PATHOPHYSIOLOGICAL EFFECT OF CImONIC AND ACUTE STRESS IN ATLANTIC SALMON, SALMO SALAR (ACTINOPTERYGII: SALMONIFORMES: SALMONIDAE)
Armin KOUSHA
1*,Reidar MYKLEBUST
2,and Rolf Erik OLSEN
3 1Norwegian College of Fishel)! Science, Faculty of Biosciences, Fisheries, and Econom.ics,The Arctic University of Norvvay, 9037 TromSRJ,Norway
2Molecular and Im.aging Center (MIG), University of Bergen, Norway
3Institute ofA1arine Research, Matre Aquaculture Research tation, A1atredal, Norway
Kousha A., Myklebust R., 01en R.E. 2013. Pathophy iological effect of chronic and acute stress in Atlantic salmon, Salmo sa/ar (Actinopterygii: SalmonifOlmes: Salmonidae). Acta Ichthyol. Piscat.
43 (4): 299-305.
Abstract. The knowledge on the effect of different stress factors on Atlantic salm.on?Satmo alar Linnaeus, 1758, is far from complete and therefore we decided to find out how the low water level stress could effect patho- physiological parameters such as: plasma c0l1isollevel, haemoglobin, haematocrit, chloride (Cn, sodium (Na+), osmolality, lactate, and glucose in this fish; and how this stressor affects the gut morphology. Two hundred and four juvenile Atlantic salmon were randomly distributed into six fibreglass tanks and divided into two groups:
group .1(control) and group 2 (low water level tress). The low water level stre did not affect growth perfonn- ance and the pathophysiological parameters. Light- and n-ansmission electron microscopy evaluations of the pyloric caeca and the distal intestine revealed that chronic tress had no effect on gut morphology. Low water level stress had no clear effects on pathophysiological parameter and gut m.orphology of Atlantic almon.
Keywords: chronic and acute stress, Atlantic salmon, haematological parameters, gut, light microscopy, trans- mission electron microscopy
It i well known that fi h are expo ed to stre or in the wiLd as well as in captivity, pm1icuLarly during handling in commerciaL aquacull1.u· . Num rou paper have r pOlted that chronic and acute stress may be associated with bio- chemical and physical disturbances and vm'ious physioLogi- cal effects (Kubilay and Uluk6y 2002, Vijayan et al. 2009).
Plasma cOl1isol is reported to increase in fish after stress exposure and tbis is well documented in several studies (Balton et al. 1988, Mu tafa and MacKinnon 1999,01 net al. 2002, 2005, 2008, Askarian and Kousha 2009, Santos et a1. 2010). COltisoL can also affect growth and metabo- lism of fish dming chronic and acut he (Fa t et al. 2008, Vijayan et al. 2009).
Fridell et al. (2007) r port d increa ed COJti 01 1 vel dming chronic sU'ess response in the hyperoxic group (reduced water flow) of Atlantic salmon, Salm.o salar Linnaens 1758, reared in fre bwater. In juvenile rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), the level of COl1isoLincreased dming acute stress, but after 1 h it regained its normal level (Fa t et al. 2008, Mmtin z- Porcbas et a1. 2009).
Olsen et al. (2008) subjected Atlantic cod, Gadus morhua Linnaeu , 1758, to an acute tre (exhau tiv
exerci ) and tbi cau ed an increa e in blood baematocrit and pLasma C0I1isoL,glucose, chlOlide, osmolality, and lactate. Other I1.ldi hav repOlted that acut str ss inflicted by high fi h densities, induced a ignificant increase in glucose and Lactate in netted European seabass, Dicen.trarchus labrax (Linn.aeus 1758) (see Santos et al. 2010) and elevating Level of plasma cortisoL and lactate in Atlantic salmon exposed to tbe infectious ana mia (01en t at. 1992) and acut str ss respon e in Atlantic cod (OLsen et al. 2008). The first aim of the pre ntly r port. d study was to valuat wbetb r I.owerin.g the water I vel affj ct the growth p rformance and tb pathophysiological parameters of salmon. Information is availabL of th effect of noi on. growth performan.c smelting rates, parasite effects, and disease resistance (Terhune et al. 1990, Mustafa and MacKinnon 1999, Wysocki et al. 2007, Davi.dson t al. 2009), but to Oll\' knowledge no paper on tbe effect of low water stress on gut morpbology ha been publisbed.
Morphologi.cal evaluation of tbe ga troin.t stinaL tract by light microscopy (LM) and transmission eLectron microscopy (TEM) have been suggested to be an impor- tant tool to valuate wh. n. fi h. are expo ed to tr s
• Correspondence: Annin KOllsha, Fakullel for biovilenslmp, liskeri og okonomi, HiT Norges arkli ke 11lliversilel, 9037, TronlSo, Non ay, phone: (+47) 91008263, e-mail: anninkonsha@gmaiIcom_
(Olsen et al. 2002). In thr e previous shldies, Olsen et al.
(2002, 2005, 2008) focused on the effect of stress response on intestinal lining of Atlantic almon, inte tinal function of rainbow h'out., and in fed- and food deprived Atlantic cod, Gadus II/.orhua.According to t.hese shldies, acute stress had marginal effect on gut morphology, junc- tional complexes damage (zone between epit.helial cells), and increased cellular detachment in Atlantic salmon and rainbow trout.. The econd aim of th pI' sently r pOli d shldy was therefore to address t.he effect of low water st.ress on morphological changes in pyloric caeca and dis- tal intestine of Atlantic salmon.
The experiment. started in January 2011 and last.ed until May 2011-for a total of 65 day. Juvenile of Atlantic salmon, Salmo salar, fi'om a single population were obtained fi'om the lnst.ihlt.e of Marine Research, Matre Research Station, Matr dal, NOJway. Two hundr d and four fish were randomly distributed int.o 6 fibreglass tanks 34 fish per tank, with a total average wet weight of approximately 7 kg per tank. Th 400-L tank wer up- plied with sea water (temperature 8-IO°C, water flow 20 L . min-I). The fi h weI' acclimatized to exp rim ntal condition for two days prior to the xp riment. The indi- vidual fish weight (mean ± st.andard deviat.ion) ranged from 104±4.9 to 354 ±6.1 g whil individual fork I ngth (mean ± standard deviat.ion) ranged fi'om 220 ± 1.5 to 300 ± 1.6 mm. The fish were divided int.o two groups:
group 1 (control) and group 2 (low water I vel tre ). Th experiments (in two groups) were cani.ed out in tJi.plicate for 65 days. During that time the fish were fed Fiskefor pel- lets (Skretting Ltd., 50 mg, 3 mm) at 1000 hand 1400 h;
2.5% of wet body weight per day. Faeces and uneat.en food in the tanks were collected 1 h after each feeding.
Cleaning ofrearing tank i a normal procedme in com- mercial aquaculhu'e and this chronic stJ'ess may affect. the fish. In order to evaluat how fi h r act to thi tr ; th water level in three tanks were lowered (reduced tolO em) for 30 min twice a day at 0900 hand 1300 h t.hroughout the 65 days. After 65 days of rearing, all the groups w I' exposed to low water level for 30 min (acut.e stress).
Specific growth rate (SGR) and feed conversion ratio (FCR) were calculated u ing th formulae de crib d by De Silva and Anderson (1995) as follows:
SGR =100 [In Wc- InWi] .[1
where: ~ and Wfwere the initial and final body weight [gJ and t,the time in days.
FCR =FC . WG-1
where: WG=wet weight. gain, FC=dry feed consumed [gJ.
Fish sampled for blood analysis were anesthet.ized u iog tJ-icaine methane ulfonate (MS-222), 70 ppm for 3 min, to avoid bleeding stress. Blood amples from 30 fish of each treatment were taken from the caudal artery using commercially produced h parini ed n die.
Haematocrit was measured using heparinised microcap- illary rubes and a Compur MII00 haematocrit centrifuge according to 01 en et.al. (2002, 2008). Blood (l00 f-lL) w I' transfened to a 0.5 mL Eppendorft.ubes and frozen in liq- uid nitrogen and flUih r stor d at -80°C plioI' to analy i
of haemoglobin, which was est.imat.ed tl ing the ReflotronPlus auto analyzer (Roche, Mannheim, Germany) according to 01 eu et al. (2008). Blood osmo- lality wa mea ured using freeze point determination (Advanced Micro Osmomet.er Model 3300, Advanced In trum nt Inc., NOlwood MA USA). Plasma was pre- pared from the remaining blood sample by immediate centrifugat.ion at. 11 000 rpm for 1 min (Jouan A14), frozen in liquid nitrogen and tor d at -80°C until ana- lyzed. Plasma chloride concentrat.ions (CI-), lactate, Na+
and K+ weI' analysed by ion-selective el ctrod (Cobas C.llI). Plasma cOltisol was measured by ELISA as described by Barry et al. (2001).
Fi hued for analy ing biochemical parameters were al 0 used for LM and TEM evaluations of the gut.
Abdomens were carefully opened aseptically and samples from pyloric ca ca and distal iute tine were immediately fixed in formalin and Kamovsky fixative as described by Olsen et. al. (2002). To determine morpbol.ogi.cal. differ- uc ofth variou treatm nt 10 randomly selected LM and TEM images from each segment were sampled from five fi h of each tJ- atment group.
The morphologi.cal chang s weI' valuated in the terms of full and empty goblet cells, length of microvilli [11m], disintegrated mi.crovil1i. number ofvacuol.es oedema and cell damage, and number of granulocytes in lamina pro- pria. Differences were ranked according to Ring0 et al. (2007b); 0=no ob IV d, 1=low (1-3 out of 10 images) 2 = moderate (4-6 out of 10 images), 3 = high (7 or more out of 10 images).
Stati tical analy of th re ults w I' p lformed using MS Excel software. Numerical results were exhibited as the mean ±standard deviation. Th Pearson's product-moment COlTlation co ffi.cient wa cal.culated and t ted for signjf- icance against the no conelation zero hypothesis through th proc dm conelati.on. Data perfOlID d to analysis of variance (ANOV A) test to observed variance in a pmticu- lar vati.able. The significance level was chosen atP<0.05.
Normality and vat'i.ance homogen ity of data were ca.nied out by using NOlm-Quant software and F-test in MS Excel, respectively. Student's I-test was run as post hoc test when ANOV A wa valid for data t.
The experiment was approved by the Food Safety Authotity, FaTS, orway (No. 2907: Etabl.ering av k.ro- ni k tre mod 11).
Throughout the experiment no mortalities were ob rved. Th pre n.t1.yr ported tudy show d that str had not ignificantly affected growth dming the experi- ment.; 65 days (Table 1). The specific growth rates (SGR) ranged betwe n 0.95 and 0.97 fe d conver ion ratio (FCR) ranged from 0.46 to 0.54, and weight gains were between 6050 and 6277 g.
Re ult of blood cOlti 01 ampl. d from the control. group (not exposed to stress; 65 days) and after chronic stress and acute stress are presented in Table 2. The results of blood COlii ollevel (ng' rnL-1) ofth two tr atm nt group: con.- trol (group 1), and low water level stress (group 2) showed no ignificant differ nee (P> 0.05). Table 3 display the
result of haematocrit, haemoglobin, glucose, lactate, K, Na, el,and osmolality and show that these parameters were not affected by the different treatments.
Morphological evaluations of pyloric caeca and distal intestine of the control group and group 2 exposed to chronic tress a.re hown in Table 4. No clear diffi .r nc with regard to full and empty goblet cells, number ofvac- uoles and granulocytes within lamina p.rop.ria were observed. Mean lengtb of microvilli in pyloric ca ca; e ti- mated by light microscopy, were shorter but not signifi- cantly different; in tbe control group; 3.9 ± 0.59 11m,com- pared with the low water level group; 4.6 ± 0.75 ~lm.
A similar trend was observed for the microvilli length in distal intestine being borter in tbe control group com- pared to tbe otber u'eatment groups, but the results showed no significant differences between the groups (P> 0.05).
Histological evaluation of pylo.ric caeca and di tal intestine of Atlantic salmon exposed to one hour of acute stress by LM and TEM are sbown in Table 5. No loosen- ing of enterocytes fi'om ba al membrane in the treatm nt groups was observed (results not shown). A high score (3) of both full and mpty goblet cell we.re noticed in group 1 (control) compare to group 2 (low water level; core 1).
Disintegrated microvilli determined by TEM, but to less extent were only detect din di tal inte tine oftbe contJOI
group. Some oedema or cell damages were noticed in tbe gastl'OintestinaL tract of group 2, but no clear effect was noticed betw en the treatment . The pre eoces of granu- locyte in lamina propria in pyloric caeca and distal intes- tine were generally high in most of the gut segments of the treatm nt group amp led on hoLU'po t acute tr s except for distaL intestine of group 1; estimated by TEM and group 2; estimated by LM. Mean. length of mi.crovilli timated by LM in pylori.c caeca; were h0l1 r in the con- troL compared to the low water level group; 5.5± 1.30 11m but th r ult bow d no significant djffer uc s between the groups (p> 0.05).
With regard to the microvilli length the only signifi- cantly difference (f> < 0.05) ob elved was between in distal intestine of group 2 (chronic stress) vs. distal inte - tine of group 2 (acute stress) and pyloric caeca of group 2 (chronic tre ) v . pylO1ic caeca of group 2 (acut tr s ).
LM evaluation of pyloric caeca and distal intestine of salmon exposed to chronic stress (I.ow water I.evel) after 65 day how d normal appearauc (Fig. 1). TEM evalu- ations of pyloric caeca and distal intestine of this group w re al 0normal (r ult not hown). TEM evaluation of di tal int tine of almon xpo d to low wat r level stress for 65 days (Fig. 2), showed no major influence on the eoterocytes of the di.staLiot stine.
Table 3
Table 2 Blood COJ1i011 vel of Atlantic almon,
Sa/mo salar, after 65-day exposure
to low water level acute str ss and 1 b po t-acut tre Table 1 Specific growth rat , fi ed conve.r ion ratio, initial and final weight per treatment, and weight gain of Atlantic salmon, Salmo safar, during 65-day exposure
to low water level acut tr s
Values are mean ± standard deviation; Group 1= control, Group 2 =low water level tr atment; C I =blood cortisol level before acute stress, C2 = blood c0l1isoilevei one hour post-acute stress; n = 30 (in each treatment); cFish not exposed to chronic stress.
Group 2 36.6 ± 6.4 32.5 ± 3.6 9.18±1.9 8.84 ± 0.9 4.08 ± 0.6 4.71 ± 0.4 3.01 ± 0.8 3.62±0.8 3.5 ± 0.9 4.3±0.5 149 ± 8.7 164 ± 3.4 126 ± 7.5 140 ± 4.5 327.8±6.1 349.2± 6.1 Group I
33.1±4.8c 35.5±4.7 9.60 ±2.2c 9.79± 1.6 4.32 ± 0.5c 4.70±0.7 3.41 ± 0.7c 5.52 ± 1.8
3.8± 0.5e 4.3 ±0.5 154±2.3c 160±9.3 127±2.9c 133±8.7 329.9 ±7.6c 355±6.3 Parameter
Hctl [%]
Hct2 [%]
Hb1 [g'dL-1]
Hb2 [g' dL-1]
Glul [mm'L-1]
Glu2 [mm'L-1]
LactJ [mm -L-I]
Lact2 [mm -L-I]
KI [lmn' -1]
K.2[lmn' -1]
a1 [mm'L-I]
a2 [mm' L-1]
Cl1 [lmn'L-I]
CI2 [lmn' L-I]
Osl [MOsm' kg-I]
Os2 [MOsm' kg-I]
Treatment Blood parameters of Atlantic salmon,
Salmo sa/aI',after 65-day xpo w' to low wat r I vel acute stress and 1 b post-acute stress
Values are mean ± standard deviation; Group I.=control, Group 2 =low water level treatment; n=30 (in each treat- ment); cfish not exposed to chromc stress; Hct=haematocrit, Hb = haemoglobin, Glu = glucose, Lact = lactate, K = potas- sium, Na = sodium, CI = chloride, Os = osmolality; Numbers J and 2 following individual parameters denote parameter level before (e.g., Hctl) and 1 h (e.g., Betl) after acute stress.
Group 2 12.01 ± 3.42 171.33±67.2 Treatment
Group 1 11.01 ±29.01 160.87±65.2 Treatment
Parameter [ng'mL-I]
CI C2
Group 1 Group 2
(control) (low water level)
SGR 0.95 ± 0.ll7 0.97 ± 0.079
FCR 0.46±0.0005 0.54±0.0001
lnjtial weight [g] 6903.3 7131.3
Final weight [g] 12954 13408.7
Weight gain [g] 6050.7 6277.4
Values are mean ± standard deviation; weight gain =final weight - injtial weight; n = 102 (in each treatment);
SGR = specific growth rate, FCR = food conversion ratio.
Table 4 HistoLogicaL evaLuation of pyLoric caeca and distaL intestine of AtLantic saLmon,Salmo sCilar,
after 65 days ofrearing; with no tre (control; group 1) and lowered of water level (group 2)
Group] Group 2
IEM
1
o o
I Distal intestine Pyloric caeca
Distal intestine Pyloric caeca
Characteristics
LM TEM LM T 'M LM I M LM
Full goblet cells 1 2 1 1 1 3 1
'Tnpty goblet cells 0 0 1 0 0 ] 0
No. of vacuoles 1 1 1 1 2 3 2
Granulocytes in LP 2 2 ] 2 2 2 1
MicrovilLi length 3.9 0.59 3.8 1.14 4.6 0.75 4.1 0.40
Group I=control, Group 2=low water level treatment; LM=light microscopy, 'IEM =transmission electron microscopy;
LP
=
lamina propria; Microvilli length values [11m] are mean±standard deviation; Scores of semi-sections for LM and IEM are based on LOrandomly taken images from each gut segment per treatment; Evaluations were ranked according to Ring0 et al. (2007a): 0=no observed, 1=low (1-3 out of 10 images), 2=moderate (4 out of 10 images), 3=high (7 or more out of 10 images).Table 5 HistoLogicaL evaluation of pyloric caeca and distal intestine of Atlantic salmon Salmo sCilar,
expo d to 1 h acut tr
Characteristics
Group 1
Pyloric caeca Distal intestine
Group 2
Pyloric caeca Distal intestine IEM
2 1
o
3 I 2
LM IEM LM IEM LM IEM LM
Full goblet cells 3 0 2 0 I I I
Empty goblet cells 3 0 ] 0 1 ] 1
Disinl. microvilli 0 0 0 1 0 0 0
No. of vacuoles 3 2 2 I 3 3 3
OCD 0 0 1 0 1 1 1
Granulocytes in LP 2 2 2 0 3 3 0
Microvilli length 3.4 ± 0.54 4.2 ± 1.10 5.5 ± 1.3 3.1 ± 0.60
Group I=control, Group 2=low water level treatment; LM=light microscopy, IEM =transmission electron microscopy, Disint. microvilli =disintegrated microvilli, OCD=0 d ma or cell damage, LP=lanuna propria; Microvilli length values [11m] are mean ± standard deviation; Scores of semi-sections for M and IEM are ba ed on 10 randomly taken images from each gut segment p r treahnent; Evaluations were rank d according to Ring0 t al. (2007a): 0 =no observed, 1= low (1-3 out of ] 0 lmages), 2 = moderate (4-6 out of 10 images), 3 = high (7 or more out of 10 Images).
Bi.ron and Benfey (1994) showed a short-tenn increas- ing of plasma cOTti0],haematocrit, and gluco e level on diploid and triploid brook trout, Salvelinus fontinalis (Mitchill, 1814), exposed to 5 min acute handling stress.
In contrast, FTidell et al. (2007) repOJt dan increa ing of cor- tisol level during chronic stress response in the hyperoxic group of Atlantic salmon held in fre hwater. In th pre nt- ly repOlted study, group 2 with decrease of oxygen in water circulation, the cortisol leveL elevated simultaneously and reduced the erythropoie i activity compare to control.
Previous studies illu trated blood parameters uch as raised haematocrit, lactate level, glucose, osmolality, and plasma cOlti0] concentration following acute tr in Atlantic salmon (Olsen et al. 2002, Sundh et al. 2009), rain- bow trout, Oncorhynchus mykiss (see Olsen et al. 2005);
Atlantic cod, Gadus morhua ( ee 0] en et al. 2008); and European sea bass, Dicentrarchus labrax (see Santos et al. 2010), respectively. The I' ult ohhe pre ently report-
ed study are in. accordance these observati.ons but the
I' pon appear d to b somewhat higher in th control.
and low water level groups while haematocrit and haemo- globin had low r r spons in.group 2 (Table 4).
01 en t al. (2008) I' port d that increasing I vel of pLasma glucose in cod is caused by extended glycogenol- y i and g]uconeogen i. through the degradation of gLycogen, a process that may reLate to the prolonged eLe- vated levels of plasma cortisol, which will stimulate these proc se. Ob ervation of iocrea ed plasma g]llCO fo]- lowing group one in the presently reported study are in accordance wi.th the resul.ts of Bartoo. and Iwama (1991) and OL n t al. (2002). Although w di.d observed lower level of glucose in group 2 compare to the control, this finding was marginal. and not si.gnifi.cantly different.
Our tudy how d I vati.on of I.actate level after acut post stress in all treatments. A smaller decrease of lactate in group 2 after 65 day of chronic tress compar wi.th
Fig. 1. Light microscopy micrographs of the gastrointestinal tract of Atlantic salmon Sa/ilia salar, exposed to low water level for 65 day; A pyloric ca ca; B di tal iut tine; MY =microvil.li, GC=gobl t c II, V=vacuole
Fig.2. TEM micrograph of tbe di tal iot tine of Atlantic almon Safmo safar 65 days' MY =microvilli, D=desmosome, TJ=tight junction
for
acute situation and control o'eatment was noticed. Lactate levels in group 2 were lower compare witb control in acute post stress and might indicate that fish exposed to low water level are better adapt d to tre . Howev r, thi hypothesis merits further investigations.
Olsen et a1. (2002, 2005) repolted cell damage of Atlantic salmon and rainbow trout intestinal enterocytes during stress. In contrast to these results, no clear effect on cell damage was noticed in the pr sent study, indicat- ing that Atlantic salmon can adapt to chronic stress.
In the presently reported study, no morphological gut changes were ob erved. Widening of intJacellulaJ pac was noticed in fish exposed to low water level and vibra- tion sO·ess. These results are in contra t to tho e publish d by Olsen et al. (2002,2005), who repolted increa ed inter- cellular gap in rainbow trout and Atlantic salmon exposed to handling and fasting tr ,r pectively.
Our study demonso'ated that stress did not have any major effect on enterocytes in distal intestine of Atlantic
almon. This ob ervation i. in accordance with tho reported by Olsen et al. (2002, 2005).
The effect of stress responses on gut microbiota and the immune system wa inve tigated in endothermic animal (Kight and Swaddle 2011, Gonzalez-Rodriguez et aL.2013).
In fish, evaluation of the gut microbiota, gut immunology, and challenges studie are impoltant parameter to evalu- ate in stress experiments. As these tests were not can;ed out in our study we r commend that the e topic aJ
included in futur stJC' studies becau e the gastrointe ti- nal tract is one oftbe major infection routes in fish (Ringe et aL.2007a, 2007b).
ACKNOWLEDGMENTS
W would lil< to thank Prof. Einar Ring" at University of Trom Ii1for his valuable helps and advising. We also appreciate the help of Ivar Helge Matre, Grethe Thorsheim, Ton Vags tb, Mari.ta Lars 0, Stian Mork 0, and Lisse Dyrhovden of the Matre Research Station in technical issues and in fish handling. Tbanks to Anne Nyhaug and Ingvild Wend lbo of tbe 01. cular and Imaging Center University of Bergen, Norway for their help on Light and
I ctron microscopy valuation of gut samples.
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Rec ived: 19 Sept mb r 2013 Accepted: 24 October 2013 Pubtished el.ectronicaUy: 31 December 2013
