls. The fetuses of pregnnt femles exposed to either hypoxi or hyperoxi did not differ from the fetuses of pregnnt femles exposed to normoxi in their red cell NTP concentrtions or NTP/H vlues (Fig. 5). This corresponds to the lck of effect of mient oxygen levels on fetl red cell P 5 (Fig. 3).

6 266 J. K. HERMAN AND R. L. INGERMANN Tle 1. Initil nd finl (14 dys) vlues for men corpusculr hemogloin concentrtion nd hemtocrit Hypoxi Normoxi Hyperoxi Initil Finl Initil Finl Initil Finl Mle MCHC (mmol l 1 ) 4.28± ± ± ± ± ±.39 Hct (%) 25.6±7.2* 21.6±4.5* 25.7± ± ±2.4* 19.2±2.5* Nonpregnnt femle MCHC (mmol l 1 ) 4.81± ± ± ± ± ±.49 Hct (%) 22.3± ± ± ± ±3.6** 23.1±2.9** Pregnnt femle MCHC (mmol l 1 ) 5.23± ± ±.48 Hct (%) 23.8± ± ±3.6 Fetus MCHC (mmol l 1 ) 3.83± ± ±.3 Hct (%) 23.6± ± ±2.5 *P<.5 (initil versus finl); **P<.1 (initil versus finl). MCHC, men corpusculr hemogloin concentrtion (hemogloin expressed s the tetrmeric form); Hct, hemtocrit. Vlues re mens ± 1 S.D.; N=5, except for pregnnt femle nd fetuses for hypoxi nd hyperoxi where N=6. However, the fetuses of hyperoxi-exposed femles did hve greter red cell NTP concentrtion (ut not NTP/H) thn those of hypoxi-exposed femles (Fig. 5). reltive to their initil individul vlues. This chnge ws not, however, ssocited with chnges in either lood hemogloin concentrtions (Fig. 6) or MCHC vlues (Tle 1). Oxygen-crrying cpcity Exposure of snkes to 14 dys of hypoxi or hyperoxi resulted in few significnt chnges in prmeters relted to oxygen-crrying cpcity (Tle 1; Fig. 6A,B). Reltive to normoxic controls, there were no differences in lood hemogloin concentrtion, MCHC or hemtocrit, expressed s finl vlues or s percentge chnges from initil vlues, for mles nd nonpregnnt femles. Similrly, there were no differences in these finl prmeters etween normoxic controls nd hypoxi- or hyperoxi-exposed pregnnt femles or their fetuses. Significnt chnges were oserved in mles exposed to hypoxi or hyperoxi for 14 dys reltive to their initil individul vlues. In ech experiment, lood hemogloin concentrtions decresed significntly (from 1.9±.33 to.94±.25 mmol l 1, N=5, for hypoxi; from 1.9±.3 to.87±.11 mmol l 1, N=5, for hyperoxi), s did lood hemtocrit (from 25.6±7.2 to 21.6±4.5 %, N=5, for hypoxi; from 25.8±2.4 to 19.2±2.5 %, N=5, for hyperoxi), ut in neither experiment ws there significnt difference etween initil nd finl vlues of MCHC (Tle 1). Hyperoxi resulted in decresed hemtocrit (from 26.4±3.6 to 23.1±2.9 %, N=5) in the nonpregnnt femles Fig. 5. NTP concentrtion nd NTP/H rtio for pregnnt femles nd fetuses fter 14 dys of exposure to hypoxi, normoxi nd hyperoxi. (A) Red cell NTP concentrtions (mmol l 1 ) for pregnnt femles (verticl stripes) nd fetuses (horizontl stripes). (B) NTP/H rtio for oth pregnnt femles nd fetuses. Like letters indicte significnt difference (P<.5) etween groups. Numers of individuls re s indicted in Fig. 3. Vlues re mens + 1 S.D. [NTP] (mmol l 1 ) NTP/H rtio 2 A B c Hypoxi Normoxi c Hyperoxi

7 Effects of mient O 2 on snke lood O 2 ffinity A Fetl wet mss (g) 2 1 [H] (mmol l 1 ) B Hypoxi Normoxi Hyperoxi Fig. 7. Averge fetl wet mss fter 14 dys of mternl exposure to hypoxi, normoxi or hyperoxi (N=5 for ech group.) Like letters indicte significnt difference (P<.5) etween groups. Vlues re mens + 1 S.D. 1. hypoxi-exposed femles were primrily in Zehr stges 35 36, while the control fetuses rnged from stges 35 to 37 nd the fetuses of the hyperoxi group were in stges Hypoxi Normoxi Hyperoxi Fig. 6. Blood hemogloin concentrtions (in mmol l 1 ) following 14 dys of exposure to hypoxi, normoxi nd hyperoxi. (A) Mle (open columns) versus nonpregnnt femle (crosshtched columns) (N=5, ech group). (B) Pregnnt femle (verticl stripes) versus fetl (horizontl stripes) snkes (N=5 for normoxic nd N=6 for hypoxic nd hyperoxic groups). There were no significnt differences etween the hypoxi versus normoxi versus hyperoxi groupings. Vlues re mens + 1 S.D. Mortlity nd development During the time course of the study, no dult snkes died in the hypoxi or normoxi experiments; one dult died during hyperoxi-exposure fter 2 dys. There ws no ovious fetl mortlity in pregnnt femles exposed to normoxi or hyperoxi. Of six pregnnt femles exposed to hypoxi, two hd pprently norml litters. The remining four pregnnt femles contined litters with % ded fetuses. (Blood ws only collected from live fetuses.) The verge wet mss of the live fetuses from hypoxiexposed femles ws lower thn the verge mss of normoxic control fetuses (Fig. 7). The verge wet mss of fetuses in the hyperoxic group did not differ from the verge mss of the control fetuses ut ws, however, greter thn tht of the fetuses from femles exposed to hypoxi. The fetuses from Discussion Oxidtive phosphoryltion is the primry energy source in the nucleted red cells of non-mmmlin vertertes (Bouverot, 1985). Thus, decrese in oxygen vilility my limit oxidtive phosphoryltion nd led to decrese in red cell ATP concentrtions, while n increse in oxygen vilility my ccomplish the opposite. Since nucleoside triphosphtes re importnt llosteric regultors of hemogloin function in the red cells of most ectotherms, oxygen vilility my influence red cell oxygen ffinity in these orgnisms. Dt from fishes pper to support link etween oxygen vilility nd red cell function. Fish show decresed red cell orgnic phosphte content nd n incresed red cell oxygen ffinity with hypoxic cclimtion (e.g. Greney nd Powers, 1978; Soivio et l. 198; Wells et l. 1989). Red cells incuted in vitro under metolic inhiition show similr chnge (Greney nd Powers, 1978; Tetens nd Lykkeoe, 1981), while trout red cells cutely exposed in vitro to hyperoxi increse their NTP concentrtions (Lne nd Di, 1992). However, not ll orgnisms with nucleted red cells respond s do the fishes to ltered oxygen vilility. The oxygen ffinity of red cells of mphiins does not pper to exhiit cler pttern of response when nimls re chroniclly exposed to low mient oxygen levels (Pinder nd Burggren, 1983; Wells et l. 1989), while red cells of irds pper unresponsive under these conditions (McGrth, 1971; Pionetti nd Bouverot, 1977). Whether the nucleted reptilin red cell is responsive to the oxygen vilility of orgnism is not known. However, it my e expected on the sis of the findings of Ogo et l. (1993), which demonstrted correltions

8 268 J. K. HERMAN AND R. L. INGERMANN etween oxidtive phosphoryltion, intrcellulr ATP production nd hemogloin oxygen ffinity from studies on isolted mitochondri nd hemogloin from Bothrops lterntus red cells. Consequently, this study exmined the response of the grter snke T. elegns to chnges in the mient P O. Blood NTP concentrtions of crp nd trout respond to chnges in wter oxygen tension, nd full cclimtion of the lood requires pproximtely 1 week or more (Soivio et l. 198; Weer nd Lykkeoe, 1978). Therefore, the initil experiments of this study exmined the temporl response of the nonpregnnt dult snke to chnges in mient oxygen tension. Except for the nonpregnnt femle exposed to hyperoxi, the grter snke exhiited its pprent mximl chnge in red cell oxygen ffinity fter 2 weeks of exposure to the ltered oxygen environment. Mles nd femles tended to respond to hypoxi with n increse nd to hyperoxi with decrese in red cell oxygen ffinity (Figs 1, 2). Generlly, corresponding to the chnge in oxygen ffinity ws n pprent chnge in the red cell NTP concentrtion (rther thn in the molr NTP/H rtio) (Fig. 4). Nonetheless, corresponding to wht my e considered mjor chnges in oxygen vilility were reltively limited responses in orgnic phosphte levels nd oxygen ffinity: generlly chnges of 2 % or less. Therefore, while the snke ppered to respond to low nd high mient oxygen levels in predicted directions, the mgnitude of the response ws reltively modest. The mgnitude of this response to hypoxi in the snke ppered to e intermedite etween the slight chnges in red cell oxygen ffinity oserved for the dult tod Bufo mrinus (Wells et l. 1989) or ullfrog Rn ctesein tdpoles (Pinder nd Burggren, 1983) nd the 35 % chnge in red cell oxygen ffinity of dult R. ctesein (Pinder nd Burggren, 1983). Further, the mgnitude of this response in nonpregnnt femle T. elegns suggests tht ny hypotheticl increse in oxygen vilility (for exmple, due to hyperventiltion nd/or greter pulmonry perfusion) during pregnncy my contriute to, ut cnnot ccount for, the pregnncy-ssocited rise in red cell NTP levels nd decrese in red cell oxygen ffinity seen in this species (Ingermnn et l. 1991). To test further the possiility tht the pregnncy-ssocited rise in red cell NTP levels is relted to chnged oxygen vilility during pregnncy, this study exmined the effect of 14 dys of hypoxi or hyperoxi exposure on pregnnt T. elegns. Pregnnt snkes exposed to hypoxi hd higher red cell oxygen ffinity thn pregnnt snkes exposed to normoxi (Fig. 3). Corresponding to this incresed oxygen ffinity ws lowered red cell NTP concentrtion nd NTP/H rtio (Fig. 5A,B). Hypoxic exposure did not reduce the red cell NTP concentrtion to nonpregnnt vlues, gin suggesting, s ove, tht oxygen vilility cnnot lone ccount for the pregnncy-ssocited rise in the NTP concentrtion of the dult red cell. Hyperoxi did not induce significnt chnge in the red cell oxygen ffinity in the pregnnt femle. Nonetheless, the pregnnt femle fter 14 dys of hyperoxi did hve slightly elevted red cell NTP concentrtion reltive to tht of the normoxic controls, lthough this elevtion ws not reflected in n ltered NTP/H rtio. Therefore, in terms of oxygen ffinity nd red cell NTP levels, pregnnt femles ppered to respond similrly to chnges in oxygen vilility s did nonpregnnt dults, especilly mles. This response, however, does not pper to e of sufficient mgnitude to ccount for the pregnncy-ssocited rise in red cell NTP levels. Upon irth, red cell NTP concentrtions of the neonte rise nd oxygen ffinity decreses to nonpregnnt dult levels within out 6 h (Ingermnn et l. 1991). Further, in vitro incution of neontl red cells with metolic inhiitors suggests tht oxygen vilility underlies these rpid red cell trnsitions t prturition (Ingermnn et l. 1991). These dt further imply tht the grter snke fetus is reltively hypoxic prior to irth. In mmmls, the oxygen tension tht the pregnnt femle rethes influences oxygen vilility to the fetus (Kitnk et l. 1989; Wood et l. 1971). If the snke fetus is lso sensitive to the oxygen tension rethed y the mother, hyperoxic exposure my reduce puttive fetl hypoxi (prompting rise in red cell NTP level), while mternl hypoxi my excerte such hypoxi (perhps prompting decrese in NTP level). However, fter 14 dys of mternl exposure, neither red cell oxygen ffinity nor NTP levels of fetl grter snkes hd responded to chnges in mternl oxygen vilility reltive to normoxic controls. Although not significnt, there did pper to e n upwrd trend in the fetl P 5 vlues with mternl hyperoxi, Fig. 3. The only significnt response ws etween the extremes of 5 % nd 1 % oxygen. Fetl grter snkes exposed to these conditions differed in their red cell NTP concentrtions (Fig. 5A), lthough neither vlue differed from tht of the normoxic control. Tht fetuses not only experienced the ltered oxygen environment ut were lso ffected y the lowered oxygen tension ville to the mother is supported y the increse in fetl mortlity nd the decrese in fetl wet mss in the hypoxic group (Fig. 7). A reduction in fetl irth mss with mternl hypoxi hs een noted in humns (e.g. Kruger nd Aris- Stell, 197; Lichty et l. 1957; Moore et l. 1982). These dt indicte tht the fetuses did experience chnges in their oxygen vilility, t lest during mternl hypoxi, despite lck of chnge in red cell oxygen ffinity or NTP levels, chnges tht re ssocited with irth (Ingermnn et l. 1991). These oservtions suggest two principl possiilities. (1) Conceivly, the fetus of the hyperoxic femles did not experience n increse in oxygen vilility commensurte with tht occurring upon irth (e.g. perhps due to vsculr shunting of lood from the fetus). (2) Alterntively, there is no direct link etween oxygen vilility nd red cell NTP levels, i.e. the mrked increse in red cell NTP concentrtions which occur t irth re not due simply to sudden increse in oxygen vilility ssocited with the onset of irrething; some other or dditionl stimulus or interction my e responsile for this chnge t irth. This ltter possiility is supported y the oservtion tht the effect of hypoxi on the red cell NTP levels of intct fish occurs t oxygen tensions well ove those required to sturte cytochrome oxidse nd cn

9 Effects of mient O 2 on snke lood O 2 ffinity 269 e mimicked in vitro only y incution of red cells under noxi (Greney nd Powers, 1978; Tetens nd Lykkeoe, 1981). Therefore, s suggested y Jensen et l. (199) nd Nikinm (199), the influence of oxygen vilility on the NTP concentrtions of nucleted red cells my not e due to direct effect on oxidtive phosphoryltion ut rther involve or require dditionl input from the orgnism s whole. As such, events ssocited with irth (other thn the initition of ir rething) my llow the red cells of the neonte to respond to incresed oxygen levels with chnge in red cell NTP level nd oxygen ffinity. Hypoxic cclimtion in fishes nd mmmls is ssocited with n increse in the oxygen-crrying cpcity of the lood (e.g. Bouverot, 1985; Soivio et l. 198; Wells et l. 1989; Wood nd Johnsen, 1972). Exposure to hyperoxi in the trout results in decrese in the lood hemogloin concentrtion (Jewett et l. 1991; Lne et l. 1991). We predicted similr responses in these grter snkes, yet no snkes in the hypoxic, hyperoxic or control groups showed ltered oxygen-crrying cpcities when compred with initil vlues. Tht there were no differences mong the test groups nd the controls suggests tht mient oxygen levels do not lter hemogloin/red cell production during the durtion of these experiments. The unresponsiveness of the grter snke s oxygen-crrying cpcity to low oxygen tension ws similr to tht reported for Bufo mrinus (Wells et l. 1989) ut differed from the response of Rn ctesein, which increses its oxygencrrying cpcity (Pinder nd Burggren, 1983). Like the lood of R. ctesein, the oxygen-crrying cpcity of the lood of the grter snke ppered to e unresponsive to hyperoxi. The results of the present study of the grter snke indicte tht this terrestril ectotherm generlly shows modest chnges in red cell oxygen ffinity nd NTP concentrtion ut not in lood oxygen-crrying cpcity in response to hypoxi or hyperoxi. The mximum response of oxygen ffinity nd NTP concentrtions to oxygen vilility ppers to require severl weeks to develop, nd chnges occur in directions consistent with those noted especilly in fishes. Finlly, oxygen vilility does not pper to e le to ccount directly for the pregnncy-ssocited rise in red cell NTP levels, stedystte fetl red cell NTP levels or the rise in NTP levels in the red cells of the neonte upon irth. We re grteful to Drs T. A. McKen nd F. R. Rgsdle for comments on the concepts, dt nd mnuscript. This study ws supported in prt y grnt from the Idho Stte Bord of Eduction. References BAYLISS, D. A., MILLHORN, D. E., GALLMAN, E. A. AND CIDLOWSKI, J. A. (1987). Progesterone stimultes respirtion through centrl nervous system steroid receptor-medited mechnism in ct. Proc. ntn. Acd. Sci. U.S.A. 84, BERNER, N. J. AND INGERMANN, R. L. (1988). Moleculr sis of the difference in oxygen ffinity etween mternl nd foetl red lood cells in the viviprous grter snke, Thmnophis elegns. 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Effects of hyperoxi on some fctors influencing the oxygen-crrying cpcity of trout lood. Am. Zool. 31, 73A (Astrct). LICHTY, J. A., TING, R. Y., BURNS, P. D. AND DYAR, E. (1957). Studies of ies orn t high ltitude. I. Reltions of ltitude to irth weight. Am. med. Ass. J. Dis. Child. 93, MCGRATH, J. J. (1971). Acclimtion response of pigeons to simulted high ltitude. J. ppl. Physiol. 31, MOORE, L. G., ROUNDS, S. S., JAHNIGEN, D., GROVER, R. F. AND REEVES, J. T. (1982). Infnt irth weight is relted to mternl rteriole oxygention t high ltitude. J. ppl. Physiol. 52, NIKINMAA, M. (199). Verterte Red Blood Cells. Berlin: Springer- Verlg. NIKINMAA, M. AND SOIVIO, A. (1982). Blood oxygen trnsport of hypoxic Slmo girdneri. J. exp. Zool. 219, OGO, S. H., BERNARDES, C. F., GLASS, M. L., TORSONI, M. A. AND VERCESI, A. E. (1993). Functionl mitochondri in snke Bothrops lterntus erythrocytes nd modultion of HO 2 ffinity y mitochondril ATP. J. comp. Physiol. B 163, PINDER, A. AND BURGGREN, W. (1983). 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10 27 J. K. HERMAN AND R. L. INGERMANN ltitude on oxygen ffinity nd orgnic phosphte concentrtions in pigeon lood. Life Sci. 2, PÖRTNER, H. O., MACLATCHY, L. M. AND TOEWS, D. P. (1991). Metolic responses of the tod Bufo mrinus to environmentl hypoxi: n nlysis of the criticl P O. Physiol. Zool. 64, RAGSDALE, F. R., IMEL, K. M., NILSSON, E. E. AND INGERMANN, R. L. (1993). Pregnncy-ssocited fctors ffecting orgnic phosphte levels nd oxygen ffinity of grter snke red cells. Gen. comp. Endocr. 91, REGENSTEINER, J., WOODARD, W. D., HAGERMAN, D. D., WEIL, J. V., PICKETT, C. K., BENDER, P. R. AND MOORE, L. G. (1989). Comined effects of femle hormones nd metolic rte on ventiltory drives in women. J. ppl. Physiol. 66, SOIVIO, A., NIKINMAA, M. AND WESTMAN, K. (198). The lood oxygen inding properties of hypoxic Slmo girdneri. J. comp. Physiol. B 136, TETENS, V. AND LYKKEBOE, G. (1981). Blood respirtory properties of rinow trout, Slmo girdneri: Responses to hypoxi cclimtion nd noxic incution of lood in vitro. J. comp. Physiol. B 145, TUCKER, V. A. (1967). Method for oxygen content nd dissocition curves on microliter lood smples. J. ppl. Physiol. 23, WEBER, R. E. AND LYKKEBOE, G. (1978). Respirtory dpttions in crp lood influences of hypoxi, red cell orgnic phosphtes, divlent ctions nd CO 2 on hemogloin oxygen ffinity. J. comp. Physiol. 128, WELLS, R. M. G., GRIGG, G. C., BEARD, L. A. AND SUMMERS, G. (1989). Hypoxic responses in fish from stle environment: lood oxygen trnsport in the ntrctic fish Pgotheni orchgrevinki. J. exp. Biol. 141, WELLS, R. M. G., TREUMAN, B. J. AND BRITTAIN, T. (1989). Orgnic phosphte hemogloin interctions pper non-dptive in the hypoxic tod, Bufo mrinus. Comp. Biochem. Physiol. 92B, WOOD, C., HAMMOND, J., LUMLEY, J. AND NEWMAN, W. (1971). Effect of mternl inhltion of 1 % oxygen upon the humn fetus. Aust. N.Z. J. Ostet. Gynec. 11, WOOD, S. C. AND JOHANSEN, K. (1972). Adpttion to hypoxi y incresed HO 2 ffinity nd decresed red cell ATP concentrtions. Nture 237, WOOD, S. C., JOHANSEN, K. AND WEBER, R. E. (1975). Effects of mient P O on hemogloin oxygen ffinity nd red cell ATP concentrtions in enthic fish, Pleuronectes pltess. Respir. Physiol. 25, ZEHR, D. R. (1962). Stges in the norml development of the common grter snke, Thmnophis sirtlis sirtlis. Copei 1962,