Great Basin Naturalist Great Basin Naturalist

Volume 57 Number 3 Article 11

7-31-1997

Effects of myofibrogranuloma on serum calcium levels in walleye Effects of myofibrogranuloma on serum calcium levels in walleye

((Stizostedion vitreumStizostedion vitreum) )

Craig A. Shoemaker University of North Dakota, Grand Forks, North Dakota

Harry L. Holloway Jr. University of North Dakota, Grand Forks, North Dakota

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Recommended Citation Recommended Citation Shoemaker, Craig A. and Holloway, Harry L. Jr. (1997) "Effects of myofibrogranuloma on serum calcium levels in walleye (Stizostedion vitreum)," Great Basin Naturalist: Vol. 57 : No. 3 , Article 11. Available at: https://scholarsarchive.byu.edu/gbn/vol57/iss3/11

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Great Basin Naturali~t 57(3), e 1997, pp. 268-272

EFFECTS OF MYOFIBROGRANULOMA ON SERUM CALCIUMLEVELS IN WALLEYE (STIZOSTEDION VITREUM)

Craig A. Shoemaker1,2 and Harry L. HoUowa)'. Jr. l

AnSTllACT.-The effect of myofibrognmuloma (skeletal muscle degeneration) on serum calcium levels in spawningwaJley!';! (Stizostedion vitreum) wa~ examined. Mean serum calcium levels for healthy male walleye (11.7 ± 1.5 mg/1OO ml:-iel'll/n) ww,: significantly lower (P < 0.05) than calcium levels for healthy female walleye (15.4 + 1.8 mg/IOO mt serum).Significant increases (P < 0.1) in serum calcium were seen between healthy male and myofibrogranuloma-diseasedmale walleye (13.6 ± 2.1 mg/lOO ml serum) and between healthy and myofibrogranuloma.diseased female walleye (20.2± 3.7 lng/IOU ml serum). Elevations seen in mean serum C'J.ldum levels suggest the muscle degeneration and subse­quent gnlOll)oma formation in later stages of myofibrogranuloma bave a significant effect on serum calcium.

Key words: calciurn, waUeye, myofibrQgranuloma, colorimetric determination, Stizostedion vitreulU.

Calcium (Ca) levels of osteichthyan extra­cellular fluids are regulated to a finer degreethan those of more primitive fishes (Dacke1979). Hormones from the pituitary, ultimo­branchials, stannius corpuscles, and gonadsaffect osteichthyan Ca metabolism (Dacke 1979),Since Ca concentration is essential for cellmembrane permeability to \vater, neuromus­cular irritability and blood clotting, growth andenzyme reactions, it is less variable than otherions in serum (Urist and Van de Putte 1967).

My06brogranuloma (MFG) is a unique formof skeletal muscle degeneration recognizedonly in adult walleye (Stizostedion vitreum;Mayes 1976, Economon 1978, Holloway andSmith 1982). This myopathy is characterizedby profound alterations of the trunk muscula­ture produced by extensive hypertrophy of themuscle fibers (Economon 1978). Holloway andSmith (1982) noted 2 degenerative processes.The lst and most pronounced lesion consistedof coagulation necrosi.s of muscle fibers accom­panied by an inflammatory response and theformation of granulomas (muscle tumors), The2nd was noninflammatory and characterizedby focal areas of acute myolysis, Mineralizationor calcification was evident in the central por~bon of some fibers in the more granular stageof degeneration (Economon 1978). Kelly et al.(1987) showed an increase in muscle Ca associ­ated with my06brogranuJoma (55 times normalmuscle 6bers). Histochemical staining indicated

relatively more Ca-positive fibers in normalmuscle of MFG-positive fish and MFG-positivetissue than in muscle from healthy walleye(Kelly et al. 1987). Holloway and Shoemaker(1993) used X-ray technology to demonstrateincreased opacity, presumably due to concen­trated Ca in MFG-diseased tissue, The causeof MFG is unknown. Fisheries managers andanglers are concerned about the disease be­cause the involved muscle has a yellow colorwith sandy texture. Anglers discard these fish.

Adult osteichthyans (bony fishes) exhibitseasonal variations in plasma ea levels associ­ated with breeding cycles (Dacke 1979). Sexdifferences in plasma Ca levels of fish werefirst reported by Hess et al. (1928). Booke (1964)found differences in the Ca levels of spawningmale and female brook trout (Salvel;""s font!­nalls). Huon (1972) presented values of vari­ous fishes, including spawning male walleye,from the upper Mississippi River. Our objectivewas to establish Ca values for spawning male.female, and MFG-diseased walleye using asimple colorimetric procedure.

MATERIALS AND METHODS

Spawning walleye were collected by gill andframe net from Merrit Reservoir, Nebraska(1991 and 1992), and Lake Sakakawea, NorthDakota (1991). Only walleye greater than 500mm total length were sampled to eliminate

ll)"i"<lrt",,,nt "f Binl~<. Univeuity tl! Nm lh D~kct;o, Boa<. 9019 Urti-'",oity Station, Gl am! Forb, !'IO 5B202.Z">'C$el>1 <ll.11I1O.:ti<: U!>DA. ARS F'l:s11 DiSC;lSCS;and PlmlSilc5 Reo;wn;h I...oWoratory, PO BM 0052,. Aubum, AL 36831.

268

1997] ,\!YOFIBROGRANULOMA AND SERUM CALCIU>1 269

killing smaller fish that would not show macro­scopic signs of disease. Blood was sampled viacardiac puncture with heparinized lO-rol sy­ringe and 21-gauge needle. Centrifugation wascarried out within 24 h to minimize samplehemolysis. Serum was placed in dry ice fortransport and stored at _80 0 C until analyzed.Heavily hemolyzed samples were discarded.Walleye were filleted and examined with un­aided eye for MFG. MFG-diseased fish weredetermined by observing yellow-colored, sandy­textured muscle.

The procedure for measuring serum Ca con­centration was modified from Fales (1953) byOser (1965). We used the latter protocol, butserum and reagent volumes were halved dueto small sample volume. A 1% stock Ca standardsolution was prepared by dissolving 2.497 gcalcium carbonate in 6 N hydrochloric acid andevaporating on a steam bath to dryness. Theresidue (CaClz) was then dissolved in distilledwater to make 100 ml. A Ca standard (0.1 mg!ml) was prepared by diluting 1 ml of the stockCa to 100 ml with 1.4 N sodium chloride. Alight-sensitive stock solution of murexide wasprepared by dissolving 0.25 g ammonium pur­purate in 5 ml distilled water and 25 ml 95%ethanol. The working murexide solution wasprepared by adding stock murexide solution to100 ml 0.05 N sodium chloride until an aD of0.39-0.48 was reached at 620 nrn. The titrantwas prepared by mixing 18 volumes of theworking murexide solution, 1 volume of 0.14N sodium chloride, and 1 volume of stock di­sodium dihydrogen ethylene-diaminetetraaceticacid (EDTA) solution (0.005 M EDTA). Titrantwas mixed prior to each set of determinations,kept in brown bottles, and used within 1.5 h.

The principle of the procedure as describedby Oser (1965) is that the Ca in serum mixedwith murexide forms a red-colored complex inequilibrium with free Ca ions. Titration of theCa-murexide complex with EDTA chelatesfrees Ca ions, causing the release of Ca ionsfrom the complex. As the murexide ion: calciummurexide ratio increases, there is a shift incolor from red to purple (endpoint). The pro­cedure followed is described briefly: 0.25 mlof 0.14 N sodium chloride was placed in ablank cuvette, and equal amounts (0.25 ml) ofserum and working Ca standard vvere placedin test and standard cuvettes, respectively.Then, 2.25 ml of working murexide and 0.5 mlof titrant were added to each cuvette and

mixed. The spectrophotometer (Bausch andLomb Spectronic 20) was set at 50% transmit­tance at 620 nm with the blank, and the testand standard Clivettes were then read. Afterthis, 0.25 ml of titrant was added to eachcuvette and the spectrophotometer reset at50% transmittance with the blank, beforereading the test and standard. This step (titra­tion) was repeated and followed colorimetri­cally until the endpoint was reached (allserum Ca bound to EDTA). Calculations werethen carried out using the following formula asdescribed by Oser (1965) to determine serumCa level:

Volume of titrant for serumx 10 = mg CaiIDO ml serum

Volume of titrant for standard

A Student's t test compared mean serumCa levels of healthy male and female walleye.Significance was determined at P < 0.05. Dueto small sample size and differences betweenvariances, healthy male walleye and MFG-posi­tive male walleye as well as healthy femalewalleye and MFG-positive female walleye werecompared by unequal variance t test (Sakaland Rohlf 1981). The critical significance levelwas P < 0.10 for tests between MFG-positiveand healthy male and female walleye becauseof the low numbers of MFG-positive fish (6males and 4 females).

RESULTS AND DISCUSSION

Selected serum Ca titration curves (standard,spawning female [NB20l], spawning male[0066], MFG-positive spawning female [0043J,and MFG-positive spawning male [0252]) areshown in Figure 1, and serum Ca concentra­tions are described in Table 1 for healthy andMFG-positive walleye. A significant differ­ence was found between serum Ca levels ofmale and female walleye (P < 0.05). Serum Calevels of male walleye differed significantlyfrom mean serum Ca levels of MFG-positivemale walleye (P < 0.10). A significant differ­ence was found between serum Ca levels ofhealthy female walleye and MFG-positivefemale walleye (P < 0.10).

Serum Ca values were higher in this studythan in others (Table 2). One explanation maybe species differences. Hunn (1972) examinedspawning male walleye from the upper Missis­sippi River and found serum Ca lower (9.52

270 GREAT BASIN NATURALIST [Volume 57

.....ig. 1. Selected eu titration CUT\'eS of senlln collectedfrom spawning walleye (Stizostediun vitreum) usin~ a col­orimetric assay (standard = 0.1 mg C,~!ml, NB201 =healthy female, 0066 = healthy male, 0043 = MFe-posi­tive female, 0252 = MFG-positiv0 male).

mg/l00 ml) than our mean value (11.68 mg/100 mIl for spawning male watleye. This maybe a result of the blood-sampling procedure(caudal peduncle puocture vs. cardiac punc­ture), which is known to influcnce other bloodparameters (e.g., serum enzyme levels; Hille1982), or simply differences between river andreservoir fish. Low serum Ca levels in paddle­fish (Polyodon spath"la) su~est physiologicalhypocalcemia, 'illd in this respect paddlefishshow phylogenetic affinity to sturgeons (Grantet al. 1970). The low levels seem to supportUrist and Van de Putte's (1967) suggestion thathypocalcemia is related to the ahsencc of a bonyskeleton; however, gravid female white sturgeon(Adpenser transm-ontanus) exhibit increasedserum Ca. Low serum Ca determined for chan­nel catfish (lctal"ros ponclaltlS; 9.2 mg/l00ml) may he a result of obtainin~ fish from cul­ture ponds rather than from natural waters(Warner and Williams 1977).

Various techniques were used to detennineserum Ca levels, which may have resulted indifferences noted. Methods of Ca detennina­lion include automated methods (Warner andWilBams 1977), ci>lorimetric methods (Field et

85

8'

77

73

§.;1 69

~ 85,!!!-c 61~•"-

57

53

49

450.0

• standard

+ NB2O.

.­[]0043

X0252

+

• ,.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0rrl titnlIrt

a1. 1943, Shell 1961, Booke 1964), and atomicabsorption spectrophotometry (Grant et al. 1970,Hunn 1972). The colorimetric method describedby Falcs (1953) detem.ined total serum Ca (ionicplus protein-hound), and he found excellentagreement with the method of Clark and Col­lip (1925), which also measured lotal Ca. inaddition, the method is inexpensive (low costof equipment [Spectronic 20] and reagents).

The difference hetween serum Ca levels ofhealthy male and female walleye was due toegg production. Sex differences in Ca levels inosteichthyan fish were first reported by Hesset aI. (1928), who found a range of 9-12.5 mg/100 ml serum in male cod (GadtlS mOTh"a),while mature females had a range of 12.7-29mg/l00 mI serum. Dacke (1979) suggested thathypercalccmia is related to the influence ofestrogen and ovarian follicle maturation, whichstimulate synthesis of yolk protein and henceaD increase in protein-bound plasma Ca. Uristand Van de Pulle (1967) found similar resultsfOt" spawning sturgeon. Spawning brook troutfemales also exhibitcd increased levels of Ca(Booke 1964). Hunn et aL (1992) found Ca lev­els were significantly higher in gravid femalethan in male golden trout (OncoThynchtlSaguubonita).

Dacke (1979) stated that Ca levels through­out the osteichthyan subphylum arc regulatedwithin narrow limits. This is accomplished. inpart, by exchange with Ca in the aquatic envi­ronment and by exchange with hone Ca. Oser(1965) slated sBght variations in normal levelsof serum Ca are indicative of pathology such asbone abnormalities and muscle tumors. Eventhough our sample sizes of MFG-positive fishwere small. elevations were seen in Ca levels ofMFG-positive male and female walle)'e. Theseclevations su~~est Ca is highly regulated inwalle)'e and that MFG has a significant effecton serum Ca levels, probahly resulting fromacute myolysis (Holloway and Smith 1982)leading to increased extracellular Ca.

ACK~OWU·:DCMENTS

We acknowledge Federal Aid Sport FishRestoration fnnds from Nchraska Game andParks Commission (Project F-77-R), NorthDakota Game and Fish Department (ProjectF-2-R), and South Dakota Game, Fish and ParksDepartment (Project F-14-R; HLH, principalinvestigator).

1997] MYOFIBROGRANUWMA AND SERUM CALCIUM 271

TABLE 1. Serum Ca (mg/loo ml serum) of healthy and MFG-positive spawning male and female walleye (Stizostedicnvitreum) from Merrit Reservoir, E. and Lake Sakahwea, NO.

Fish Sample size Mean ± $1 Bange

Healthy female 20 15.4 ± 1.8'1 11.9-19.5Hellhhy male IB 11.7 ± 1.511,1 9.1-14.6MFG-positive female 4 20.2 ± 3.71 17.1-25.4MFC-positive male 6 13.6 ± 2.1+ 11.5-16.8

I MClIll~ with different <llphabutiel\l letter ~urerscripts were significantly diflerenll"lt'-tween iI>"XC' at P < O,OS. '111., llle>\ll' marked wilh diffen,nl 'l'ml)l>!~ wt:m.j!:nilk-~nt!l' dilTercnt within the S<l1ll1l ,;ex ~.e .. h<~lllhy female (Xllllparl;)(l to MFe-positive female ""d hCllllhy mnJe compared I" MFG-po,itive lllnl~) ut P < 0.10.

TAULE 2. Puhlished serum Ca values (mg/loo ml senun) for freshwater fishes.

Autho"s)

Wurner and Williams (1977)

Field el a!. (1943)

SheU (1961)

Grnnt et at. (1970)

Grant et al. (1969)

Urist and Van de Putte (1967)

Hunn (1972)

Booke (1964)

Sample sizeSpecies

107Ictdurus plJtl.Ctat.u.y

5-10C!JPrinus carpW

30 (pooled sample)Micropteros dolomieui

3 female8 malePolyodon spathula

14 malesCarassius aumtus

6 gravid females4 maleinongravid femaleAcipenser transmontanus

9 maleStizostedion vitreum

femalesmalesSalnelinus fontUudis

Mean ±&

9.2 ± 2.65

11.5

7.467.72

12.6

18.47.2

9.52± 0.3

Hange

3.9-4.5

9.45-14.77

10.2-19.5

5.54-27.796.34-10.65

LITERATURE CITED

DooK!!:, H.E. 19('>4. Blood serum pmtein and calcium levelsin yearling brook trout. !'wgressive Fish-Culturist26,107-111.

Cl....RK, E.P., AND J.B. COLLlJ.'. 1925. A study of the Tisdallmethod for the determination of blood serum calciumwith suggested modification. Journal of BiologicalChemistry 63:461.

DACKE, C.G. 1979. Calcium l'egulation in submammalianvertebrates. Academic Press Inc., LJndon. 222 pp.

ECONOMON, P.p. 1978. A muscular dystmphy like anomalyof walleye. American Fisheries Society Special Pub·lication 11:226-234.

F...LES, F,W 1953. A micromethod for the determination ofserum calcium. Joumal of Biological Chemistry 204:577-565.

FlEW, J.B., C.A. ELVElljl::M. AND C. JUDAY. 1943. A studyof tbe blood constihlents of carp and troul Journalof Biological Cllemi~try 148:261-269.

GRANT, B.E, P.M. MEHIlI.E, AND T.n. RUSSEL. 1970. Serumcharacteristics of spawning paddlefish (P.olyodvnspatlwla). Comparative Biochemishy and Physiology37,321-330.

GRANT, B.I<:, P.K.T PANG, AND RW. GRlFFITH. 1.969. Thetwenty-four houl' seminal hydration response in gold­fish (Carassius aUl"fLtus) I. Sodium, potassium, cnl­cium> magnesium, chloride ,md osmolality of senllnand seminal fluid. Comparative Biochemi.~try alLdPbysiology 30:273-280.

HRSS, A.E, C.E. BILU" M. WEIN~TOCK, AND II. RIVKIN.

1928. Differences in calcium level of blood betweenthe male and female cod. Proceedings of the Societyfor Experimental Biology and Medicine 25:349-359.

HILLE, S. 1982. A litemhll'tl review of the blood chemistryof rainbow trout, Salmo gairdneri. Rich. Journal ofFish Biology 20:535-569.

HOlLOWAY, H.L., JR., AND C.A. SHOEMAKER. 1993. Roent·genology of myofihrogranuloma in walley!;, Sti.wste·dion mreunl- Canadian Journal ofZoology 71:219-221.

272 GREAT BASIN NATURALIST [Volume 57

HOLLOWAY, ttL., 1Il., AND C.E. $MITli. 1982. A myopathyin North D-akota waUeye, Stizostedion vitreum(Mitchill). Journal offish Diseases 5:527-530.

HUNN, J-B. 1972. Blood chemistry vaJlIes for some fishesof the upper Mississippi River. Joumal of the Min­nesota Academy of Science 38:19-21.

HUNN, J.B.. fl.]. WlEDMEYER, I.E. GREER, AKD A.W. GRADY.

1992. Blood chemistry of laboratory-reared goldentrout. Journal ofAquatic Animal Health 4:21&-222.

KELLY, R.K.. J.F. K1AVIERKA~P RY. HUNl; MD O. NlELSEN.1987. Chemical analysis of muscle from walleye (Sti­zostedion vitreum tJweum) with myo6brogranuloma,a chronic myopathy. Canadian Journal of Fisheriesand Aquatic Sciences 44: 1425-143l.

M....YES, M.A. 1976. Muscular necrosis of the walleye Sti­ZQstedion vitreum in Nebraska. Transactions of theAmerican Microscopical Society 95:269--270.

OSER, B.L. 1965. Hawk's physiological chemistry. TheBlakiston Division, McCraw-Hill Book Company,New York. 1472 pp.

SHELL, £.\Y.1961. Chemical composition of blood of smaJl­mouth bass. United States Fjsh and W.iJdlife Service,Research Report 51. U.S. Government Printing Office.Washington, DC. 36 pp.

SOKAL, RH., AND EJ. ROHLF. 1981. Biometry. W.H. Free­man and Company, New York. 859 pp.

URJST M.H., AND KA. VAN DE PtTJTE. 1967. Comparativebiochemistry of the blood of fishes: identification offishes by chemical composition of serum. Pages207-217 in R.E Mathewson and D.E. Rall, editors,Sharks, skates, and rays. The Johns Hopkins Press,Baltimore.

WARNER. M.G., AND R.W WIUJAMS. 1977. Comparison 00­N'een serum values of pond and intensive racewaycultured channel catfish, lctalurus pun.ctatus (Rafines­que). Journal offish Biology 11:385-395.

ReceitJed 1 April 1996Auepled 17 March 1997