Electrophoretic proteinogrm reference intervl from Argentin Northestern cptive cimns (crocodyli: Alligtoride) COPPO, J.A. 1 ; MUSSART, N.B. 1 ; BARBOZA, N.N. 1 ; ZEINSTEGER, P.A. 1 ; PRADO, W.S. 2 SUMMARY The purpose of the study ws to estlish the reference intervl for serum electrophoretic proteinogrm from cptive Northestern Argentinen cimns, s well s to detect physiologicl vritions ttriutle to species, sex, ge, feeding nd yer seson. Smples from 223 helthy su-dults, oth sexes specimens of Cimn ltirostris (n=109) nd Cimn ycre (n=114), were otined. Totl protein (4.01±0.61 g/dl) ws determined y spectrophotometry. Vlues from lumin (0.96±0.20 g/dl) nd gloulins lph (0.74±0.16 g/dl), et (0.87±0.18 g/dl) nd gmm (1.39±0.27 g/dl), were otined y electrophoresis on cellulose cette nd densitometric vlution. Presence of pre-lumin on nd comprised etween 20-66 kd, were detected y electrophoresis on polycrilmide gel. Reptile lumin registered moleculr weight from 69 kd. Protein vlues were higher in mles thn in femles, eing significntly higher (p<0.05) for gmm gloulin. All the studied prmeters were lower in C. ltirostris thn in C. ycre. Growing produced significnt increse of totl protein, lumin, nd lumin/gloulin rtio. On the contrry, these prmeters were lower during winter. Hndling nd feeding systems used with frmed reptiles cused totl protein nd lumin vlues higher thn those registered in zoo specimens. Otined dt re pplicle for cimn s nutritionl control, s well s for dignosis nd illness prevention. Key words: (Cimn ltirostris), (Cimn ycre), (electrophoretic proteinogrm), (reference intervl), (physiologicl vritions). 1 Cátedr de Fisiologí, Fcultd de Ciencis Veterinris, UNNE, Srgento Crl 2139, Corrientes (3400), Argentin. E-mil: jcoppo@vet.unne.edu.r 2 Refugio «El Cchpé» (Chco), Fundción Vid Silvestre Argentin. * Proyecto 16/Q-267 (CIDET, UNAM). Reciido: myo 2005. Aceptdo: diciemre 2005. Versión on line: diciemre 2005. ISSN(ppel): 1514-6634 129 coppo.pmd 129 25/10/2006, 22:52
Intervlo de referenci pr el proteinogrm electroforético de cimnes del nordeste rgentino en cutiverio (Crocodyli: Alligtoride) RESUMEN El propósito del estudio fue estlecer el intervlo de referenci pr el proteinogrm electroforético sérico de cimnes rgentinos en cutiverio, sí como detectr vriciones fisiológics triuiles especie, sexo, edd, limentción y estción del ño. Se empleron 223 ejemplres snos, su-dultos de mos sexos, de ls especies Cimn ltirostris (n=109) y Cimn ycre (n=114). Ls proteíns totles (4,01±0,61 g/dl) fueron determinds por espectrofotometrí. Medinte electroforesis en cetto de celulos y vlorción densitométric, se otuvieron vlores pr lúmins (0,96±0,20 g/dl) y gloulins lf (0,74±0,16 g/dl), et (0,87±0,18 g/dl) y gmm (1,39±0,27 g/dl). Por electroforesis en gel de policrilmid se verificron los pesos moleculres de pre-lúmins (20-66 kd) y lúmins (69 kd). Los vlores proteicos fueron myores en mchos que en hemrs, significtivmente pr gmm gloulins (p<0,05). Todos los prámetros fueron más jos en C. ltirostris que en C. ycre. El crecimiento produjo umentos significtivos de proteíns totles, lúmins y relción lúmins/gloulins, prámetros que resultron más jos en invierno. Los sistems de mnejo y limentción plicdos en un cridero produjeron vlores de proteíns totles y lúmins superiores los registrdos en un zoológico. Los dtos otenidos son plicles l control nutricionl, sí como l dignóstico y prevención de enfermeddes de estos reptiles. Plrs clve: (Cimn ltirostris), (Cimn ycre), (proteinogrm electroforético), (intervlo de referenci), (vriciones fisiológics) INTRODUCTION Plsm protein concentrtion widely vries in different species, etween rnges from 15 g/dl in cephlopods to 0.1 g/dl in mollusks. By electrophoresis, proteins cn seprte in different frctions, minly pre-lumin, lumin nd lph, et nd gmm gloulins; some species possess lumin concentrtion higher thn totl gloulins, nd vicevers 2. Some dt out serum totl protein nd lumin vlues were reported for Argentinen northestern utochthonous cimns; tough they were otined y different techniques thn those used in the present work 5,18,19 (fst methods), nd considering lower smple mount. Correct interprettion of reptile physiologicl phenomen, s well s pproprite illness dignosis nd nutritionl control, require deep knowledge concerning serum proteins, s they re responsile of importnt functions such s trnsport, nutrition, clotting, immunity, enzymtic control, cid-se lnce, internl environment viscosity nd coloidosmotic pressure mintennce 2,8,10. The ojective of this study ws to otin the reference intervl for serum protein electrophoreticl frctions from cptive Cimn ltirostris nd Cimn ycre, s well s to estlish eventul differences ttriutle 130 ISSN(ppel): 1514-6634 coppo.pmd 130 25/10/2006, 22:52
to species, sex, ge, feeding nd seson of the yer. MATERIALS AND METHODS Experimentl sujects. During 2 yers (2003-2004), 223 su-dults 20 cimns, 50% ech sex, cliniclly helthy, from C. ltirostris (n=109) nd C. ycre (n=114) species, were studied. Helth stte ws tested y exms of generl stte, dynmism, vivcity, ppetite, skin nd mucous colortion, nd sence of externl lesions. Some of them (n=29) were mintined t the Zoo of Corrientes City, in smll ponds, without roof, with running wter, which ws constntly renovted; they were fed on chicken viscer nd fish. Remining nimls (n=194) were housed t the «El Cchpé» frm (Chco, northestern Argentin), in roofed tnks with underground wter, which ws renewed dily nd styed t 27 ± 3ºC (heted y gs), fed on met flour supplemented with vitmins nd minerls; spordiclly they received ovine viscer. To evlute growth, reptiles were divided in 3 development stges, considering live weight nd corporl longitude. Blood smpling. Hemtic nd morphometric studies were performed 4 times/ yer, in ech seson, during morning hours (8-9 h.m.), fter 12 h fst. Live weight ws otined using portle lnce nd corporl dimensions were evluted y mens of metllic tpe mesure. Blood smpling ws crried out y mens of post occipitl venous sinus punction 9, while reptile ws mnully held nd his jw remined tied. Blood ws plced in glss tues nd once cogulted it ws centrifuged to otin serum, which ws mintined cooled t 5ºC until its nlysis, which ws mde efore 3 hours post-extrction. Lortory techniques. Totl protein concentrtion ws determined in L.Mnnheim 4010 spectrophotometer (iuret method, 540 nm, Wiener L regents) 10. Protein frctions were seprted y electrophoresis on cellulose cette (Cellogel) nd quntified t 610 nm in n utomted Citocon CT-440 densitometer 10,13, equipped with microprocessor for protein frction clcultions, nd printer module. Electrophoresis on polycrilmide gel (SDS- PAGE under reductive conditions, Sigm) ws lso mde 13. All iochemicl determintions were checked y qulity control system, using reference ptterns nd moleculr weight mrkers, s ovine serum lumin (66 kd), ovlumin (45 kd), glicerldehyde-3- phosphte-dehydrogense (36 kd), cronic nhydrse (29 kd), tripsinogen (24 kd), trypsin inhiitor (20 kd), nd lphlctlumin (14.2 kd). Sttisticl nlysis. A totlly rndomized design ws used. Distriutive normlity ws verified y Wilk-Shpiro test (WS). Prmetric sttistics included mesures from centrl tendency (rithmetic men, x) nd dispersion (stndrd devition, SD). Fiduciry proility ws evluted y confidence intervls (CI±95%). Homogeneity of the vrince ws verified y Brtlett test. Anlysis of vrince (ANOVA) ws mde y one-wy liner model. Men comprisons were crried out y Tukey test. Coefficient of linel ssocition ws evluted y correltion (Person test). Clcultions were mde with the id of sttisticl softwre (Sttistix 1996). For ll inferences 5% significnce ws specified, elow which the equlity null hypothesis ws rejected. RESULTS Glol vlues otined for oth reptile species re shown in Tle 1. WS coefficients revel n pproximtely norml distriution. ISSN(ppel): 1514-6634 131 coppo.pmd 131 25/10/2006, 22:52
Dt dispersion (SD) did not exceed the limits recommended y prmetric sttistics. Confidence intervls were djusted round rithmetic mens, ut individul rnges were wide. Proteinogrms on cellulose cette otined in C. ltirostris nd C. ycre re shown in Figure 1. They revel the presence of lumin nd gloulins lph, et nd gmm, ut not of pre-lumin. Slight differences in migrtion velocity were registered etween species. In Figure 2, serum frctions from oth crocodile species otined y polycrilmide gel electrophoresis, re compred to those from reference ptterns, s well to those registered in humn eings. Low pre-lumin quntities re evidenced. Confronting simultneously ser versus moleculr weight mrkers, differences etween humn eings nd reptiles stnd out, ut differences mong reptiles re not registered. Vlues otined on ech studied species re shown in Tle 2. Totl protein, et nd gmm gloulins were significntly higher in C. ycre (p < 0.05). Gmm gloulin concentrtion ws higher in mles (Tle 3). When growth progressed, increses of totl protein, lumin nd lumin/gloulin rtio were registered. Significntly higher totl protein nd lumin levels were registered in spring nd summer; concentrtions decresed during utumn nd reched the lowest vlues in winter (Tle 4). Food supplied in the frm promoted vlues of totl protein, lumin nd lumin/gloulin rtio higher thn those registered in the zoo. Person test reveled high liner ssocition degree (p<0.05) etween live weight nd vriles s totl longitude (r = 0.90), muzzle-til longitude (r = 0.83), hed longitude (r = 0.79), hed wide (r = 0.86), nd thorcic perimeter (r = 0.88). Considering growth stges, live weight correlted to totl Figure 1. Cellulose cette electrophoresis from C. ltirostris (1, 3, 5) nd C. ycre (2, 4, 6). Slight differences on migrtion velocity were registered on ech species protein frctions. Figure 2. Polycrilmide gel electroforesis from C. ltirostris (2, 3, 4), C. ycre (6, 7, 8) nd humn eings (1, 9). In chnnel 5 run reference pttern, which included (from top to ottom), mrkers of 66, 45, 36, 29, 24, 20 nd 14,2 kd. Pre-lumin presence ws evident in ll cses (rrows). protein (r = 0.92; p = 0.01), lumin (r = 0.89; p = 0.03) nd lumin/gloulin rtio (r = 0.94; p = 0.001). Increse of totl longitude lso correlted to the rise of sme proteic vriles. 132 ISSN(ppel): 1514-6634 coppo.pmd 132 25/10/2006, 22:52
Tle 1. Otined vlues in totl studied popultion (n = 223) Prmeter ξ ± SD W S CI ± 95% Rnge Totl protein (g/dl) 4.01 ± 0.61 0.995 3.91-4.08 2.30-5.84 lumin (g/dl) 0.96 ± 0.20 0.981 0.93-0.99 0.50-1.65 lph gloulin (g/dl) 0.74 ± 0.16 0.985 0.72-0.77 0.34-1.29 et gloulin (g/dl) 0.87 ± 0.18 0.988 0.84-0.90 0.42-1.41 gmm gloulin (g/dl) 1.39 ± 0.27 0.988 1.34-1.44 0.50-2.50 totl gloulins (g/dl) 3.02 ± 0.22 0.987 2.89-3.14 1.97-4.16 lum./gloul. rtio 0.31 ± 0.06 0.986-0.32 0.14-0.51 ξ: rithmetic men, SD: stndrd devition, WS: Wilk-Shpiro distriutive normlity test (criticl vlue: 0.947, α = 0.05), CI±95%: confidence intervl. Tle 2. Otined vlues on ech species totl Prmeter protein (g/dl) ξ ± C. ltirostris ( n = 109) C. ycre (n = 114) SD C I ± 95% ξ ± SD C I ± 95% 3.88 ± 0.62 3.76-3.99 4.10 ± 0.59 3.99-4.22 lumin (g/dl) 0.98 ± 0.19 0.92-1.01 0.95 ± 0.20 0.91-0.99 lph gloulin (g/dl) 0.75 ± 0.14 0.71-0.79 0.73 ± 0.15 0.70-0.76 et gmm totl gloulin (g/dl) gloulin (g/dl) gloulins (g/dl) 0.82 ± 0.18 0.77-0.86 0.93 ± 0.18 0.89-0.96 1.33 ± 0.25 1.26-1.39 1.46 ± 0.27 1.38-1.54 2.92 ± 0.23 2.83-3.04 3.13 ± 0.25 3.02-3.24 lum./gloul. rtio 0.32 ± 0.07 0.31-0.34 ± 0.05 0.28-0.31 ξ: rithmetic men, SD: stndrd devition, CI±95%: confidence intervl. In ech line, different letters indicte significnt differences (Tukey test, p < 0.05). Tle 3. Vritions ccording to sex, live weight, nd totl longitude in oth species (ξ) Prmeter S ex L ive weight (kg) Totl longitude (cm) mle totl protein (g/dl).03 lumin (g/dl).97 lph gloulin (g/dl).75 et gloulin (g/dl).84 gmm gloulin (g/dl).44 totl gloulins (g/dl).04 lum./gloul rtio.33 4 8 0 5 0 7 0 8 1 6 3 2 0 0 feml e < 3. 5 3,6-5, 0 > 5 < 99 100-110 > 110 3.9 3.90 3.96 4.18 3.92 3.94 4.15 0.9 0.90 0.92 1.16 0.94 0.92 1.13 0.7 0.78 0.76 0.71 0.74 0.73 0.73 0.8 0.84 0.89 0.88 0.87 0.88 0.85 1.3 1.38 1.37 1.41 1.36 1.39 1.41 3.0 2.99 3.03 3.00 2.96 3.01 2.98 0.3 0.37 0.29 0.36 ξ: rithmetic men. In ech line, different letters indicte significnt differences (Tukey test, p < 0.05). ISSN(ppel): 1514-6634 133 coppo.pmd 133
Tle 4. Vritions ccording to yer seson nd feeding system in oth species (ξ) Prmeter spring summe r Yer seson utum n winter frm Feedin g c totl protein (g/dl) 4.19 4.15 3.88 3.71 4.12.93 ξ: rithmetic men. In ech line, different letters indicte significnt differences (Tukey test, p < 0.05). zoo 3 c lumin (g/dl) 1.17 1.13 0.82 0.69 1.08.88 0 lph gloulin (g/dl) 0.73 0.77 0.74 0.75 0.80 0.78 et gloulin (g/dl) 0.87 0.88 0.84 0.88 0.83 0.84 gmm gloulin (g/dl) 1.37 1.33 1.45 1.42 1.38 1.41 totl gloulins (g/dl) 2.98 2.99 3.02 3.04 3.00 3.02 lum./gloul. rtio 0.37 0.36 0.28 0.24 0.35.29 0 DISCUSSION Eventul vritions due to postprndil effect nd circdin rhythm were excluded from the experimentl design, ecuse smples were tken during fst, in uniform morning hours. In crocodiles, fst lck cuses hyperlipemi nd interferes with photometric determintions 12. Wide individul rnges verified in this study should e ttriuted to reptilin physiologic prticulrities, ecuse its lood prmeters fluctute considerly due to feeding system, hitt, climte nd sex 19. Blood vlues from other qutic nimls, such s mphiins, lso register greter oscilltions due to their scrce regultion mechnisms, nd to higher tolernce to hemodilution nd hemoconcentrtion 8. Cellulose cette electrophoresis llowed to seprte lumin nd gloulins lph, et nd gmm, ut not pre-lumin. The ltter ws evidenced y polycrilmide gel electrophoresis, which lso permitted to know tht the lumin of C. ltirostris nd C. ycre possess moleculr weight similr to those reported for humn eings lumin (69 kd) 13, ecuse it ws locted nery to the 66 kd mrker. Reptile pre-lumin ws locted etween the nds from 20 to 66 kd, in consonnce to those of humn eings, which stys elow 61 kd 13. In humn eings, pre-lumin includes importnt proteins s ntitrypsin, cid glycoprotein, thyroxine-inding-prelumin (T4 hormone crrier), nd others; pre-lumin increse is uncommon, it occur in certin nephrosis; decrese of this frction indictes heptic insufficiency or tissue destructions 10. Men vlues otined in the present study re lower thn those reported from juvenile nd dult specimens of cptive C. ltirostris, for totl protein (5.01±1.14 g/dl), lumin (2.42±2.33 g/dl) nd gloulins (3.1±3.1 g/dl) 18. Other uthors lso found high vlues for the sme prmeters, from 5.06±0.87 g/dl, 1.66± g/dl, 3.37±0.85 g/dl nd 0.53±0.21 respectively, on 12 juvenile frmed C. ltirostris specimens 19. On the other hnd, from 5 C. ycre cptive specimens, lst uthors otined vlues of totl protein (4.28±0.29 g/dl), lumin (0.95±0.22 g/dl), gloulins (3.33±0.24 g/dl) nd lumin/ gloulin rtio (0,29±0,07) similr to those found in the present tril. Concentrtions of totl protein otined on 65 frmed specimens from C. ltirostris (3.80±0.14 g/dl) 5 resulted similr to those 134 ISSN(ppel): 1514-6634 coppo.pmd 134
determined in the present study on the sme species, ut remining proteinogrm vlues were very different. On 33 cptive C. ycre specimens, sme uthors otined vlues of totl protein (4.48±0.20 g/dl), lumin (1.43±0.05 g/dl) nd lumin/gloulin rtio (0.50±0.07), which were higher to those found y us on sme species, s long s gloulin levels resulted similr (3.08±0.19 g/dl). On exotic Crocodylus porosus helthy yerling specimens, vlues of totl protein (4.1-7.0 g/dl), lumin (1.4-2.3 g/dl), nd gloulins (2.7-5.0 g/dl) were otined 12, which resulted higher thn those found here. For the sme prmeters, vlues of 6.2±1.1 g/dl, 1.8±0.3 g/dl, nd 4.4±0.9 g/dl respectively, were determined in Alligtor mississippiensis 15. In other trils, totl protein vlues from 5.1 g/dl nd 6.5 g/dl were registered respectively on A. mississippiensis nd Crocodylus cutus 17. On Crocodylus niloticus, totl protein vlues of 5.3 g/dl 6 nd 5.0 g/dl 21, s well s lumin concentrtion of 1.9 g/dl nd gloulin level of 3.1 g/dl 6, were reported. Severl uthors mentioned ove do not descrie the techniques used in their works. Other investigtors employed dry chemistry methods (rective strips) 19. Some of them otined gloulin vlues y difference etween totl protein nd lumin 18. According to ech cse, heprinized plsm or serum were employed. None of them used electrophoreticl techniques. At the light of this reserch is very difficult to mke comprison especilly ecuse in certin group of studies, reptiles were not divided ccording to sex, ge, or yer seson. Photocolorimetric technique used to determine lumin not lwys gives vlues coincident to those otined y electrophoresis nd densitometry; the ltter re trustworthier ecuse the sum of protein frctions djusts exctly to the totl proteinemi vlue 10. Blood extrction technique, not lwys detiled in consulted studies, could e nother cuse of vlues vrition 9. Totl protein vlues otined in present tril were lower in C. ltirostris thn in C. ycre, contrry to dt reported in nother investigtion 19. Autochthonous reptiles studied did not register significnt differences ttriutle to sex (except gmm gloulin), in opposition to dt reported for other crocodile species 16. In frogs of qutic life, which re phylogeneticlly relted to reptilins, serum protein concentrtion is higher in mles thn in femles, significntly for totl protein (4,41 versus 4,32 g/dl, p <0,05), nd not significntly for lumin nd gmm gloulin 3. Growth cuses considerle chnges on certin crocodile lood prmeters, just s it ws checked for Crocodylus porosus from 1 yer old versus 2-4 yers old 1,12. Our findings on Cimn genus endorse this fct, eing corroorted y high liner ssocition coefficients registered etween growth progress nd increses of totl protein, lumin nd lumin/gloulin rtio. In this study, rise of proteinemi should necessrily e ttriuted to increse of lumin nd gmm gloulin, ecuse lph nd et gloulins registered low nd irregulr oscilltions. Gloulin gmm serum increse my e responds to increment of ntiodies synthesis promoted y the immunologic system progressive mturtion, just s it hppens in other species 2. Grdul lumin increse, which ws prllel to cimn development, could e compred to the ontogenetic increse registered in mphiins; in tdpoles (qutic life) lumins re lmost sent, ut they pper in mture tod due to necessity of retining intrvsculr wter during terrestril life 8. In coincidence, on 300 Rn ctesein ISSN(ppel): 1514-6634 135 coppo.pmd 135
specimens controlled from 9 to 21 months of ge, growth correlted to significnt increses of totl protein (9 months: 3.81 g/dl versus 21 months: 4.90 g/dl), lumin (1.38 versus 1.80 g/dl), nd gloulins et (0.65 versus 0.89 g/dl) nd gmm (1.17 versus 1.52 g/dl) 3. Our results indicte tht cold climte influenced unfvorly on proteinemi nd luminemi from cptive reptiles, even on those protected y heting. On frmed ullfrogs, cese of feeding during winter lethrgy lso cuses depletion of serum proteins, minly lumins 3. Wintry consumption of tissulr reserves cn cuse severe mlnutrition on crocodiles 15. A group of C. ltirostris mintined during 2 months t higher environmentl temperture (22ºC) thn control group (18ºC), otined higher live weight nd corporl longitude 14. Zoo limentry system cused lower totl protein nd lumin rtes thn those registered on frmed cimns. Alimentry indequcies re reflected on serum protein concentrtions 2. Totl protein nd lumin serum vlues from R. ctesein were directly proportionl to dietry protein quntity nd qulity 3. The chnge from hitul food (ovine met) to new diet (flour of met nd lood) produced serum lumin increse in cimns from northestern Argentin 5. Cimns, s ll crnivorous reptiles, require high qulity dietry protein during cptivity, le to replce the preys tht constitute their nturl feeding; in wrm climtes, crocodile mlnutrition is more deleterious due to its simultneous metolic increse 4. Crocodile hypoproteinemi is relted with immunitry depression, ecuse mino cids deficit interfere with the pproprite inmunogloulin synthesis 11. Preys cptured y reptiles in nturl life provide dequte levels of essentil mino cids, lipids, vitmins, minerls nd trce elements, contrry to diet given in cptivity 7. In such wy, iochemicl vlues re useful to evlute crocodile physiologicl stte nd to erly detect diseses 19. To optimize cimns reeding systems, it is ffirmed tht feeding improvement is the current priority 5. In conclusion, C. ltirostris nd C. ycre serum protein reference intervl is estlished strting from numerous smple, y mens of trustworthy techniques sujected to qulity control. Severl differences re verified etween species, ut vritions re scrce etween sexes. Growth progress correltes to increses of totl protein, lumin nd lumin/ gloulin rtio. Serum protein chnges ttriutle to feeding type nd climte re verified; lowest totl protein nd lumin vlues re registered in winter. Otined vlues re useful to control nutritionl nd metolic stte of cimns. ACKNOWLEDGMENTS To Dr. L.C. Leiv (Fcen, UNNE), for her importnt technicl collortion. BIBLIOGRAPHY 1. CANFIELD, P.J. 1985. Chrcteriztion of the lood cells of Austrlin crocodiles (Crocodylus porosus nd C. johnstoni). Zentrll Vet Med C Ant Histol Emryol 14:269-288. 2. COPPO, J.A. 2001. Fisiologí Comprd del Medio Interno. Ed. Dunken, Buenos Aires, 297 p. 3. COPPO, J.A.; MUSSART, N.B.; FIORANELLI, S.A. 2004. Impcto de l limentción, clim y sistem de mnejo del cridero sore el peso y ls proteíns plsmátics de Rn ctesein. Vet Arg 21:101-116. 4. DONOGHUE, S.; LANGERBER, J. 1996. Nutrition. In: MADER, D.R. (ed.): Reptile Medicine nd Surgery. Sunders, Phildelphi, p. 148-173. 136 ISSN(ppel): 1514-6634 coppo.pmd 136
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