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Research ArticleSeasonality Influence on Biochemical and HematologicalIndicators of Stress and Growth of Pirarucu (Arapaima gigas)an Amazonian Air-Breathing Fish

Rosiely Felix Bezerra1 Maria do Carmo Figueiredo Soares2

Athiecirc Jorge Guerra Santos2 Elba Verocircnica Matoso Maciel Carvalho1

and Luana Cassandra Breitenbach Barroso Coelho1

1 Universidade Federal de Pernambuco (UFPE) Centro de Ciencias Biologicas Departamento de BioquımicaRua Professor Nelson Chaves sn Cidade Universitaria 50670-901 Recife PE Brazil

2 Universidade Federal Rural de Pernambuco (UFRPE) Departamento de Engenharia de Pesca Rua DomManoel de Medeirossn Dois Irmaos 52171-900 Recife PE Brazil

Correspondence should be addressed to Luana Cassandra Breitenbach Barroso Coelho lcbbcoelhogmailcom

Received 13 August 2013 Accepted 14 November 2013 Published 21 January 2014

Academic Editors C Cameselle K F Chang and A DrsquoAnnibale

Copyright copy 2014 Rosiely Felix Bezerra et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Environmental factors such as seasonal cycles are themain chronic stress cause in fish increasing incidence of disease andmortalityand affecting productive performance Arapaima gigas (pirarucu) is an Amazonian air-breathing and largest freshwater fish withscales in the world The captivity development of pirarucu is expanding since it can fatten up over 1 kg per month reaching 10 kgbodymass in the first year of fatteningThis work was conducted in three periods (April to July 2010 August to November 2010 andDecember 2010 toMarch 2011) defined according to rainfall andmedium temperatures Seasonality effect analysis was performed onbiochemical (lectin activity lactate dehydrogenase and alkaline phosphatase activities) and hematological (total count of red bloodcells hematocrit hemoglobin and hematimetric Wintrobe indexes) stress indicators as well as on growth and wellbeing degreeexpressed by pirarucu condition factor developed in captivity All biochemical and hematological stress indicators showed seasonalvariations However the fish growth was allometrically positive condition factor high values indicated good state of healthiness incultivationThese results reinforce the robust feature of pirarucu and represent a starting point for understanding stress physiologyand environmental changes during cultivation enabling identification and prevention of fish adverse health conditions

1 Introduction

Pirarucu Arapaima gigas (Shinz 1822) is the largest Ama-zonian and freshwater fish with scales in the world It isconsidered an air-breathing fish and constitutes a specieswith great potential for farming due to interesting featuressuch as excellent taste of meat and high growth rate withextraordinary weight development [1] in captivity the fishcan fatten up over 1 kg per month reaching 10 kg body massin the first year of fattening [2] Pirarucu has been a targetof fishermen due to its occurrence in lakes and isolatedenvironments which allows a disorderly intense explorationdepreciating their natural stocks [3] The expansion of

the creation of pirarucu in captivity around the world forcommercial exploitation was too an alternative to reduce therisk of extinction

The increase of fish farming has developed a growinginterest by producers in respect to the search for solutionsto avoid the losses caused by mortality and productionproblems Stress caused by common farming practices (acutestress) as well as environmental factors (chronic stress) suchas seasonal changes increases the incidence of disease andmortality affecting the productive performance of animals[4] The seasonal cycles can affect fishes in several biologicalactivities such as behavior nutrition metabolism immu-nity and reproduction [5] The immunocompetence is often

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 541278 6 pageshttpdxdoiorg1011552014541278

2 The Scientific World Journal

affected by seasonal variations and in general biochemi-cal hematological and immunological parameters such aslevels of various blood cells hematocrit percentages [6]lysozyme activity [7] respiratory burst levels of head-kidneymacrophages [8] and lectin activity [9] The biochemicaland hematological parameters are useful tools to determinethe characteristics of fish blood in different situations suchas stress or normality The relative robustness or degreeof wellbeing from a fish is expressed by the coefficient ofcondition or condition factor (CF) considered basically asthe quotient between observed mass and theoretical massestimated through the length-mass relationship Variationsin fishrsquos coefficient of condition primarily reflect the state ofsexual maturity and degree of nourishment they provide rel-evant information about physiological and health character-istics of individual or population which are very importantin captive fish to their management and maintenance [10]

The aimof this workwas to analyze the effects of seasonal-ity (temperature and rainfall) on biochemical (lectin activitylactate dehydrogenase and alkaline phosphatase activities)and hematological parameters (hematocrit hemoglobin andhematimetric indexes of Wintrobe) that can be used as phys-iological indicators of stress Also the influence of seasonalityon growth of pirarucu developed in captivity as well asthe state of wellbeing of the fish was analyzed This is afirst study that relates chronic stress with biochemical andhematological indicators of seasonal stress as well as healthand growth in A gigas fish farming

2 Material and Methods

21 Weather Data Obtention Rainfall and TemperatureWeather data was provided by the Instituto Nacional deMeteorologia (INMET Brazil) collected fromweather station82900 (08∘031015840S 34∘571015840W) Recife PE Brazil according tointernational standards of the World Meteorological Orga-nization Medium rainfall (mm3) as well as medium tem-perature (∘C) was calculated to each period respectively999mm3 and 2614∘C to period 1mdashP1 (April to July 2010)274mm3 and 2546∘C to period 2mdashP2 (August to November2010) 58mm3 and 2694∘C to period 3mdashP3 (December 2010to March 2011) (Figure 1)

22 Obtaining Fish Blood Fish were provided by the EstacaodeAquiculturaContinental Prof Johei KoikeDepartamento dePesca da Universidade Federal Rural de Pernambuco (UFRPE)and developed in earth pond The juvenile animals (119899 = 6)were anesthetized by hypothermia on ice immediately afterblood collection procedure the fishes returned to earth pondBlood was obtained from caudal vein with syringes 5mL21 G 23G or 25G needles (BD Precision Glide PN Brazil)according to fish size To obtainwhole blood tubes containingEDTA 18mgmL as anticoagulant (Vacuette Greiner bio-one Brazil) were used Serum was collected from tubeswithout anticoagulant the blood was centrifuged at 3000timesgfor 10min at 4∘C

23 Lectin Activity and Protein Evaluation Serum lectin acti-vity (LA) was evaluated as specific hemagglutinating activity

02468

1024

26

28

lowast

lowast

lowast

Period 3Period 1 Period 2

Med

ium

tem

pera

ture

(∘C)

Med

ium

rain

fall

(mm

3)

Medium temperature (∘C)Medium rainfall (mm3)

024681024

26

28

Figure 1 Medium rainfall and medium temperature for eachperiod Period 1 (AprilndashJuly 2010) Period 2 (AugustndashNovember2010) Period 3 (December 2010 to March 2011) Data were obtainedfrom Instituto Nacional de Meteorologia (INMET Brazil) (lowast) Signif-icant difference in rainfall among the periods (119875 lt 005)

inmicrotiter plates with 96 wells [11] Specific hemagglutinat-ing activity was defined as the ratio between titer and proteinconcentration (mgmL) and expressed with hemagglutinat-ing activity units for protein milligrams (HAUmg) Proteinconcentrations were determined by Bradford [12]

24 Lactate Dehydrogenase Activity Lactate dehydrogenaseactivities (LDH) were determined following the oxidation ofNADH (340 nm 25∘C) The reaction mixture contained atotal volume of 1mL 50mM imidazol 1mM KCN buffer pH74 at 25∘C 013mM of NADH and different concentrationsof pyruvate for LDH saturation plots One unit of enzymeactivity is defined as the amount of enzyme using 1 120583mol ofsubstrate per min (340 nm 25∘C) Each value represents themean of three measurements

25 Alkaline Phosphatase Activity The serum alkaline pho-sphatase activity (AP)was performedwithmodifications [13]Briefly enzyme activity was measured using p-nitrophenylphosphate (pNPP) as substrate (50mM) in 1M diethanol-amine (pH 98) containing 1mM MgCl

2(405 nm 25∘C

1min)

26 Total Count of Red Blood Cells To determine the totalcount of red blood cells (RBC) a 1 in 1000 dilution wasmade in 002Mphosphate saline buffer (PBS pH 73) Countswere carried out using a Neubauer haemocytometer (INLABBrazil) and expressed as cellmm3 [14]

27 Hematocrit Hemoglobin and Hematimetric Indexes ofWintrobe The hemoglobin (Hb) levels were obtained usinga kit for determination of hemoglobin in whole blood(Doles Brazil) following the manufacturerrsquos instructionsThe hematocrit (Htc) was determined by the microhema-tocrit technique and result was expressed as percentage

The Scientific World Journal 3

Period 1 Period 2 Period 30

5

10

15

20

25

30

35

40

lowast

Season

Protein (mgmL)Lectin activity (HAUmg)

Lect

in ac

tivity

(a)

Period 1 Period 2 Period 3Season

lowast

lowast

lowast

lowast

lowast

lowast

LDH (UL)AP (UL)

0

50

100

150

200

250

300

350

400

450

LDH

and

AP

activ

ities

(b)

Figure 2 Variations in serum of A gigas in Period 1 (999mm3 2614∘C) Period 2 (274mm3 2546∘C) and Period 3 (58mm3 2694∘C) tolectin activity (specific hemagglutinating activity) and serum protein concentration (a) LDH lactate dehydrogenase activity and AP alkalinephosphatase activity (b) (lowast) Significant (119875 lt 005)

of erythrocytes compared to whole blood Hematimetricindexes of Wintrobe were calculated as follows MCV (meancorpuscular volume) = HtcRBC times 10 (fl) MCH (meancorpuscular hemoglobin) = HbRBC times 10 (pg) and MCHC(mean corpuscular hemoglobin concentration) = HbHtc times100 (gdL)

28 Condition Factor Mass (g) and length (cm) were usedto determine CF and constant regression (119887) which revealsthe rate of growth in mass Empirical point ratios 119872119871(masslength) for each period were submitted to regressionanalysis and adjusted by power function119872 = 119886119871119887 where119872is the dependent variable 119871 is the independent variable ldquo119886rdquois the CF and ldquo119887rdquo is the constant associated with the type ofgrowth inmass of animalsThese constants were estimated bylinear regression of the transformed equation119872 = log 119886+119887timeslog 119871 where119872=mass (g) 119871= total length (cm) 119886= constantand 119887 = growth exponent or constant regression [10]

29 Statistical Analysis Data shown represent the meanvalues of each parameter in the specified periods Statisticalsignificance of data between groups (mean plusmn se) wasdetermined with analysis of variance (ANOVA) and Tukeytest using OriginPro 80 (OrginLab Corporation USA) Avalue of 119875 lt 005 was considered significant

3 Results

Significant difference was observed to serum lectin in theperiods (119875 lt 005) activity increased in P2 (2845HAUmg)while in P1 (826HAUmg) and P3 (128HAUmg) val-ues were lower indicating some seasonal influence on LA

(Figure 2(a)) The activity of the serum enzyme LDH wassignificantly higher in P3 (4440UL) than in P1 (1840 UL)and P2 (1150UL) in addition therewas a greater decrease inLDHactivity in P2 than in P1 (Figure 2(b)) SerumAPactivityin the studied periods showed a gradual increase (583UL P1130UL P2 and 180UL P3) and was significantly differentfrom one period to another (Figure 2(b))

RBC (124 times 106mm3 P1 114 times 106mm3 P2 and148 times 10

6mm3 P3) Hb levels (106 gdL P1 855 gdL P2and 97 gdL P3) and Htc percentages (246 P1 1875 P2and 279 P3) revealed values decreasing in P2 howeverthis decrease was significant to Hb and Htc and not to RBC(Figure 3(a)) MCV showed seasonal variation with valuessignificantly lower in P2 (1984 fl P1 1644 fl P2 and 1885 flP3) no significant difference was showed between the otherperiods MCH decreased gradually from P1 to P3 (855 pg P17505 pg P2 and 655 pg P3) MCHC (4308 gdL P1 456 gdLP2 and 3475 gdL P3) was significantly lower in P3 while P1to P2 did not reveal significant difference (Figure 3(b))

The constant values of linear regression ldquo119887rdquo obtained in P1(119887 = 312) P2 (119887 = 333) and P3 (119887 = 346) revealed a positiveallometrically growth to pirarucu this can be observed by aprogressive increase in body mass in grams (936times 103 plusmn024P1 1248 times 103 plusmn 127 P2 and 1796 times 103 plusmn 141 P3 meanvalues per period) throughout the periods (Figure 4(a)) Thevalues of the constant linear regression ldquo119887rdquo may vary from250 to 350 [15] This wide variation of ldquo119887rdquo is a function ofbiotic and abiotic factors when 119887 = 3 growth is isometric119887 gt 3 is positive allometric and 119887 lt 3 is negative allometricIsometric growth (119887 = 300) suggests an increase in massand length at the same rate which is theoretically ideal forfish especially in cultivation CF average showed high values0891 plusmn 0038 in P1 0909 plusmn 0058 in P2 and 0926 plusmn 0124 in



lowast

lowast

Season

Red blood cell counts (times105m3)

Hemoglobin (gdL)Hematocrit ()

RBC

Htc

and

Hb

02468

1012141618202224262830

(a)


20

40

60

80

100

120

140

160

180

200

lowast

lowast

lowast

Hem

atim

etric

inde

xes o

f Win

trobe

Season

MCV (fL)MCH (pg)MCHC (gdL)

(b)

Figure 3 Variations in serum of A gigas in Period 1 (999mm3 2614∘C) Period 2 (274mm3 2546∘C) and Period 3 (58mm3 2694∘C) toRBC red blood cell counts Hb hemoglobin and Htc hematocrit (a) hematimetric indexes of WintrobemdashMVC MCH and MCHC (b) (lowast)Significant (119875 lt 005)

8

10

12

14

16

18

20

22

b = 346

b = 333

b = 312

Season

(


Mas

s (times103

g)

936 times 103 g plusmn 024)

(1248 times 103 g plusmn 127)


(a)

99102105108111114117120123126129132

CF = 0926 plusmn 0124

CF = 0909 plusmn 0058

CF = 0891 plusmn 0038

Period 3Period 2Period 1Season

Leng

th (c

m)

(1155 cm

(10986 cm

(1016 cm

plusmn 267)

plusmn 298)plusmn 094)

(b)

Figure 4 Variations inmass and estimated values for the regression constant ldquo119887rdquo rate of growth inmass (a) variations in length and conditionfactor (CF) (b) to A gigas in Period 1 (999mm3 2614∘C) Period 2 (274mm3 2546∘C) and Period 3 (58mm3 2694∘C)

P3 progressive increase in fish length (cm) over the periodswas observed (1016plusmn094 P1 1098plusmn267 P2 and 1155plusmn298P3 mean values per period) (Figure 4(b))

4 Discussion

Lectins are proteins or glycoproteins involved in innateimmunity and are therefore considered as the first line ofimmune defense of fish [16] These proteins have been foundin serum mucus skin and eggs of several fish species [17 18]

The seasonal variation found for A gigas LA in P2can be displayed in response to increased concentrationsof specific potential pathogens in the environment due tolower rainfall and lowering temperature Some pathogenshave higher occurrence in winter although the presence ofpathogens generally is higher with increasing temperature insummer [19] Stress situations increased the levels of lectinin this case it can function as an acute phase protein [20]Seasonal variation too was found in activity of lectin presentin serumofColossomamacropomum showing higher activityin summer and lower activity in winter [9]


The enzyme lactate dehydrogenase (LDH EC 11127) hasbeen approached in several studies with fish since it is directlylinked to the glycolytic pathway and anaerobic metabolismresponsible for environmental stress responses In this workthe levels of LDH in pirarucu serum were significantly lowerin P2 (119875 lt 005) that demonstrates the relation withenvironmental change processes such as changes in oxygenlevels even for air-breathing fish The highest LDH levelswere found in P3 showing that probably the enzyme activityin pirarucu ismore sensitive to variation on temperature thanon rainfall Alkaline phosphatase (EC 3131 AP) has beenapproached as a potential indicator of stress in the epidermalmucus of Atlantic salmon (Salmo salar) high levels of APwere observed in the mucosa of Atlantic salmon infectedwith the ectoparasite Lepeophtheirus salmon [21] HoweverAP activity in pirarucu could be related with the fish growthHuman serum AP may provide an index of bone formationrate and probably plays a role in the mineralization of newlyformed bone [22]

The seasonal variation observed in the RBC Hb and Htcin pirarucu may be due to a compensatory effect betweenrainfall and temperature Thus with more oxygen availablein water fewer red blood cells are needed to carry oxygenaround the fish body consequently the hematocrit andamount of hemoglobin available are lower [23] Similarresults were obtained for RBC of Oncorhynchus mykiss [23]however with higher rates probably due to higher seasonalvariations in temperature experienced in the northern hemi-sphere as well as the type of A gigas breathing Hemat-ocrit values for O mykiss were low in the summer (highertemperatures) and high in winter (lower temperatures) [24]demonstrating the differences in the physiological responseto environmental stimuli into distinct species Decrease inMCV in P2 is strongly related to low hematocrit percentage atthis period indicating the possible development of anemia inresponse to seasonal variation a chronic stress situationThelow Htc levels exhibit hemoconcentration as a help to copewith the stress related to oxygen demand corroborating withthe results obtained by Gomes [25] Lower levels of dissolvedoxygen should not be detrimental to the creation of pirarucuespecially juveniles which during its development shouldprovide breathing air due to a change in its swim bladder[26] This may be explained since A gigas is considered anobligatory air-breathing fish however 10 of its breath stilldepends on the oxygen dissolved in water [24]

The type of growth observed for pirarucu was allometricpositive (119887 gt 3) indicating a greater increase in mass thanin length Growth-type isometric was observed to A gigas insemi-intensive fish farming in central Amazonia [27] Studiessuggest that allometry should be used to characterize thedifferent growth strategies of fish associated with ecologicalbehavioral and physiological characteristics [28] CF is anindex widely used in the bioecology of fish it reflects thephysiological status of the animal conditioned to interactionof biotic and abiotic factors [29] The values of CF inthis work did not show seasonal variation however thevalues were high indicating a good state of fish healthinessin cultivation Furthermore the progressive increase in CFhaving the highest value in P3 may be an indicator of onset

gonadal development in A gigas since it can also be used asan indicator of sexual maturation [10]

5 Conclusions

Lectin activity decreases in period of lower medium rainfalland temperature LDH activity is more sensitive to changesin temperature than rainfall AP could be related to bonegrowth of fish since length (cm) body mass (g) and enzymeactivity increased progressively Hb Htc and RBC decreasedin response to dissolved oxygen in water and reflect changesin hematimetric indexes of Wintrobe Pirarucu growth wasallometrically positive good healthiness in cultivation wasindicated by CF

Regulation of seasonal effects in fish has not yet beensatisfactorily elucidated but surely constitutes a complexdefense mechanism in these animals A gigas physiologicalinformation under stress caused by environmental factorsbeside expanding the biological knowledge of the speciesmay also be useful in developing better techniques to increasethe success of cultivation and improve fish production indifferent periods of the year

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Authors express their gratitude to the Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) forresearch grants postdoctoral support (EVMMC) and fel-lowship (LCBBC) They are also deeply grateful to MariaBarbosa Reis da Silva for technical assistance The Estacao deAquicultura Continental Prof Johei Koike Departamento dePesca da Universidade Federal Rural de Pernambuco (UFRPE)is acknowledged for fish availability also the meteorologicaldata from the Instituto Nacional de Meteorologia (INMETBrazil) is acknowledged

References

[1] E A Ono R Roubach and M F Pereira ldquoPirarucuproductionmdashadvances in Central Amazon Brazilrdquo GlobalAquaculture Advocate vol 6 pp 44ndash46 2003

[2] F H Dos Santos Fogaca E G De Oliveira S E Q Carvalhoand J F De Seixas Santos ldquoYield and composition of pirarucufillet in different weight classesrdquo Acta Scientiarum vol 33 no 1pp 95ndash99 2011

[3] E G Oliveira A B Pinheiro V Q Oliveira et al ldquoEffectsof stocking density on the performance of juvenile pirarucu(Arapaima gigas) in cagesrdquo Aquaculture vol 370-371 pp 96ndash101 2012

[4] G B Skomal and JWMandelman ldquoThephysiological responseto anthropogenic stressors in marine elasmobranch fishes areview with a focus on the secondary responserdquo ComparativeBiochemistry and Physiology vol 162 no 2 pp 146ndash155 2012


[5] R F Bezerra M C F Soares A J G Santos E V MMacielCarvalho and L C B B Coelho ldquoSecondary indicators ofseasonal stress in the amazonian pirarucu fish (Arapaimagigas)rdquo in Advances in Environmental Research J A DanielsEd vol 28 pp 233ndash244 Nova Science Publishers New YorkNY USA 2013

[6] F Pascoli G S Lanzano E Negrato et al ldquoSeasonal effectson hematological and innate immune parameters in sea bassDicentrarchus labraxrdquo Fish and Shellfish Immunology vol 31 no6 pp 1081ndash1087 2011

[7] T J Bowden ldquoModulation of the immune system of fish by theirenvironmentrdquo Fish and Shellfish Immunology vol 25 no 4 pp373ndash383 2008

[8] S M Reppert and D R Weaver ldquoCoordination of circadiantiming in mammalsrdquo Nature vol 418 no 6901 pp 935ndash9412002

[9] E V M Maciel Carvalho R F Bezerra R S Bezerra etal ldquoDetection of the first lectin with antimicrobial activitypresent in Serum of the Amazonian Fish Tambaqui (Colossomamacropomum)rdquo Fisheries Science vol 78 no 4 pp 866ndash8792012

[10] A A Ayoade and A O O Ikulala ldquoLength weight relation-ship condition factor and stomach contents of Hemichromisbimaculatus Sarotherodon melanotheron and Chromidotilapiaguentheri (Perciformes Cichlidae) in Eleiyele Lake South-western Nigeriardquo International Journal of Tropical Biology andConservation vol 55 no 3-4 pp 969ndash977 2007

[11] M T S Correia and L C B B Coelho ldquoPurification ofa glucosemannose specific lectin isoform 1 from seeds ofCratylia mollis mart (Camaratu Bean)rdquo Applied Biochemistryand Biotechnology vol 55 no 3 pp 261ndash273 1995

[12] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[13] G N Bowers Jr and R B McComb ldquoA continuous spectropho-tometric method for measuring the activity of serum alkalinephosphataserdquo Clinical Chemistry vol 12 no 2 pp 70ndash89 1966

[14] M Saravanan K Prabhu Kumar and M Ramesh ldquoHaema-tological and biochemical responses of freshwater teleost fishCyprinus carpio (Actinopterygii Cypriniformes) during acuteand chronic sublethal exposure to lindanerdquo Pesticide Biochem-istry and Physiology vol 100 no 3 pp 206ndash211 2011

[15] S Dirican and S Cilek ldquoCondition factors of seven Cyprinidfish species from Camligoze dam lake on central AnatoliaTurkeyrdquo African Journal of Agricultural Research vol 7 no 31pp 4460ndash4464 2012

[16] G R Vasta M Nita-Lazar B Giomarelli et al ldquoStructuraland functional diversity of the lectin repertoire in teleost fishrelevance to innate and adaptive immunityrdquoDevelopmental andComparative Immunology vol 35 no 12 pp 1388ndash1399 2011

[17] M A S Lino R F Bezerra C D C Silva E V M MacielCarvalho and L C B B Coelho ldquoFish lectins a brief reviewrdquoin Advances in Zoology Research O P Jenkins Ed vol 5 pp95ndash114 Nova Science Publishers New York NY USA 2013

[18] A F S Santos T H Napoleao R F Bezerra et al ldquoStrategiesto obtain lectins from distinct sourcesrdquo inAdvances inMedicineand Biology L V Berhardt Ed vol 63 pp 33ndash60 Nova SciencePublishers New York NY USA 2013

[19] A A Khidr A E Said O A A Samak and S E AShere ldquoThe impacts of ecological factors on prevalence mean

intensity and seasonal changes of the monogenean gill parasiteMicrocotyloides sp infesting theTerapon puta fish inhabitingcoastal region of Mediterranean Sea at Damietta regionrdquo TheJournal Basic Applied Zoology vol 65 no 2 pp 109ndash115 2012

[20] B Magnadottir B Gisladottir S S Audunsdottir B T Bra-gason and S Gudmundsdottir ldquoHumoral response in earlystages of infection of cod (Gadus morhua L) with atypicalfurunculosisrdquo Icelandic Agricultural Sciences vol 23 no 1 pp23ndash35 2010

[21] N W Ross K J Firth A Wang J F Burka and S CJohnson ldquoChanges in hydrolytic enzyme activities of naiveAtlantic salmon Salmo salar skin mucus due to infectionwith the salmon louse Lepeophtheirus salmonis and cortisolimplantationrdquo Diseases of Aquatic Organisms vol 41 no 1 pp43ndash51 2000

[22] J R Farley S L Hall D Ilacas et al ldquoQuantification ofskeletal alkaline phosphatase in osteoporotic serum by wheatgerm agglutinin precipitation heat inactivation and a two-siteimmunoradiometric assayrdquoClinical Chemistry vol 40 no 9 pp1749ndash1756 1994

[23] J Cerezo Valverde and B Garcıa Garcıa ldquoSuitable dissolvedoxygen levels for common octopus (Octopus vulgaris cuvier1797) at different weights and temperatures analysis of respi-ratory behaviourrdquo Aquaculture vol 244 no 1ndash4 pp 303ndash3142005

[24] P R Rauta B Nayak and S Da ldquoImmune system and immuneresponses in fish and their role in comparative immunity studya model for higher organismsrdquo Immunology Letters vol 148 no1 pp 23ndash33 2012

[25] LD CGomes ldquoPhysiological responses of pirarucu (Arapaimagigas) to acute handling stressrdquo Acta Amazonica vol 37 no 4pp 629ndash634 2007

[26] G V F Drumond A P deAlmeidaCaixeiroM Tavares-Dias JL Marcon and E G Affonso ldquoBiochemical and hematologicalcharacteristics of the pirarucu Arapaima gigas Schinz 1822(Arapaimidae) from semi-intensive culture in the AmazonrdquoActa Amazonica vol 40 no 3 pp 591ndash596 2010

[27] M Tavares-Dias C S O Araujo A L S Gomes and S MS Andrade ldquoWeight-length relationship and relative conditionfactor (Kn) of the pirarucu Arapaima gigas Schinz 1822 (Ara-paimidae) in semi-intensive culture in Amazonas State BrazilrdquoRevista Brasileira De Zoociencias vol 12 no 1 pp 59ndash65 2010

[28] S Dutta SMaity A Chanda A Akhand and S Hazra ldquoLengthweight relationship of four commercially important marinefishes of Northern Bay of Bengal West Bengal Indiardquo Journalof Applied Environmental and Biological Science vol 2 no 1 pp52ndash58 2012

[29] E O Ahmed M E Ali and A A Aziz ldquoLength-weightrelationships and condition factors of six fish species in atbarariver and khashm el-Girba Reservoir Sudanrdquo InternationalJournal Agricultural Science vol 3 no 1 pp 65ndash70 2011

Submit your manuscripts athttpwwwhindawicom

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OceanographyInternational Journal of


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ClimatologyJournal of


affected by seasonal variations and in general biochemi-cal hematological and immunological parameters such aslevels of various blood cells hematocrit percentages [6]lysozyme activity [7] respiratory burst levels of head-kidneymacrophages [8] and lectin activity [9] The biochemicaland hematological parameters are useful tools to determinethe characteristics of fish blood in different situations suchas stress or normality The relative robustness or degreeof wellbeing from a fish is expressed by the coefficient ofcondition or condition factor (CF) considered basically asthe quotient between observed mass and theoretical massestimated through the length-mass relationship Variationsin fishrsquos coefficient of condition primarily reflect the state ofsexual maturity and degree of nourishment they provide rel-evant information about physiological and health character-istics of individual or population which are very importantin captive fish to their management and maintenance [10]

The aimof this workwas to analyze the effects of seasonal-ity (temperature and rainfall) on biochemical (lectin activitylactate dehydrogenase and alkaline phosphatase activities)and hematological parameters (hematocrit hemoglobin andhematimetric indexes of Wintrobe) that can be used as phys-iological indicators of stress Also the influence of seasonalityon growth of pirarucu developed in captivity as well asthe state of wellbeing of the fish was analyzed This is afirst study that relates chronic stress with biochemical andhematological indicators of seasonal stress as well as healthand growth in A gigas fish farming

2 Material and Methods

21 Weather Data Obtention Rainfall and TemperatureWeather data was provided by the Instituto Nacional deMeteorologia (INMET Brazil) collected fromweather station82900 (08∘031015840S 34∘571015840W) Recife PE Brazil according tointernational standards of the World Meteorological Orga-nization Medium rainfall (mm3) as well as medium tem-perature (∘C) was calculated to each period respectively999mm3 and 2614∘C to period 1mdashP1 (April to July 2010)274mm3 and 2546∘C to period 2mdashP2 (August to November2010) 58mm3 and 2694∘C to period 3mdashP3 (December 2010to March 2011) (Figure 1)

22 Obtaining Fish Blood Fish were provided by the EstacaodeAquiculturaContinental Prof Johei KoikeDepartamento dePesca da Universidade Federal Rural de Pernambuco (UFRPE)and developed in earth pond The juvenile animals (119899 = 6)were anesthetized by hypothermia on ice immediately afterblood collection procedure the fishes returned to earth pondBlood was obtained from caudal vein with syringes 5mL21 G 23G or 25G needles (BD Precision Glide PN Brazil)according to fish size To obtainwhole blood tubes containingEDTA 18mgmL as anticoagulant (Vacuette Greiner bio-one Brazil) were used Serum was collected from tubeswithout anticoagulant the blood was centrifuged at 3000timesgfor 10min at 4∘C

23 Lectin Activity and Protein Evaluation Serum lectin acti-vity (LA) was evaluated as specific hemagglutinating activity

02468

1024

26

28

lowast

lowast

lowast

Period 3Period 1 Period 2

Med

ium

tem

pera

ture

(∘C)

Med

ium

rain

fall

(mm

3)

Medium temperature (∘C)Medium rainfall (mm3)

024681024

26

28

Figure 1 Medium rainfall and medium temperature for eachperiod Period 1 (AprilndashJuly 2010) Period 2 (AugustndashNovember2010) Period 3 (December 2010 to March 2011) Data were obtainedfrom Instituto Nacional de Meteorologia (INMET Brazil) (lowast) Signif-icant difference in rainfall among the periods (119875 lt 005)

inmicrotiter plates with 96 wells [11] Specific hemagglutinat-ing activity was defined as the ratio between titer and proteinconcentration (mgmL) and expressed with hemagglutinat-ing activity units for protein milligrams (HAUmg) Proteinconcentrations were determined by Bradford [12]

24 Lactate Dehydrogenase Activity Lactate dehydrogenaseactivities (LDH) were determined following the oxidation ofNADH (340 nm 25∘C) The reaction mixture contained atotal volume of 1mL 50mM imidazol 1mM KCN buffer pH74 at 25∘C 013mM of NADH and different concentrationsof pyruvate for LDH saturation plots One unit of enzymeactivity is defined as the amount of enzyme using 1 120583mol ofsubstrate per min (340 nm 25∘C) Each value represents themean of three measurements

25 Alkaline Phosphatase Activity The serum alkaline pho-sphatase activity (AP)was performedwithmodifications [13]Briefly enzyme activity was measured using p-nitrophenylphosphate (pNPP) as substrate (50mM) in 1M diethanol-amine (pH 98) containing 1mM MgCl

2(405 nm 25∘C

1min)

26 Total Count of Red Blood Cells To determine the totalcount of red blood cells (RBC) a 1 in 1000 dilution wasmade in 002Mphosphate saline buffer (PBS pH 73) Countswere carried out using a Neubauer haemocytometer (INLABBrazil) and expressed as cellmm3 [14]

27 Hematocrit Hemoglobin and Hematimetric Indexes ofWintrobe The hemoglobin (Hb) levels were obtained usinga kit for determination of hemoglobin in whole blood(Doles Brazil) following the manufacturerrsquos instructionsThe hematocrit (Htc) was determined by the microhema-tocrit technique and result was expressed as percentage



5

10

15

20

25

30

35

40

lowast

Season


Lect

in ac

tivity

(a)


lowast

lowast

lowast

lowast

lowast

lowast

LDH (UL)AP (UL)

0

50

100

150

200

250

300

350

400

450

LDH

and

AP

activ

ities

(b)





3 Results








lowast

lowast

Season



RBC

Htc

and

Hb

02468

1012141618202224262830

(a)


20

40

60

80

100

120

140

160

180

200

lowast

lowast

lowast

Hem

atim

etric

inde

xes o

f Win

trobe

Season


(b)


8

10

12

14

16

18

20

22

b = 346

b = 333

b = 312

Season

(


Mas

s (times103

g)




(a)

99102105108111114117120123126129132

CF = 0926 plusmn 0124

CF = 0909 plusmn 0058

CF = 0891 plusmn 0038


Leng

th (c

m)

(1155 cm

(10986 cm

(1016 cm

plusmn 267)


(b)



4 Discussion








5 Conclusions





Acknowledgments


References




































Journal of












Volume 2014

Advances in









Geophysics
















5

10

15

20

25

30

35

40

lowast

Season


Lect

in ac

tivity

(a)


lowast

lowast

lowast

lowast

lowast

lowast

LDH (UL)AP (UL)

0

50

100

150

200

250

300

350

400

450

LDH

and

AP

activ

ities

(b)





3 Results








lowast

lowast

Season



RBC

Htc

and

Hb

02468

1012141618202224262830

(a)


20

40

60

80

100

120

140

160

180

200

lowast

lowast

lowast

Hem

atim

etric

inde

xes o

f Win

trobe

Season


(b)


8

10

12

14

16

18

20

22

b = 346

b = 333

b = 312

Season

(


Mas

s (times103

g)




(a)

99102105108111114117120123126129132

CF = 0926 plusmn 0124

CF = 0909 plusmn 0058

CF = 0891 plusmn 0038


Leng

th (c

m)

(1155 cm

(10986 cm

(1016 cm

plusmn 267)


(b)



4 Discussion








5 Conclusions





Acknowledgments


References




































Journal of












Volume 2014

Advances in









Geophysics
















lowast

lowast

Season



RBC

Htc

and

Hb

02468

1012141618202224262830

(a)


20

40

60

80

100

120

140

160

180

200

lowast

lowast

lowast

Hem

atim

etric

inde

xes o

f Win

trobe

Season


(b)


8

10

12

14

16

18

20

22

b = 346

b = 333

b = 312

Season

(


Mas

s (times103

g)




(a)

99102105108111114117120123126129132

CF = 0926 plusmn 0124

CF = 0909 plusmn 0058

CF = 0891 plusmn 0038


Leng

th (c

m)

(1155 cm

(10986 cm

(1016 cm

plusmn 267)


(b)



4 Discussion








5 Conclusions





Acknowledgments


References




































Journal of












Volume 2014

Advances in









Geophysics



















5 Conclusions





Acknowledgments


References




































Journal of












Volume 2014

Advances in









Geophysics













































Journal of












Volume 2014

Advances in









Geophysics














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