Effect of dietary willow herb, epilobium hirsutum extract on growth performance, body composition, haematological parameters and aeromonas hydrophila challenge on common carp, cyprinus carpio

Effect of dietary willow herb, Epilobium hirsutum extract on growth performance, body composition, hydrophila challenge on common carp, Cyprinus carpio Somayeh Pakravan, Abdolmajid Hajimoradloo & Rasool Ghorbani Fisheries Department, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran Correspondence: A Hajimoradloo, Fisheries Department, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, a consequent e¡ect on the industry’s economic devel-opment (Yunxia, Jianzhong & Guoliang 2001). The The present study was conducted to evaluate the use use of disinfectants and antimicrobials has shown of Epilobium hirsutum extract in diet of common carp, limited success in preventing or curing aquatic Cyprinus carpio (20 Æ 2 g). Di¡erent levels of plant extract (0%, 0.5%, 1, 3% and 3%12% multivitamin The organism Aeromonas hydrophila is a gram nega- (2 g of multivitamin per kg diet) were spread on com- tive facultative anaerobic short bacillus. It is motile and mercial diet. The feed was o¡ered by 8 weeks. Results grows wide range of temperature11.2^40.5 1C (Biradar, showed that ¢sh fed experimental diets had no signi¢- Goud, Neogi & Saunya 2007). Aeromonas hydrophila is a cant di¡erence (P40.05) in speci¢c growth rates, con- widespread, opportunities pathogen, causes high mor- dition factor, feed conversion ratios and survival tality of cultured and feral ¢sh (McDaniel1979). It is the compared with control. There were no signi¢cant dif- causative agent of the disease known as ‘haemorrhagic ference (P40.05) in moisture, crude protein, crude septicemia’,‘ulcer disease’, or ‘red-sore disease’. Aeromo- lipid and ash content of common carp fed diets con- nas hydrophila is generally found in the gastrointestinal taining various levels of plant extract. The mortality tract of ¢sh is considered an opportunistic pathogen.
of ¢sh challenged by Aeromonas hydrophila have been Most of bacteria, which are termed ‘opportunistic’ recorded for 30 days after challenging, results showed usually, do not cause disease unless other factors are that mortality decreased signi¢cantly (Po0.05) in ¢sh involved. Aeromonas hydrophila is always capable of fed diet containing plant extract compared with the producing disease if given the chance. Outbreaks of control. The lowest mortality observed in group fed the disease are usually associated with a change in en- diet containing 3% plant extract. Haematological vironmental conditions, such as stress, overcrowding, a parameters showed that white blood cells had signi¢- sudden change of temperature, transfer of ¢sh, mis- cantly (Po0.05) increased in infected and uninfected handling, poor water quality, high nitrite and carbon groups compared with the control. Red blood cells, dioxide levels (Adanir & Turutoglu 2007).
haemoglobin and haematocrit had no signi¢cant Motile aeromonad septicemia, caused by various change (P40.05) in infected and uninfected groups strains of A. hydrophila, is one of the most common and challenging disease in freshwater ¢sh (Cipriano,Bullock & Pyle 1984; Choudhury, Pal, Sahu, Kumar, Keywords: A. hydrophila challenge, body composi- Das & Mukhrejee 2005; Kumari & Sahoo 2005;Yildiz, tion, common carp, extract, growth performance Bekcan, Karasu Benli & Akan 2005).
Resistance to antimicrobial agents and emergence of multiple drug resistant strains in a wide variety of pathogens pose a serious threat to the management Disease outbreaks were recently identi¢ed as a major of infectious diseases (Tomin & Tomasz 1986), render- constraint to aquaculture production and trade, with ing the traditional antibiotic and chemotherapeutic E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
treatments less successful (Takashima, Aoki & Kitao into common carp, Cyprinus carpio diets on growth performance, body composition, haematological Herbal medicine is a growing ¢eld of alternative parameters and Aeromonas hydrophila challenge.
medicines nowadays. Many active ingredients inmanufactured drugs are derived from plant com- pounds and have a wide range of use. Plants andplant extracts more safe than chemical products whereas natural products is becoming more popular, The common carp, C. carpio were obtained from Sha- since drugs of synthetic origin may have a negative hid Marjani’s ¢sh propagation and breeding center, Go- impact on the environment and parasite resistance lestan province, Iran. Fish were kept under the same to poisonous chemicals can develop after repeated environmental conditions and placed in 420 L circular applications (Magi & Sahk 2003).
¢berglass tanks (350 L water volume and 30 ¢sh per Plant-derived phytomedicines have great promise tank) for 2 weeks as an acclimation period to the in the treatment of infectious disease and thus repre- laboratory condition and they fed a commercial diet sent a vast tapped source, which has the potential to [Starter food Kutum (SFK)]. The proximate composi- combat pathogen infections in aquaculture.
tions of the commercial diet (wet basis %) contained A potential added bonus to the chemotherapeutic 8.7% humidity, 32% protein,10.5% lipid and 11.2% ash.
agents isolated from plant origin is the observationthat these extracts have also been shown to enhancegrowth of cultured ¢sh (Abutubul, Golan-Gldhirish, Bavazani & Zilberg 2004; Rao & Chakrabarti 2005; Di- Epilobium (E. hirsutum) ethanolic extract was ob- vyagnanswari, Christy Babita & Dinakaran Michael tained from Giah Essence Company, Gorgan, Gole- 2007; Sahu, Das, Mishra, Pradhan & Sarangi 2007).
Epilobium hirsutum (willow herb) is a medical plant In midsummer, E. hirsutum were collected from is found all over Europe, Asia apart from the tropical Grow and Industry Farm of Giah Essence. Aerial parts islands, Africa and America, Australia,Tasmania and of this plant were washed, dried at room temperature New Zealand. The medical parts of E. hirsutum are and ground. One kilogram powdered sample was the herb and the roots. The chemical compositions extracted by percolation with 6 L methanol (40%).
of this plant consist of £avonoids (in particular guaia- The resulting extract was concentrated over a rotary verin, quercetin-3-0-beta-D-glucuronide, and querci- vacuum evaporator and then frieze-dried. Then 6 g trin), steroids (in particular beta-sitosterol and its concrete was dissolved in 100 mL absolute ethanol.
ester, including among other beta-sitosterol caproate)and tannins (Gruenwald, Brendler & Jaenicke 1998).
Willow herb is reported to have antiphlogistic and antiexudative e¡ects. A watery infusion revealed a Multivitamin was obtained from a veterinary phar- signi¢cant inhibitory e¡ect on oedemas in rat paws macy. Ingredients of the multivitamin used in this Antibacterial e¡ects have also been demonstrated.
A suspension of fresh drug in ethanol stunts thegrowth of the bacteria of Pseudomonas pyocyanea.
Tincture and the extract work antimicrobially againstCandida albicans, Staphylococcus albus and Staphylo- Aeromonas hydrophila (AT118) was obtained from coccus aureus. The dried residue of a maceration, Faculty of Veterinary Medicine, Shiraz University, Iran.
which is ¢xed on ¢lter paper, showed a weak e¡ectagainst Bacillus subtilis, Escherichia coli, Mycobacter- ium smegmatis, Shigella £exneri, Shigella sonnei and S.
aureus. In spite of many reports about Epilobium spe- Zones of growth inhibition were determined on cies, there are not su⁄ciently studies about Epilobium Mueller^Hinton agar (MHA) surface inoculated to hirsutum in ¢sh (Gruenwald et al.1998; Battinelli,Tita, yield a con£uent A. hydrophila lawn that was auto- claved for 15 min [121 1C at 1.05 kg cm À 2 (15 psi)], The objectives of the present study were to evalu- poured in sterile condition onto a sterile petri dish ate the e¡ect of E. hirsutum at di¡erent levels (diameter 510 cm), and cooled to room temperature.
r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
Table 1 Ingredients of the used multivitamin in this ex- tively). The ¢fth group (E4) was fed with a commercial diet contained plant extract (3% of diet), added 2%multivitamin (2 g of multivitamin per kg diet). Diet of each treatment was poured in dish and the necessaryamounts of ethanolic plant extract (and multivitamin for E4 group) were spread in the diet thoroughly (by using of a small perfume glass) half an hour before each feeding time. Feeding was done manually and ob- served that almost all of diets were eaten by ¢sh imme- At the end of feeding trial 10 ¢sh each tank were ran- domly taken and their weights and lengths weremeasured. Speci¢c growth rates (SGR), condition fac- Each petri dish was inoculated with a diluted A. hy- tor (CF), feed conversion ratios (FCR) and survival drophila culture at 1.5  108 colony forming unit (CFU)/mL (McFarland No. 0.5) onto the surface of SGR ¼100  ðLn final weight À Ln initial weight=dayÞ MHA and distributed evenly with a sterile L-shaped CF ¼ ½weight ðgÞ=ðlength ðcmÞÞ3Š  100 glass rod. Then, sterile paper discs (Whatman No. 1) FCR ¼ feed intake ðgÞ=weight gain ðgÞ previously impregnated with herbal extract for Survival ¼100Âðinitial fish numberÀdead fish numberÞ 10 min were placed on the MHA medium. Ethanol- impregnated disc and tetracycline-impregnated discserved as positive and negative controls respectively.
The experiments were performed in triplicate. After the plates were incubated at room temperature for24 h, the inhibition zones around the discs where no At the end of feeding period, three ¢sh from each tank growth occurred were measured in millimeters (Har- were sampled for proximate composition analysis 24 h after the last feeding. Sampled ¢sh were anaesthetizedby using clove powder (100 ppm). Then chemical com-positions of whole body of ¢sh (moisture, protein, lipid and ash) were determined following the Association of After 2 weeks of acclimatization period, healthy C.
O⁄cial Analytical Chemists (AOAC) methods. The carpio (20 Æ 2 g) were divided into ¢ve groups (con- proximate compositions of whole body of ¢sh were trol and treatment named E1^E4), and there were analysed based on the standard methods of the Asso- three replicates for each treatment arranged ran- ciation of O⁄cial Analytical Chemists (AOAC 1995).
domly. Each replicate contained 30 individuals in cir- Moisture was determined by drying in oven (Binder, cular ¢breglass tanks (420 L capacity with 350 L Tuttlingen, Germany), at 105 1C for 24 h. Crude pro- water volume). Fish were fed at the rate of 2% of their tein was determined by using a Kjeldal system (Ger- body weight per day in the period of experiment for 8 hardt, type VAP.40, Konigswinter, Germany). Crude weeks. The daily ration was subdivided into two lipid was determined with ether extraction in a Soxh- meals at 10:00 and 18:00 hours. Water temperature let extractor (Gerhardt, type SE-416), and ash was was 23 Æ 1 1C. Fish on each tank were weighed determined using a mu¥e furnace (Nabertherm, Li- monthly and the amounts of given feed were read- lienthal, Germany), at 550 1C for 8 h.
justed according to increase in body weight.
After 8 weeks of feeding trial diets, 10 ¢sh (34 Æ 2 g) Four experimental groups (Control and E1^E3) were from each tank were transferred into the same other fed with a commercial diet (SFK) contained di¡erent le- tanks and were challenged by intraperitoneal injec- vel of plant extract (0%,0.5%, l% and 3% of diet respec- tion (IP) with pathogenic A. hydrophila diluted in r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
distilled water (1mL 5 3 Â 108 CFU). During the Table 2 Bacterial inhibition zone (mm) of plant extract, challenge test, the ¢sh were not fed. Mortality of the tetracycline and methanol on Aeromonas hydrophila challenged ¢sh was noted up to 30 days. The survived¢sh after 30 days post-challenge and unchallenged ¢sh were sampled for haematological studies.
Disc di¡usion test. Values are expressed as mean Æ SD. Mean the same letter is not signi¢cantly di¡erent (Po0.05).
Ten ¢sh from each infected (challenged) treatment and10 ¢sh from each uninfected (unchallenged) treatmentwere anaesthetized with clove powder (100 ppm).
tract have been shown in Table 3. Between treat- Blood was sampled individually by peduncle severance ments survival, SGR, CF and FCR value had no and immediately used for haematological examination.
signi¢cant di¡erence (P40.05). All diets were ac-cepted by the ¢sh and survival of ¢sh fed the experi- Red blood cells (RBC) and white blood cells (WBC)counts were counted manually, using Neubaurhaemocytometer (Paul Marienfeld Gmbh, Lauda, Koenigshofen, Germany) after diluting blood samples The proximate chemical compositions of whole body by adding Daice solution [Made based on laboratory of common carp fed diets containing di¡erent levels methods of ¢sh pathology,Roberts (1989)]. Haemoglo- of plant extract have been shown in Table 4. Results bin concentration (Hb: g/dL) was measured spectro- in Table 4 indicated that there was no signi¢cantly photometrically (Libra S12 biochrom, Cambridge, di¡erence between treatments (P40.05). Therefore, England) at 540 nm with cyanomethemoglobin body compositions were not a¡ected by di¡erent le- method. Haematocrit (Ht: %) was measured with mi- crocentrifuge method (IEC MB centrifuge, Needham,MA, USA), using standard heparinized microhaema-tocrit capillary tubes (75 mm at 3000 g for 5 min) (Harikrishnan, Balasundaram & Heo 2010).
The survival rate of C. carpio fed with experimental dietafter challenging with A. hydrophila have been shown in Table 5. In the period of 4 days post-challenge, survi- Statistical analysis was performed by one way ANOVA.
vals had no signi¢cant di¡erence (P40.05) between Di¡erence means were tested at the 5% probability groups (Control and E1^E4 treatments). In the period level using Duncan test. All the statistical analyses of 30 days post-challenge, survivals were signi¢cantly were carried out by using SPSS program version16.Va- di¡erent between groups (Po0.05). At the end of 30 lues are expressed as mean Æ standard deviation.
days post-challenge, the survival percentages werefound highest (96 Æ 6%) in the group E3 and lowest(75 Æ 5%) in control group (Fig.1).
Ethanolic plant extract were tested against A. hydrophi- la. The inhibitory zone diameters obtained by the discdi¡usion test have been shown in Table 2. Inhibitory The blood parameters of infected ¢sh fed diets con- zone diameter of plant extract was equal to tetracycline.
taining di¡erent levels of plant extract have beenshown in Table 6. The RBC count decreased signi¢-cantly (Po0.05) in the E2 group.
The WBC count increased signi¢cantly (Po0.05) The survival rates (per cent), SGR, CF and FCR of C.
between treatments when compared with control.
carpio fed diet containing di¡erent levels of plant ex- In contrast, there were no signi¢cant di¡erences in r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
Table 3 SGR, CF, FCR and survival of Cyprinus carpio fed diets containing di¡erent levels of Epilobium hirsutum extract Table 4 Proximate chemical analysis (% wet basis) of whole body of common carp fed diets containing di¡erent level of plant Table 5 Survival rate of Cyprinus carpio fed diets containing di¡erent levels of plant extract at the end of 4 days and 30 days Values are expressed as mean Æ SD. Mean the same letter in the same row is not signi¢cantly di¡erent (Po0.05).
Table 6 Hematological parameters of infected ¢sh fed diet containing di¡erent levels of plant extract Values are expressed as mean Æ SD. Mean the same letter in the same row is not signi¢cantly di¡erent (Po0.05).
r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
Table 7 Hematological parameters of uninfected ¢sh fed diet containing di¡erent levels of plant extract Values are expressed as mean Æ SD. Mean the same letter in the same row is not signi¢cantly di¡erent (Po0.05).
lymphocyte, monocyte and neutrophil count be- tween treatments and control (P40.05). The Hb con- centration of all groups did not show any signi¢cant di¡erence (P40.05). The Ht percentage had signi¢- cant di¡erence between treatments (Po0.05). The Survival % 40
highest Ht percentage was in E2 treatment.
Treatment
The blood parameters of uninfected ¢sh fed diets di¡er- ent levels of plant extract have been shown in Table 7.
In the uninfected groups the RBC, Hb and Ht levels did Figure 1 Survival rate of Cyprinus carpio fed diets con-taining di¡erent levels of plant extract at the end of 4 days not have signi¢cant di¡erence between treatments and 30 days post-challenge. Mean the same letter is not when compared with the control (P40.05).
The WBC count increased signi¢cantly (Po0.05) between treatments when compared with the con-trol. There was no signi¢cant di¡erence in lympho- dica showed inhibitory e¡ect on growth of A. hidrophi- cyte, monocyte and neutrophil count between la. The highest antibacterial e¡ect of the ethanolic treatments and control, too (P40.05).
extract of E. hirsutum may be due to its high contentof £avonoids, tannins and steroids. In fact these com-pounds are known for their strong antimicrobial activ- ity (Cushnie, Hamilthon & Lamb 2003; Martini, During recent decades many substances have shown Katerere & Elo¡ 2004; Rahman, Rana, Zaman, Uddin, to enhance the immunity of ¢sh and the route of ad- Uddin & Akter 2010). Many biological activities and ministration of them have di¡erential e¡ects on the antibacterial promoting e¡ects have been reported for immune system (Chritybapita, Divyagnaneswari & plant tannins and £avonoids (Haslam 1989; Scalbert Dinakaran Michael 2007). Hence, the present work 1991; Chung, Wong, Wei, Huang & Lin 1998). Tannins focuses on the administration of E. hirsutum extract- are polyphenols that are obtained from various parts spread on the diet, to C. carpio, a ¢sh species of grow- of di¡erent plants (Gajendiran & Mahadevan 1990).
Tannin can be toxic to bacteria, ¢lamentous fungi In the present study, E. hirsutum was screened for its and yeast (Harborne1973). The mechanism of antibac- inhibitory activity against A. hidrophila. The result in- terial action of £avonoids remains largely unknown.
dicated that ethanolic extract of E. hirsutum inhibited However, results of recent studies suggested that inhi- the growth of pathogen in agar plates. This result is bition of nucleic acid synthesis may be the primary consistence with earlier studies of Harikrishnan and cause of the antibacterial character of at least some of Balasundaram (2008) who reported that the extracts these compounds (Ulanowska, Tkaczyk, Konopa & of Curcuma longa, Ocimum sanctum and Azadirachta in- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
Survival of the ¢sh was not signi¢cantly a¡ected by groups signi¢cantly increased (Po0.05) when com- the experimental diets. This result is in agreement with pared with the control. This may explain the e⁄cacy the studies of Cho, Lee, Park, Ji, Lee, Bae and Oh (2007), of E. hirsutum in terms of the health status that low- who reported that green tea had no e¡ect on survival of ered the mortality rate in ¢sh fed diets containing olive £ounder, Paralichthys olivaceus. Ji, Jeong, Im, Lee, plant extract during the post-challenge test period.
Yoo and Takii (2007) observed that medicinal herbs These observations are in agreement with the ¢nd- had no e¡ect on survival of Japanese £ounder.
ings of Goplakannan and Arul (2006) who reported In our experiment, E. hirsutum had no signi¢cant that there was an increase in the WBC count after e¡ect on FCR, SGR and CF in ¢sh fed diet containing feeding the common carp with immunostimulants plant extract compared with the control. Yu, Li, Lin, like chitin. Sahu Das, Pradhan, Mohapatra, Mishra Wen and Ma (2008) reported that FCR in white & Sarangi (2007) reported that WBC count were shrimp (L. vannamei) fed diet containing bacillus and higher in Labe rohita ¢ngerlings fed Magnifera indica medical herb had no signi¢cant di¡erence compared kernel when compared with the control.
with the control. Ji et al. (2007) found that Japanese RBC, Hb and Ht levels had no signi¢cant di¡erence in £ounder fed diet containing medical herbs had no infected and uninfected ¢sh compared with the control.
signi¢cant di¡erence in CF between groups. Some of Similar result was obtained by Dada and Ikuerowo plant extract improve SGR (Ji et al. 2007;Yu et al. 2008; (2009) who reported that there was no signi¢cant dif- Dada & Ikuerowo 2009), however, in this study, SGR ference in RBC and Hb for cat¢sh fed diets containing had no signi¢cant di¡erence between groups fed diet ethanolic extracts of Garcinia kola seeds. Ji et al.
containing di¡erent levels of E. hirsutum.
(2007) reported that Hb and Ht had no signi¢cant dif- Moisture, crude protein, crude lipid and ash in ¢sh ference on Japanese £ounder fed diet containing body did not be a¡ected by di¡erent levels of plant ex- medicinal herb compared with the control.
tract. These results are in agreement with those ob- It may be concluded that E. hirsutum acts as both an tained by Abd. Zaher, Mostafa, Hassan Ahmad, immunostimulant and a disease control agent in ¢sh. It Mousallamy and Samir (2009) who found that ¢sh may be recommended as a dietary supplement espe- body composition of Nile tilapia ¢ngerlings did not cially at 3% in order to improve aquaculture production.
be a¡ected by di¡erent levels of fenugreek seeds. Si- Further studies need to run to evaluate cost bene¢ts.
milarly, red sea breams fed diets containing medic- This work provides a new perspective for use of inal herb showed no signi¢cant di¡erence in their medicinal plants as adjuvant therapy added to ¢sh food to prevent diseases. Further studies are needed The mortality rate of ¢sh challenged A. hydrophil for 30 days was high in ¢sh fed control diet comparedwith the other groups. The lowest mortality was in E3treatment, therefore it was considered that we would be able to use the diet containing 3% E. hirsutum ex- The authors would like to thank Shahid Marjani’s ¢sh tract in common carp against A. hydrophil.
propagation and breeding center for supplying ¢sh Harikrishnan et al. (2010) observed that herbal and Dr M.H. Suleimani for preparation of plant ex- supplementation diets increase immune system of tract, Mr M. Asghari and Miss M. Abolfathi for their gold¢sh against A. hydrophil. Abd. Zaher et al. (2009) reported that mortality not observed in Nile tilapia feddiet containing di¡erent level of fenugreek and chal-lenged A. hydrophil.
Haematological indices are an index and a re£ec- tion of the e¡ects of dietary treatments on the animalin terms of the type, quality and amounts of the feed Abd.Zaher A., Mostafa M., Hassan Ahmad M., Mousallamy A. & Samir A. (2009) E¡ect of using dried fenugreek seeds ingested and were available for the animal to meet its as natural feed additives on growth performance, feed uti- physiological, biochemical and metabolic necessities lization, whole-body composition and entropathogenic (Ewuola, Folayan, Gbore, Adebunmi Akanji, Ogun- Aeromonas hydrophila-challing of monsex nile tilapia O.
niloticus (L) ¢ngerlings. AustralianJournal of Basic and Ap- WBC a¡ords protection against infectious agent caused by microbial and chemical factors. In these Abutubul S., Golan-Gldhirish A., Bavazani O. & Zilberg D.
study, the WBC level of infected and uninfected (2004) Use of Rosmarinus o⁄cinalis as a treatment against r 2011 Blackwell Publishing Ltd, Aquaculture Research, 1^9 E¡ect of dietary willow herb, Epilobium hirsutum S Pakravan et al.
Streptococcus iniae in tilapia Oreochromis sp. Aquaculture Gajendiran N. & Mahadevan A. (1990) Utilization of cate- chin by Rhizobium sp. Plant and Soil 108, 263^266.
Adanir D.O.R. & Turutoglu H. (2007) Isolation & antibiotic Goplakannan A. & Arul V. (2006) Immunomodulatory e¡ects susceptibility of Aeromonas hydrophila in a carp (Cyprinus of dietary intake of chitin, chitosan and levamisole on the carpio) hatchery farm. Bulletin of theVeterinary Institute in mmune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture 255, 179^187.
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