AACL BI OFLUX
Le gisla t ion
&
Con se r va t ion
,
Aqu a r iu m
,
Aqu a cu lt u r e
I n t e r n a t ion a l Jou r n a l of t h e Bioflu x Socie t y
Com pa r a t ive ch a r a ct e r iza t ion of blood ce lls a nd
h e m a t ologica l pa ra m e t e r s be t w e e n t he m a t u re
a n d im m a t u re Ca spia n V im ba ,
V im ba v im ba pe r sa
( Te le ost e i, Cypr in ida e )
1
Rohollah Noroust a,
2Ham ed Mousav i- Sabet
1
Depart m ent of Marine Biology, Science and Research Branch, I slam ic Azad Univ ersit y ,
Tehran, I ran; 2 Depart m ent of Fisheries Sciences, Facult y of Nat ural Resources, Univ ersit y
of Guilan, Sow m eh Sara, Guilan, I ran. Corresponding aut hor: H. Mousav i- Sabet ,
Mousav i- sabet @guilan. ac.ir
Abst r a ct. The aim of t he pr esent st udy was t o obt ain a basic knowledge of t he hem at ology of Caspian Vim ba and com par ison hem at ological par am et er s bet ween im m at ur e and m at ur e specim ens. Lym phocyt es, m onocyt es, het er ophils and eosinophils, wer e dist inguished and char act er ized. Hem at ological indices ( RBC, WBC, HCT, Hb, MCV, MCH, MCHC and leucocyt e differ ent ial count ) wer e m easur ed in one blood sam ple fr om 81 Vim ba vim ba per sa capt ur ed in Sout h coast s of t he Caspian Sea and in River - m out h of t he Sefid- r oud River in Nor t h of I r an. Twent y nine and fift y t wo of t he specim ens wer e m at ur e and im m at ur e, r espect ively. The m ean of cou nt ed r ed blood cell in im m at ur e fish was 1. 80± 0. 2 2 × 106 μl- 1
and in m at ur e fish was 1. 69± 0. 21 × 106 μl- 1
. The m ean of whit e blood cell in im m at ur e fish has been cou nt ed 5. 36 ± 0. 78 × 103 μl- 1
, and in m at ur e fish was 6. 0 2± 0. 9 6 × 103 μl- 1
. Neut r ophil in im m at ur e fish was 11. 17± 2. 55 per cent and in m at ur e was 13. 19± 2. 52 per cent , and no basophils cell has been obser ved. The r at es of clot t im e in m at ur e and im m at ur e fishes wer e 125. 38 ± 24. 9 4 and 1 86. 03± 25. 82 S, r espect ively. Significant differ ences wer e obser ved in RBC, WBC, neut r ophil and t he r at e of clot t im e bet ween t he m at ur e and im m at ur e of t he Caspian Vim ba. St at ist ical analysis r evealed t hat differ ences in hem at ological par am et ers bet ween m ale and fem ale fish wer e not significant . Com par ed t o ot her Cypr inids, t his species has higher m ean values for HCT and Hb and sim ilar value for RBC, and t he per cent of het er ophils was found t o be low in r elat ion t o t he percent of lym phocyt es. High lym phocyt e count s occurr ed in V. vim ba per sa, as com par ed t o t hese found in ot her species.
Ke y W or ds: hem at ology, Caspian Sea, HCT, Hb, RBC, WBC.
I n t r odu ct ion. The Caspian Vim ba, Vim ba v im ba persa ( Linnaeus, 1758) ( Fam ily: Cy prinidae) is dist ribut ed from t he sout hern Caspian Sea and Anzali wet land, Nort h of I ran ( Abdoli & Naderi 2009) . I t is of econom ic im port ance as food fish in freshw at er and brack ish w at er capt ure fisheries.
2004) . The haem at ological profile of som e cy prinid species are w ell docum ent ed in lit erat ure ( Groff & Zinkl 1999; Orun & Erdem li 2002; Garav ini & Mart elli 1981; Page & Row ley 1983) but t he haem at ological fact ors of m ost of t hem hav e not been report ed. This st udy report s for t he first t im e, t he ‘norm al’ haem at ological profile of m at ure and
im m at ure Vim ba v im ba persa, and com pares it wit h t hat of ot her freshw at er fish species.
The purpose of t his st udy w as t o obt ain a basic k now ledge of t he hem at ology of V. v im ba
persa. Assessm ent of hem at ological param et ers in V. v im ba persa m ight provide som e
useful inform at ion for ot her researchers t hat could be used as a biom ark er associat ed w it h st ressors agent s or as an av ailable t ool t o diagnose and m onit or disease in t his species, w hich is represent at iv e of t he icht hyich fauna in t he sout hern Caspian Sea.
M a t e r ial an d Me t h od
Sa m plin g. Mat ure and im m at ure specim ens of Vim ba v im ba persa used in t his st udy w ere capt ured from riv er- m out h of t he Sefid- rud in t he sout hern Caspian Sea, Guilan prov ince, Nort h of I ran ( 37° 28'3. 77" N, 49° 56'26. 15" E) ( Figure 1) .
Figure 1. Map of I ran ian part s of t he sout hern Caspian Sea basin, showing som e of t he m ost im port ant rivers syst em : Aras, Ku ra, At rak and Sefid- Rud ( t he st udy area) .
Eight y one fish w ere w eighed and m easured (34 fem ales: 96. 02± 25. 55 g, BW and 19. 78± 3. 31 cm , TL; 18 m ales: 92. 43± 29. 47 g, BW and 20. 26± 4. 92 cm , TL; 29 j uv enile: 64. 43± 14. 4 g, BW and 15. 95± 2. 20 cm , TL) . Careful net t ing and handling w as im plem ent ed t o m inim ize st ress. All fishes w ere considered healt hy on t he basis of t heir appearance and t he absence of obvious signs of disease. The specim ens w ere anest het ized wit h Fish Calm er ( Clov e pow der), and t he peripheral blood w as collect ed by punct ure of t he caudal v ein or heart (in sm all specim ens) wit h a heparin- coat ed 25 gauge× 0. 5 in. needle, at t ached t o an 1 m l sy ringe. Right aft er ex t ract ion, blood sam ples w ere processed for m icroscopy as follow s: a blood sm ears form ev ery fish w as fix ed in absolut e m et hanol for 3 m in at room t em perat ure or form alin v apor at 37oC for 1 h, and st ained wit h 10% Giem sa in PBS, hem at ox ylin–eosin ( Mart oj a & Mart oj a 1970; Pearse 1980) .
Herrick 1952) w ere det erm ined using a Neubauer hem ocy t om et er. Different ial w hit e cell count w as done on blood film s st ained w it h Giem sa. The different t y pes of leucocy t es w ere det erm ined on each blood sm ear and a m ean relat iv e percent calculat ed. Replicat e count s w ere m ade for each blood sam ple. Hem at ocrit v alue w as det erm ined by t he st andard m icrohem at ocrit m et hod, and ex pressed in percent age. Duplicat e blood sam ples w ere loaded int o st andard heparinized capillary t ubes, spun in a m icrohem at ocrit cent rifuge at 12, 000 rpm for 5 m in and m easured on a m icrocapillary reader. The Hb w as det erm ined using t he cy anm et haem oglobin m et hod. The following indices: m ean corpuscular hem oglobin ( MCH) , m ean corpuscular hem oglobin concent rat ion ( MCHC) and m ean corpuscular v olum e ( MCV) w ere calculat ed according t o Seiv erd ( 1964) .
St a t ist ica l an a lysis. The dat a w as ex pressed as m ean± SD differences in hem at ological param et ers bet w een m at ure and im m at ure specim ens w ere st at ist ically analy zed by
St udent ’s t t est . The differences w ere accept ed as significant if p< 0. 05.
Re su lt s
M or ph ology of t h e pe r iph e ra l blood ce lls. Eryt hrocy t es, het erophils and four t y pes of
leucocy t es: ly m phocy t es, m onocy t es, neut rophils and eosinophils w ere dist inguished by
light m icroscopy ( Figure 2) .
Figure 2. Light m icroscopic m icrographs of peripheral blood. ( A) e, ery t hrocy t e; m , m onocy t e. ( B) h, het erophile. ( C) eo, eosinophile. ( D) h, het erophilic granulocy t e.
( E) l, ly m phocy t e. Scale bars = 5 µm .
H e m a t ological a na lysis. Result s of hem at ological analysis are show n in Table 1, w hich
Table 1 Hem at ological param et ers in t he im m at ure and m at ure ( m ale and fem ale) Caspian Vim ba
V. v im ba persa, m ean± SD
Hem at ological param et ers I m m at ure Fem ale Male
RBC ( × 106 µl- 1) 1. 80± 0. 22 1. 70± 0. 24 1. 69± 0. 19
WBC ( × 103 µl-1) 5. 36± 0. 78 6. 12± 0. 89 5. 93± 1. 04
Hem at ocrit ( % ) 40. 58± 6. 70 39. 55± 4. 98 39. 83± 5. 78
Hem oglobin ( g/ dl) 11. 59± 1. 08 11. 58± 0. 98 11. 97± 1. 06
MCV ( fl) 226. 75± 54. 10 237. 60± 48. 29 235. 56± 40. 37
MCH ( pg) 67. 13± 11. 48 69. 26± 11. 13 71. 89± 11. 99
MCHC ( g/ dl) 29. 97± 4. 55 29. 63± 3. 74 30. 20± 4. 35
Ly m phocy t es ( × 103 µl- 1) 79. 00± 6. 89 79. 31± 8. 17 78. 45± 6. 75
Monocy t es ( × 103 µl- 1) 0. 14± 0. 30 0. 12± 0. 32 0. 14± 0. 34
Neut rophils ( × 103 µl- 1) 11. 17± 2. 55 12. 45± 2. 75 12. 45± 2. 64
Eosinophils ( × 103 µl-1) 0. 34± 0. 46 0. 33± 0. 47 0. 36± 0. 57
Clot t ing t im e ( s) 186. 03± 25. 82 142. 84± 34. 95 153. 79± 44. 35
A high count of ly m phocy t es is observ ed. The percent of m onocy t es and eosinophils are
found t o be high in relat ion t o t he percent of ly m phocy t es in m ale specim ens. St at ist ically
analy sis (t-t est , p< 0. 05) rev eals t hat hem at ological param et ers are not significant ly
different bet w een m ale and fem ale specim ens. Mean ery t hrocy t e count s obt ained for
m at ure and im m at ure V. v im ba persa w ere 1. 64± 0. 23 and 1. 80± 0. 22× 106 µl-1,
respect iv ely , m ean of t he WBC count s w ere 6. 45± 0. 80 and 5. 36± 0. 78× 103 µl- 1 in m at ure
and im m at ure specim ens respect iv ely, m ean of t he neut rophil count s w ere 13. 19± 2. 53 and 11. 17± 2. 55 percent , and t he m ean of clot t ing t im e w ere 125. 38± 24. 95 and
186. 03± 25. 82 second f or m at ure and im m at ure V. v im ba persa, respect iv ely ( Table 1) .
Significant differences w ere observ ed in RBC, WBC, neut rophils and t he clot t ing t im e bet w een m at ure and im m at ure specim ens ( p< 0.05) . There w as no significant difference
bet w een m at ure and im m at ure of V. v im ba persa in ot her hem at ological indices and
bet w een t he m ale and fem ale specim ens ( p≥0.05).
D iscussion. There is grow ing int erest in t he st udy of hem at ological param et ers and st ruct ural feat ures of fish blood cells regarded as im port ant for healt h and aquacult ure purposes. Ery t hrocy t es are t he dom inant cell t y pe in t he blood of t he vast m aj orit y of fish species. I t is widely accept ed t hat fishes, lik e m ost ot her v ert ebrat es, hav e a com m on leucocy t e pat t ern consist ing of granulocyt es, m onocy t es, ly m phocy t es and t hrom bocy t es ( Wat son et al 1963; Hart m an & Lessler 1964; Conroy 1972; Blax hall & Daisley 1973; Jav aid & Ak ht ar 1977; Nak am ura & Shim ozaw a 1984; Row ley et al 1988; Groff & Zinkl 1999; Hrubec et al 2000; Est eban et al 2000; Ueda et al 2001) . The k now ledge of t he hem at ological charact erist ics is an im port ant t ool t hat can be used as an effect iv e and sensit iv e index t o m onit or phy siological and pat hological changes in fishes ( Kori- Siak pere et al 2005) . Prev ious st udies on fish hem at ology hav e rev ealed t hat int erpret at ion of blood param et ers is quit e difficult since v ariat ions in t he blood are caused by int ernal and ex t ernal fact ors. I t is w ell k now n t hat blood sam pling, laborat ory t echniques, seasonal v ariat ions, size, genet ic propert ies, sex , populat ion densit y , lack of food supply, env ironm ent al st ress and t ransport at ion could affect hem at ological dat a ( Ezzat et al 1973; Dent on & Yousef 1975; Fourt ie & Hat t ing 1976; Van Vuren & Hat t ingh 1978; Hardig & Hoglung 1984; Wilhem et al 1992; Orun & Erdem li 2002; Arnold 2005; Kori-Siak pere et al 2005) .
I n t he present st udy , V. v im ba persa blood cells were charact erized m icroscopically
Er yt h r ocyt e cou n t. Generally, ery t hrocy t e count s are used as indicat ors for anaem ia.
Mean ery t hrocy t e count s ( ± SD) obt ained for t he m at ure V. v im ba persa w as 1. 64± 0. 22
( Table 2) , and w ere com parable t o t hose repor t ed for Cy prinus carpio ( Groff & Zinkl
1999) and Carasius aurat us ( Gr off & Zink l 1999) , but w as less t han v alues of 1. 74± 0. 12 report ed for Capoet a t rut t a ( Orun & Erdem li 2002) ( Table 2) . Collat ed wit h ot her species,
V. v im ba persa present s sim ilar m ean v alues for RBC and higher for HCT and Hb ( Table
2) . Blax hall & Daisely ( 1973) not ed t hat fish biologist s rely m ore on haem at ocrit and haem oglobin concent rat ion est im at es as indicat ors of anaem ia.
Le u cocyt e cou n t. Leucocy t e count s are useful as indicat ors of disease condit ion or response t o infect ion, and significant ly elev at ed or depressed v alues are obt ained in
abnorm al condit ions. Mean WBC ( ± SD) obt ained for t he m at ure V. v im ba persa w as
6. 45± 0. 80× 103 µl- 1 ( Table 1) . This v alue w as m uch low er t han v alues report ed for C.
carpio ( Groff & Zink l 1999) , C. aurat us ( Gr off & Zinkl 1999) and C. t rut t a ( Orun &
Erdem li 2002) . May be at t ribut ed/ relat ed t o t he condit ions in t he habit at or t he general w ell- being of t he fishes and habit at s differences. There are wide v ariat ions in t he leucocy t e count s report ed for v arious freshw at er and brack ish w at er Cy prinids ( Table 2) . Sev eral aut hors hav e dem onst rat ed a special int erest in t he leucocy t es of t eleost fishes w it h regard t o t heir m orphology and absolut e v alues. Their inv est igat ions rev ealed a great div ersit y of m orphological aspect s and v alues in som e t y pes of leucocy t es ( Sriv ast av a 1968; Blax hall & Daisley 1973; Ezzat et al 1973; Ellis 1976; Ferguson 1976; I m agaw a et al 1989; Ueda et al 1997; Veiga 1999) . I n addit ion, t he sam e t y pe of
leucocy t e has been described wit h different nam es by different aut hors. I n V. v im ba
persa, ly m phocy t es are m ore abundant t han in ot her species ( Table 2) . The
nom enclat ure used t o describe m onocy t es in fishes is v ariable. Monocy t es hav e been t erm ed hem oblast s and m acrophages ( Barber et al 1981) , w hile ot her aut hors hav e been unable t o find m onocy t es ( Blax hall & Daisley 1973) . There are relat iv ely few m orphological st udies on fish m onocy t es ( Cannon et al 1980; Page & Row ley 1983; Ueda et al 2001; Valenzuela et al 2003) . I n fishes, granulocy t es are of t hree t y pes: het erophils and eosinophils are t he m ost com m on, w hile basophils are m uch rare. Most aut hors recognize het erophils as t he m ost frequent t ype of granulocy t e. The occurence of eosinophils, and in part icular basophils, is oft en quest ioned ( Ellis 1977; Row ley et al 1988) . I n t he blood of Oreochrom is nilot icus, Ueda et al ( 2001) ident ified all t hree t y pes of granulocy t es, how ev er Rodrigues Bit t encourt et al ( 2003) failed t o find basophils and eosinophils in t his t ilapia under sem i-int ensiv e cult ure condit ions. I n V. v im ba persa, w e report t he presence of het erophilic and eosinophilic granulocy t es, w hile basophilic granulocy t es hav e not been found. Garav ini & Mart elli ( 1981) st at ed t hat t w o t y pes t he
granules are form ed in im m at ure and m at ure goldfish het erophils. I n C. carpio, t he
subst ruct ure of het erophil granules is sim ilar t o t hat of t he eosinophil granules of C.
aurat us ( Page & Rowley 1983) . The m ost abundant t y pes of leucocy t es found in t he
peripheral blood of Vim ba v im ba persa w ere t he ly m phocy t es and het erophils ( Table 2) ,
as dem onst rat ed in Pim elodus m aculat us ( Ribeiro 1978) , Sy nbranchus m arm orat us
( Nak am ot o et al 1991) , Mugil plat anus ( Ranzani-Paiv a 1995) and Oncorhy nchus m y kiss
( Ranzani- Paiv a et al 1998) . A sim ilar pat t ern w as report ed in C. carpio and C. aurat us
( Groff & Zinkl 1999) , and C. t rut t a ( Orun & Erdem li 2002) , alt hough t he relat iv e rat io
changed according t o t he species (Table 2) . High ly m phocy t e count occurred in V. v im ba
persa com pared t o t hat found in ot her species ( Table 2) .
H a e m oglobin con ce n t r a tion. I n fish blood, oxy gen is carried in phy sical solut ion and also in com binat ion wit h haem oglobin. Haem oglobin is crucial for t he surv iv al of t he fish as it s role is direct ly relat ed t o t he oxy gen- binding capacit y of blood. Mean haem oglobin
concent rat ion v alues ( ± SD) obt ained for t he m at ure V. v im ba persa w as 11. 91± 0. 98
g/ dl. The low v alues of haem oglobin concent rat ion are report ed of ot her Cy prinids ( Table 2) . And reflect s high ox y gen carrying capacit y of t he blood, w hich is consist ent w it h t he correlat ion of haem oglobin concent rat ion w it h fish act ivit y as suggest ed by Lenfant & Johansen ( 1972) .
M e a n cor pu scula r h a e m oglobin concen t ra t ion ( M CH C). Mean corpuscular
haem oglobin concent rat ion v alues for t he m at ure V. v im ba persa w as 29. 80± 3. 63 g/ dl.
The MCHC v alues report ed for C. t rut t a w as sim ilar t o t he result s of present st udy ( Orun & Erdem li 2002) .
M e a n cor pu scula r volu m e ( M CV) a n d m ean cor pu scula r h a e m oglobin ( M CH ). There are wide v ariat ions in bot h t he m ean corpuscular v olum e and m ean corpuscular haem oglobin v alues report ed in lit erat ure for various freshw at er and brackish w at er Cy prinids (Table 2) .
Table 2 Com parison of t he norm al hem at ological indices bet w een t he m at ure Caspian Vim ba and
som e ot her Cy prinids, m ean± SD or reference int erv al
Species
Param et ers Vim ba v im ba
persa
Cy prinus carpio
Carasius
aurat us Capoet a t rut t a
RBC ( × 106 µl- 1) 1. 69± 0. 21 1. 67± 0. 08 1. 61± 0. 81 1. 74± 0. 12
WBC ( × 103 µ l- 1) 6. 02± 0. 96 37. 8± 2. 88 52. 3± 4. 88 17. 65± 2. 15
Hem at ocrit ( % ) 39. 69± 5. 38 33. 4± 1. 51 22. 3± 1. 04 26. 05± 2. 38
Hem oglobin ( g/ dl) 11. 77± 1. 02 8. 2± 0. 36 6. 7± 0. 25 7. 9± 0. 24
MCV ( fl) 236. 58± 44. 33 202± 5. 5 137± 2. 6 149. 71± 2. 28
MCH ( pg) 70. 57± 23. 12 49. 1 42± 1. 4 45. 4± 1. 8
MCHC ( g/ dl) 29. 91± 4. 04 - - 30. 32± 0. 8 ( % )
Lym phocyt es ( × 103 µ l- 1) 78. 88± 7. 46 32. 26–35. 15 26. 7± 2. 89 13. 1± 0. 01
Monocyt es ( × 103 µl- 1) 0. 13± 0. 33 0. 19–0. 76 0. 2± 0. 1 1. 9± 0. 07
Neut rophils ( × 103 µl- 1) 12. 45± 2. 69 - - -
Eosinophils ( × 103 µl- 1) 0. 34± 0. 52 0. 19–0. 38 0. 1± 0. 1 0. 075± 0. 005
Het erophils ( × 103 µ l- 1) 2. 74± 0. 61 1. 13–3. 78 2. 3± 0. 56 2. 56± 0. 12
References Present st udy Groff & Zink l
( 1999)
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The ery t hrocy t e indices ( MCV and MCH) hav e a wide range of phy siological v ariat ion. The MCV of V. v im ba persa is sim ilar t o t hat of C. carpio and higher t han C. aurat us and C.
t rut t a, w hile t he MCH is higher t han all of t hem ( Table 2) . Arnold ( 2005) argued t hat in
elasm obranches, m anual RBC lack s t he precision necessary for t he accurat e assessm ent of anem ia or f or calculat ing accurat e MCV and MCH v alues. Blax hall & Daisley ( 1973) , w ork ing wit h t eleost s, also concluded t hat m anual RBC count s w ere error prov ed and suggest ed t hat HCT and Hb m ay be bet t er param et ers for t he assessm ent of anem ia in fish.
Con clusion s. The result s of our research prov ide a cont ribut ion t o t he k now ledge of t he
charact erist ics of blood cells and hem at ological param et ers of t he cy prinid fish, V. v im ba
persa, under t he norm al condit ions em ployed in t his st udy . This inv est igat ion m ay be
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Received: 28 Sept em ber 2012. Accept ed: 30 Oct ober 20 12. Pu blished online: 07 Febr uar y 201 3. Aut hor s:
Rohollah Nor oust a, Depar t m ent of Mar ine Biology, Science and Resear ch Br anch, I slam ic Azad Univer sit y, Tehr an, I r an, em ail: r . n1361@gm ail. com
Ham ed Mousavi- Sabet , Depar t m ent of Fisher ies Sciences, Facult y of Nat ur al Resour ces, Univer sit y of Guilan, Sowm eh Sar a, Guilan, I r an, e- m ail: m ousavi- sabet @guilan. ac.ir
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