Aquaculture research, tập 42, số 6, 2011

Aquaculture Research, 2011, 42, 737 doi:10.1111/j.1365-2109.2011.02881.x Introduction to the special issue on ‘‘Development of native species for aquaculture in Latin America II’’ Gustavo M Somoza1 & Lindsay G Ross2 IIB-INTECH (CONICET-UNSAM), Chascomu¤s, Argentina Institute of Aquaculture, University of Stirling, Stirling, Scotland, UK Correspondence: L G Ross, Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA Scotland, UK E-mail: lgr1@stir.ac.uk; G M Somoza, IIB-INTECH (CONICET-UNSAM), Chascomu¤s, Argentina E-mail: somoza@intech.gov.ar Following the ¢rst special edition of Aquaculture Research on Development of native species for aquaculture in Latin America II, in which selected presentations from the First Latin American Conference on Culture of Native Fishes were published in 2008, and in view of the continued growth of research and development of aquaculture of native ¢sh species in Latin America, an international steering group was established with the objective of organizing a meeting on this topic on a regular basis The concept for this series of meetings and publications is the promotion and development of native species culture in order to minimize the introduction, distribution and transplantation of exotic species throughout and within the region The conservation of biodiversity is also taken into account as an important objective As a consequence, the Second Latin American Conference on Culture of Native Fishes was held in Chascomu¤s, Buenos Aires, Argentina between 3rd and 6th November 2009 and the ¢rst 11 articles in the present issue represents a selection of the presented papers Chascomu¤s is a small city located120 km south of Buenos Aires with a tradition in studies of the biology, culture and propagation of an emblematic species for that area, the pejerrey Odontesthes bonariensisValenciennes, 1835, Atherinopsidae This meeting was supported by CONICET (Argentine National Research Council), CIC (Committee for Scienti¢c Research of Buenos Aires Province), CFI (Federal Investment Council), the Ministry of Agricultural A¡airs of the Province of Buenos Aires and the Chascomu¤s Municipality The meeting was extremely successful and approximately 250 students, technicians and researchers attended the r 2011 Blackwell Publishing Ltd di¡erent sessions A series of ¢ve lectures on key selected topics were presented by invited speakers from Colombia, Spain, United States, France, Chile and Mexico There were also 50 selected oral presentations and 159 posters from across the continent covering a wide range of species and subject areas This meeting had fundamental importance for gathering together scientists, technicians and local farmers interested in the biology and culture of Latin American species A detailed description of the meeting is given at http://www.laccnf.com.ar/ index.php?idioma=1&laccnf=0 We would like to thank the enthusiastic e¡orts of Dr Luis F Canosa, Dr Dar|¤ o Collauti, Mr Manuel Marina, Dr Leandro Miranda (IIB-INTECH) and Lic Gustavo Berasain and Lic Marcela Velasco (Ministry of Agricultural A¡airs of the Province of Buenos Aires) It is also important to mention the help of the following persons: Andre¤s Alonso, Leandro Balboni, Daniela Campanella,TomaŁs Chalde, Mar|¤ a Victoria Crivelli, Mariano Elisio, Juan Ignacio Fernandino, Javier Garcia de Souza, AŁngela GaŁrriz, Pedro Go¤mez-Requeni, Yamila Grunblat, Mar|¤ a Ester Haspert, Mauricio Kraemer, Facundo Llompart, Gabriela Carina Lo¤pez, TomaŁs Maiztegui, Horacio Onìatibia, Mar|¤ a Rita Pe¤rez, Mar|¤ a Ine¤s Pietrantuono, Maria Emilia Scharrig and Patricio Solimano without whom the symposium could not have been possible We are looking forward to the next symposium to be held at the University of Lavras, Minas Gerais, Brazil in 2011, hoping that it will show further substantial advances in the culture of native species for Latin America 737 Aquaculture Research, 2011, 42, 738^745 doi:10.1111/j.1365-2109.2011.02835.x Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818) Juan Antonio Ramirez-Merlano,Yohana Mar|¤ a Velasco-Santamar|¤ aÃ,V|¤ ctor Mauricio MedinaRobles & Pablo Emilio Cruz-Casallas Research Group on Reproduction and Toxicology of Aquatic Organisms ^ GRITOX, Aquaculture Institute, University of the Llanos, Meta, Colombia Correspondence: P E Cruz-Casallas, Research Group on Reproduction and Toxicology of Aquatic Organisms ^ GRITOX, Aquaculture Institute, University of the Llanos, Km 12 via Puerto Lo¤pez,Villavicencio, Meta, Colombia E-mail: pecruzcasallas@unillanos.edu.co ÃPresent address: Y M Velasco-Santamar|¤ a, Institute of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Abstract The e¡ects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian ¢sh species Sexually mature ¢sh were induced to ovulation or spermiation with a carp pituitary extract A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO-10%), methanol (MET-10%) or ethylene glycol (ETG-5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour The thawing process was performed in a 35 or an 80 1C water bath The fertility was evaluated after h post fertilization The highest motility percentage (33 Æ 3%) was observed with sperm cryopreserved with DMSO, packed in mL straws and thawed at 35 1C The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 1C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET-2.5 mL (9 Æ 5%) In all the treatments, a signi¢cant decrease in the sperm quality was observed at 80 1C Sperm cryopreserved with DMSO-10% or MET-10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs Keywords: sperm cryopreservation, straw volume, cryoprotectants, thawing temperature, sperm quality, Piaractus brachypomus Introduction The use of high-quality gametes in aquaculture is an important factor to achieve the successful reproduc- 738 tion of ¢sh species under captivity (Rurangwa, Kime, Ollevier & Nash 2004) Cryopreservation is considered to be one of the most e⁄cient strategies to facilitate the handling and storage of gametes (Sarvi, Niksirat, Mojazi Amiri, Mirtorabi, Ra¢ee & Bakhtiyari 2006) The use of cryopreserved sperm is a practical alternative to increase the population size and to maintain the genetic diversity, especially in those species kept in captivity (Piironen 1994) Cachama blanca Piaractus brachypomus (Cuvier1818) is a native ¢sh of South America inhabiting the basins of the Orinoco and Amazon rivers Under farming conditions, this species shows an excellent performance due to its rusticity, omnivorous food habits, docility, meat quality, food conversion and resistance to diseases, and therefore is the most widely farmed native ¢sh species in Colombia (Arias, VaŁsquez-Torres, Orrego & Isaza 1989; HernaŁndez, Munìoz, Ferraz De Lima, De Santis, VaŁsquez-Torres, GonzaŁlez, Morales, Alcantara, Luna & Kossowski 1992) Despite the successful research on their reproductive aspects, the current ¢ngerling production of cachama blanca is still limited due to the low or even absent male gamete availability during most of the year An alternative to solve this problem is the sperm cryopreservation, a biotechnology implemented in about 200 ¢sh species (Rana 1995); however, the use of this technology in P brachypomus is restricted due to the need for high amounts of suitable sperm to achieve high fecundity in this species Sperm is traditionally packed in small straws (0.5 mL French Straws), which does not allow the fertilization of a high number of oocytes Protocols for sperm cryopreservation in straws of large volume (44 mL) have r 2011 Blackwell Publishing Ltd Aquaculture Research, 2011, 42, 738^745 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al been evaluated in some freshwater ¢sh species such as Rainbow trout Oncorhynchus mykiss (Walbaum 1972) (Wheeler & Thorgaard 1991; Lahnsteiner,Weismann & Patzner 1997), Brown trout Salmo trutta fario (Linnaeus 1758), Salmo trutta lacustris (Linnaeus 1758), Arctic char Salvelinus alpinus (Linnaeus 1758) (Lahnsteiner et al 1997; Richardson, Miller & McNiven 2000),Wels cat¢sh Silurus glanis (Linnaeus 1758) (Bart, Wolfe & Dunham 1998), Prochilodus lineatus (Valenciennes 1837) (Viveiros, Orfaìo, Maria & Allaman 2009) and Brycon amazonicus (Spix & Agassiz 1829) (Velasco-Santamar|¤ a, Medina-Robles & CruzCasallas 2006) These previous studies concluded that packing sperm in large straws or macrotubes could reduce the time required to pack and thaw the sperm and also facilitate the handling during the fertilization process Another important issue is the use of cryoprotectants, which role is to prevent cell damage during the freezing and thawing steps during the cryopreservation process Several cryoprotectants have been used for sperm ¢sh cryopreservation, including methanol, ethylene glycol and dimethyl sulphoxide (DMSO); however, DMSO is reported to be the most e⁄cient to cryopreservate ¢sh spermatozoa (Anel & Cabrita 2000; Cruz 2001) due mainly to its small molecular size, which allows it to enter and exit the spermatic cell easily (Tiersch, Williamson, Carmichael & Gorman1998;Vincent, Pruliere, Pajot-Augy, Campion & Douzou 1998) On the other hand, a wide range of temperatures used to thawed cryopreserved sperm with temperatures from refrigeration (4 1C) to 80 1C are reported (Lahnsteiner, Berger, Horvath, Urbanyi & Weismann 2000) A fast thawing temperature decreases the recrystallization e¡ect in the spermatic cells and therefore diminishes the membrane damage (Tiersch et al 1998) The use of various cryoprotectants in cachama blanca sperm has been reported previously including the most widely used DMSO, dissolved in glucose and egg yolk solution and packed in small straws (Fresneda, Lenis, Agudelo & Olivera-AŁngel 2004; Navarro, Velasco-Santamar|¤ a & Cruz-Casallas 2004; Nascimento, Maria, Pessoa, Carvalho & Viveiros 2010) The main goal of the current study was to develop an appropriate protocol for P brachypomus sperm cryopreservation, minimizing the deleterious e¡ects of the cryopreservation process, therefore contributing to increase the gamete availability outside the breeding season The e¡ects of three straw volumes, three cryoprotectants and two thawing temperatures on the post-thaw sperm quality of P brachypomus were assessed Materials and methods Experimental animals Sexually mature cachama blanca P brachypomus, 5.0 Æ 0.6 kg body weight (BW) and 64 Æ cm (total length), were used The ¢sh were reared and kept in ponds at a ratio1.5 ¢sh m À at the Aquaculture Institute of the University of the Llanos,Villavicencio, Colombia The reproductive maturity was assessed in males by the presence of sperm in the urogenital papillae (after a slight massage on the coelomic cavity) and in females by measuring, through an ovarian biopsy the oocyte diameter (41200 mm) and evaluating the migration of the oocyte germinal vesicle Gametes extraction Mature ¢sh were collected from the earthen ponds and kept in circular ponds with permanent aeration and water recirculation The males were injected intramuscularly with a pituitary carp extract (EPC, Stoller Fisheries, Spirit Lake, IA, USA) at 4.0 mg kg À BW The females were injected with 5.75 mg kg À BW distributed in three applications at h (0.25 mg kg À BW), 24 h (0.5 mg kg À BW) and 36 h (5.0 mg kg À BW) To obtain the gametes, the ¢sh were anaesthetized in a 2-phenoxyethanol solution (300 mg L À 1, Sigma Chemical Co., St Louis, MO, USA) until loss of the swimming axis was observed The sperm was collected 18 h after the hormone injection in glass graduated tubes (15 mL) Sperm samples contaminated with water, urine, faeces or blood were discarded At the ovulation time, the oocytes were collected 6^7 h after the last hormone injection The abdominal region in each ¢sh was carefully dried to avoid contact between water and the gametes Sperm quality evaluation The sperm collected was kept at room temperature (25 Æ 1C) The gross motility and duration of motility were evaluated using light microscopy ( 10) by adding 10 mL of sperm to a concave slide (1.0^1.2 mm depth, Micro Slides Premiere, Shanghai, China) and activating the motility spermatozoa with 190 mL of distilled water (1:20) Samples with o80% progressive motility were not processed for cryopreservation The duration of motility was evaluated immediately r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 739 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al after the addition of water, and registered until the sperm movements decreased to ca 5% The sperm concentration was determined using two methods The ¢rst one corresponded to an indirect known technique called spermatocrit, where sperm is placed in microcapillary tubes (75 mm length and 1.1mm internal diameter) and centrifuged at 14 000 g for The direct method was carried out through the haemocytometer method, diluting (1:1200) previously the sperm in a saline physiological solution (0.9% NaCl), kept under a humid atmosphere for 10 min, and subsequently, individual spermatozoa were counted in a Neubauer chamber (Bright Line, Optik Labor, Friedrichshofen, Germany) at  40 magni¢cation (Cruz-Casallas 2001) Sperm cryopreservation process and thawing conditions The selected sperm for cryopreservation were pooled and diluted (1:5) initially in an extender solution containing glucose as an external cell cryoprotectant (5.5%, Merck, Darmstadt, Germany) and hen’s egg yolk as both a stabilizer membrane and an internal cryoprotectant To evaluate the cryoprotectants e¡ects, 10% DMSO (Sigma Chemical Co.), 5% ethylene glycol (ETG, Sigma Chemical Co.) or 10% methanol (MET, Sigma Chemical Co.) were used After the sperm dilution in each extender, no motile spermatozoa were observed in any of the samples Afterwards, the diluted sperm was packed in di¡erent straw volumes: 0.5 mL (French Straws,130  mm, Instrument de Me¤decine Ve¤te¤rinaire, Minneapolis, MN, USA), 2.5 (140  mm) and 5.0 mL macrotubes (280  mm, Minitub, Abfˇl-und Labortechnik GmbH, Tiefenbach, Germany) The French straws were sealed with polyvinyl and the macrotubes were sealed with metallic balls The straws and macrotubes were placed vertically in a custom-made support and frozen for 30 in nitrogen vapour in a dry shipper (CP100 TaylorWharton, Theodore, AL, USA) The decrease in the temperature was monitored using a thermocouple (WBrand, Friendswood, TX, USA, precision 0.01 1C and range À 200 to 800 1C) inserted directly into the ¢lled straws or macrotubes All straws were submerged and maintained in a liquid nitrogen container at À 196 1C (35 HC, Taylor-Wharton) until further evaluation The thawing process at 35 1C was carried out by submerging the straws or macrotubes for 90 s in a water bath The thawing time at 80 1C varied according to the straw size as follows: 10 s for 0.5 mL straws and 25 s for 2.5 and 5.0 mL macrotubes 740 Aquaculture Research, 2011, 42, 738^745 Experimental design To evaluate the e¡ect of di¡erent straw volumes (0.5, 2.5 or 5.0 mL), cryoprotectant substances (DMSO, MET or ETG) and thawing temperatures (35 or 80 1C), a random experimental design with a factorial   was used The factor corresponded to the three cryoprotectants used, factor to the three straw size volumes and factor to the two thawing temperatures, with a total of 18 treatments The post-thawing sperm quality was evaluated using the gross motility, duration of motility and fertility test (n 6) The gross motility was induced using 1% sodium bicarbonate (NaHCO3, Merck) and subjectively evaluated using a scale from 1% to 100% The duration of motility (s) was recorded using a stopwatch For the fertility test, g of eggs (1640 eggs g À 1) were seminated with 400 mL of post-thawed sperm (ca 296280 motile spermatozoa egg À ratio) and the activation was carried out by adding mL of 1% NaHCO3 After semination, the hydrated eggs were placed in experimental incubators with an ascendant £ow (2 L) and permanent water until hatching As a control, the same amount of eggs was seminated with 100 mL of fresh sperm (ca 54 649 motile spermatozoa egg À ratio) The fertility in each treatment was evaluated h post semination, evaluating the proportion of fertile eggs (translucent aspect and normal embryo development) in a total known amount of eggs Statistical analysis Data are expressed as mean Æ standard error of the mean (SEM) In order to verify the data normality distribution and homogeneity of variance, Kolmogorov^ Smirnov and Bartlett’s tests were carried out In addition, the data were analysed considering the plot residual distributions Data were analysed by a multiple comparison analysis, followed by a Tukey’s test in order to evaluate the e¡ect of straw sizes, cryoprotectant substances and thawing temperatures on the sperm quality In all cases, Po0.05 was used to show signi¢cant di¡erences All statistical analysis was conducted using the SAS system for Windows software version 9.1.3 (2002^2003 by SAS Institute, Cary, NC, USA) Results The fresh sperm characteristics of the samples suitable for cryopreservation are shown in Table In all cases, the males had a seminal volume higher than r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 Aquaculture Research, 2011, 42, 738^745 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al Table Fresh sperm characteristics of cachama blanca Piaractus brachypomus (Cuvier 1818) broodstock, sampled 18 h after an intramuscular injection of a pituitary carp extract (4.0 mg kg À body weight) Table Freezing rate ( 1C À 1) of extended cachama blanca Piaractus brachypomus (Cuvier 1818) sperm packed in 0.5, 2.5 and 5.0 mL straws (n 2) Straw size (mL) Sperm characteristics Volume (mL) Motility (%) Duration of motility (s) Sperm concentration (spermatozoa mL À  106) Spermatocrit (%) 13 86 45 11 25 Æ Æ Æ Æ Æ 1 1 Temperature range ( 1C) 0.5 2.5 5.0 28 to À 20 À 20 to À 100 À 100 to À 196 Total freezing rate 286.3 63.0 3.3 6.7 22.0 18.7 4.2 7.8 9.8 17 4.8 7.6 Values expressed as mean Æ SEM (n 23) Y = 0.3867X+1.6764 Sperm concentration ( x 106 sptz µL−1) r = 0.62 Spermatocrit (%) Figure Relationship between spermatocrit (%) and sperm concentration (spermatozoa  106 mL À 1) with a 1:1200 dilution (n 23; r2 50.62) of cachama blanca Piaractus brachypomus (Cuvier 1818) sperm 10 mL with a sperm motility 480% The duration of motility £uctuated between 36 and 56 s, while the sperm concentration varied between 2.0  106 and 18  106 spermatozoa mL À On the other hand, the spermatocrit showed values from 4% to 32%, which correlated positively with the sperm concentration (r2 50.62; Po0.001) (Fig.1) Freezing rates The freezing rates were analysed considering three temperatures ranges from 28 to À 20 1C, À 20 to À 100 1C and from À 100 to À 196 1C for all three straw sizes (Table 2) In the range 28 to À 20 1C and À 20 to À 100 1C, the 0.5 mL straws showed the fastest freezing curve compared with 2.5 and 5.0 mL straws Nevertheless, the freezing temperatures in all straws were considered to be stable from À 160 1C approximately (Fig 2) Figure Freezing curves of cachama blanca Piaractus brachypomus (Cuvier 1818) sperm packed in 0.5, 2.5 and 5.0 mL straws and frozen in nitrogen vapour in a dry shipper for 30 (n 2) Sperm quality Post-thaw sperm motility The highest percentages of post-thaw motility were observed in sperm cryopreserved with 10% DMSO and thawed at 35 1C in all straw volumes In general, sperm frozen in mL straws showed the highest post-thaw motility regardless of both the thawing temperature and the cryoprotectant (Table 3) At 35 1C, the lowest sperm post-thaw motility was observed with ethylene glycol as a cryoprotectant and frozen in 2.5 mL straws (17%) On the other hand, thawing temperature at 80 1C substantially decreased the sperm post-thaw motility, with sperm packed in 0.5 mL straws being the most a¡ected This ¢nding was clearly observed in sperm cryopreserved in MET-10% and ETG-5% (both with 3% post-thaw r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 741 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al Table Post-thaw sperm motility and duration of motility of cachama blanca Piaractus brachypomus (Cuvier 1818) sperm cryopreserved with DMSO, ETG or MET, frozen in 0.5, 2.5 or 5.0 mL straws and thawed in a 35 or an 80 1C water bath Cryoprotector DMSO 10% Thawing Straw size temperature volume Motility ( 1C) (mL) (%) 35 80 ETG 5% 35 80 MET 10% 35 80 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 29 28 33 10 25 30 21 17 25 18 16 20 24 23 3 23 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ ab 2ab 3a 3bcd 6ab 3a 4abcd 2abcd 3ab 2cd 4abcd 3abcd 4abcd 4ab 6abc 1d 2d 2ab Duration of motility (s) 62 Æ 49 Æ 58 Æ 38 Æ 51 Æ 52 Æ 46 Æ 42 Æ 57 Æ 10 Æ 45 Æ 50 Æ 51 Æ 52 Æ 43 Æ 9Æ 9Æ ND Aquaculture Research, 2011, 42, 738^745 Table Fertilization percentages of cachama blanca Piaractus brachypomus (Cuvier 1818) sperm cryopreserved with DMSO, ETG or MET, frozen in 0.5, 2.5 or 5.0 mL straws and thawed in a 35 or an 80 1C water bath Cryoprotector Straw size Thawing temperature volume ( 1C) (mL) Fertility (%) DMSO 10% 35 a 1abc 10a 5abc 2bcd 5ab 2abc 16abc 4a 10a 5bc 4abc 4abc 0abc 1abc 9c 9c 80 ETG 5% 35 80 MET 10% 35 80 Control (fresh sperm) 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 0.5 2.5 5.0 89 Æ 7.3a 71 66 75 32 29 27 63 42 64 36 30 28 71 70 79 19 34 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 6abcd 8abcde 7abc 14cdef 15cdef 13def 7abcde 16abcde 6abcde 23abcdef 18ab 16def 5abcde 5abcde 3ab 11e 5f 19bcdef Values expressed as mean Æ SEM (n 6) Between rows, means with di¡erent superscripts are signi¢cantly di¡erent (Po0.05) DMSO, dimethyl sulphoxide; ETG, ethylene glycol; MET, methanol; ND, not de¢ned Between rows, means with di¡erent superscripts are signi¢cantly di¡erent (Po0.05) Values expressed as mean Æ SEM (n 6) DMSO, dimethyl sulphoxide; ETG, ethylene glycol; MET, methanol motility) and DMSO-10% (10%), thawed at 80 1C and packed in 0.5 mL straws (Po0.05) with DMSO and MET, frozen in 0.5 or 5.0 mL straws and thawed at 35 1C showed the greatest fertility percentages (higher than 71%) However, sperm cryopreserved with ETG, frozen in 2.5 mL straws and thawed at 35 1C obtained the lowest fertilization percentage (42%), although no signi¢cant di¡erences were observed with the above treatments In contrast, the thawing temperature at 80 1C showed a critical e¡ect on the fertility assay, i.e sperm cryopreserved in MET, frozen in 2.5 mL straws and thawed at 80 1C had the lowest fertility (9%, Po0.05) Sperm diluted with ethylene glycol and DMSO as a cryoprotectant and thawed at 80 1C had a higher fertility at this temperature regardless of the straw volume (between 27% and 36%) During the entire incubation process, the temperature and pH were maintained constant with values of 26.9 Æ 0.4 1C and pH 6.5 Æ 0.4 Post-thaw duration of motility The highest post-thaw duration of motility was observed with sperm cryopreserved in DMSO-10% and thawed at 35 1C (Table 3) Under these conditions, diluted sperm frozen in 0.5 and 5.0 mL straws showed the highest value (62 and 58 s, respectively) On the other hand, sperm cryopreserved with ETG-5% packed in 0.5 mL straws and MET-10% frozen in 0.5 and 2.5 mL straws, and thawed at 80 1C showed the lowest duration of motility (10 and s, respectively) Overall, frozen sperm thawed at 35 1C was the optimum condition to obtain a suitable duration of motility Fertility assays Table shows the e¡ects of cryoprotectants, thawing temperatures and straw sizes on P brachypomus egg fertility The fertilization percentage obtained with fresh sperm was signi¢cantly higher (89%) than all frozen thawed sperm (Po0.05) Sperm cryopreserved 742 Discussion The results obtained in the current study contribute signi¢cantly towards improving the protocol devel- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 Aquaculture Research, 2011, 42, 738^745 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al opment of sperm cryopreservation in P brachypomus at large scale The sperm quality of samples before cryopreservation had an optimal gross motility ca 86%, which is in agreement with previous studies on this species (Fresneda et al 2004; Navarro et al 2004) and in related species such as B amazonicus (Cruz-Casallas, Velasco-Santamar|¤ a & Medina-Robles 2006) Similarly, the seminal volume obtained was higher than the reported in other South American characidae such as Piaractus mesopotamicus (Holmberg 1887) (5.02 mL) (Fogli Da Silveira, Kavamoto & Narahara 1985) and P lineatus (2.2 mL) (Viveiros et al 2009) The sperm concentration was similar to the previously study reported by Navarro et al (2004); however, a higher sperm concentration (30 spermatozoa mL À  106) and duration of motility (1006 s) were reported in the same species by Fresneda et al (2004) Despite the low number of ¢sh used, a good and signi¢cant relationship between spermatocrit and sperm concentration was observed in this study This value is close to previous studies in B amazonicus (75%) (Cruz-Casallas et al 2006), Cod¢sh Gadus morhua (Linnaeus1758) (75%) (Rakitin, Moira, Ferguson & Trippel 1999) and Rainbow trout (65%) (Bastardo, Guedez & Leo¤n 2004) These results con¢rm the clear relationship between these two variables and therefore the spermatocrit is an important alternative to determine indirectly the sperm concentration in P brachypomus This result has a high relevance and practical application mainly when a high signi¢cantly number of samples have to be evaluated The sperm cryopreservation using nitrogen vapours is the technique most used in ¢sh (Linhart, Rodina & Cosson 2000; Cruz-Casallas et al 2006) During this process, the spermatozoa is subject to drastic physical and chemical changes such as ice crystals’ formation, mechanical and osmotic stress and destabilization of the plasmatic membrane (Lahnsteiner, Patzner & Weismann 1992; Labbe, Crowe & Crowe 1997) Straws with a small diameter such as 0.5 mL o¡er a higher freezing index than the macrotubes (2.5 and 5.0 mL) which have a smaller index with a longer plateau (Bwanga, Braganca, Einarsson, & Rodriguez-Martinez 1990; Bwanga, Einarsson & Rodriguez-Martinez 1991) Lahnsteiner et al (2000) and Cabrita, Robles, Alvarez and HerraŁez (2001) showed a higher freezing index in Cyprinids and Rainbow trout sperm packed in 0.5 mL straws (80 1C À from À to À 50 1C and 92.8 1C À from À 20 to À 100 1C respectively) The post-thaw sperm motility of P brachypomus activated with 1% sodium bicarbonate was lower than the reported in B orbignyanus (Valenciennes 1850) (Carosfeld, Godinho, Zaniboni-Filho & Harvey 2003) Navarro et al (2004) reported higher post-thaw motility using 10% DMSO (40%), 5% ETG (37%) or 10% MET (57%) as cryoprotectants, by mixing with whole milk powder and thawing at 35 1C Recently, Nascimento et al (2010) reported in P brachypomus a subjective post-thaw sperm motility of 49 Æ 22% using DMSO as a cryoprotectant and thawed at 60 1C for s, which is close to the values obtained in the current study In addition, higher post-thaw sperm motility (77 Æ 17%) was observed when methylglycol was used as a cryoprotectant; nevertheless, fertility tests were not carried out (Nascimento et al 2010) Compared with the current results, Fresneda et al (2004) obtained higher sperm motility and a higher duration of motility using DMSO and MET as cryoprotectants (80% and 78%, respectively) Cabrita et al (2001) reported in Rainbow trout a sperm motility close to 45% when the sperm was cryopreserved with 7% DMSO and thawed at 25 1C for 30 s Piaractus brachypomus cryopreserved sperm thawed at 80 1C showed low post-thaw motility, suggesting alterations in the cellular membrane integrity, mainly in sperm cryopreserved in 0.5 mL straws In contrast, Velasco-Santamar|¤ a et al (2006) reported similar sperm motility (ca 38%) in sperm cryopreserved with DMSO, packed in 1.8, 2.5 or 4.0 mL straws and thawed at 80 1C In the current study, DMSO and MET showed better results as cryoprotectants probably due to their capacity to enter and exit the sperm cell based on the gradient concentration (Tiersch et al 1998; Vincent et al 1998) and as well as to the ability to maintain the membrane integrity and the mitochondrial function during the cryopreservation process (Ogier De Baulny, Vern, Kerboeuf & Mais 1997) The signi¢cant decrease in the sperm post-thaw motility and duration of motility when the sperm was thawed at 80 1C, can be explained by the fact that high temperatures are viable within a short time since fast thawing rates can contribute towards decreasing the re-crystallization e¡ect observed in the spermatic cells and minimizing the thermal e¡ect on the membrane (Tiersch et al 1998) Folgi da Silveira et al (1985) obtained 21% fertility using Rhamdia hilarii frozen sperm packed in 0.5 mL straws and r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 743 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al thawed at 70^80 1C for 3^4 s Similarly, low fertility (o45%) was observed in B amazonicus sperm thawed at 80 1C (Velasco-Santamar|¤ a et al 2006) due to the detrimental e¡ect on ¢sh sperm Thawing temperatures at 80 1C could induce several changes in the motility, viability and fertility of the sperm cell probably a¡ecting the spermatozoa fertilizing ability (Wamecke & Pluta 2003) The fertility obtained in P brachypomus sperm cryopreserved with 10% DMSO (71%), 5% ETG (63%) or 10% MET (71%), packed in 0.5 mL straws and thawed at 35 1C was higher than that obtained in a previous study in the same species (Navarro et al 2004) High fertility using cryopreserved sperm has been reported in Rainbow trout, with percentages varying slightly with the straw sizes e.g 81% in sperm packed in 4.0 and 5.0 mL straws and thawed at 25 1C (Steinberg, Hedder, Baulain & Holtz 1995), and 75% in sperm packed in 5.0 mL straws and thawed at 60 1C (Cabrita et al 2001) In contrast to the previous studies, Cat¢sh Ictalurus punctatus (Ra¢nesque 1818) sperm cryopreserved in methanol, Hanks solution and power milk and packed in 0.5 and 1.0 mL straws and thawed at 25 1C showed no fertility (0%) (Bart et al 1998), con¢rming the relevance to optimize the sperm cryopreservation process in each species Straws with a non-traditional volume i.e 2.5 and 5.0 mL showed a signi¢cant similar fertility to those obtained with fresh sperm, despite both the possible high cell damage and the low sperm motility (o50%) reported in other species (Richardson,Wilson, Crim & Yao 1999; Yao, Crim, Richardson & Emerson 2000) In this respect, Ciereszko, Drabrowski, Lin, Chris and Toth (1999) concluded that in Muskellunge Exos masquinongy (Mitchill 1824), there is no clear relationship between the spermatic post-thaw motility and the fertility percentage Our results and those obtained in other ¢sh species highlight the importance of carrying out a fertility test to validate sperm cryopreservation protocols The signi¢cant results obtained in the present study demonstrate the practical application of large volume straws on a commercial scale in ¢sh farms, as their use will reduce the time spent freezing and thawing the straws and higher amounts of oocytes will be fertilized at the same time with fewer straws Based on the results obtained, it is possible to suggest that sperm cryopreserved in 10% DMSO or 10% MET, packed in large-volume straws i.e 2.5 or 5.0 mL and thawed at 35 1C are the most suitable conditions to retain the sperm quality in P brachypomus having optimal sperm motility, duration of motility as well as 744 Aquaculture Research, 2011, 42, 738^745 high fertility percentages close to the values obtained with fresh sperm Acknowledgments This research was supported by Instituto de Investigaciones de la Orinoqu|¤ a Colombiana (IIOC), University of the Llanos,Villavicencio, Meta, Colombia References Anel I & Cabrita E (2000) E¡ect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm Theriogenology 13, 623^635 Arias C.J.A., VaŁsquez-Torres W., Orrego J & Isaza M (1989) Avances en la reproduccio¤n inducida de Piaractus brachypomus, cachama blanca Bolet|¤ n Red deAcuicultura 3, 9^10 Bart A.N., Wolfe D.F & Dunham R.A (1998) Cryopreservation of blue cat¢sh spermatozoa and subsequent fertilization of Channel cat¢sh eggs Transactions of the American Fisheries Society 127, 819^824 Bastardo H., Guedez C & Leo¤n M (2004) Caracter|¤ sticas del semen de trucha arco iris de diferentes edades, bajo condiciones de cultivo en Me¤rida, Venezuela Zootecnia tropical 22, 277^288 Bwanga C.O., Braganca M.M., Einarsson S & RodriguezMartinez H (1990) Cryopreservation of boar semen in mini and maxi-straws Zentralblatt fur Veterinaermedizin 37, 651^658 Bwanga C.O., Einarsson S & Rodriguez-Martinez H (1991) Cryopreservation of boar semen: II E¡ect of cooling rate and duration of freezing point plateau on boar semen frozen in mini and maxi-straws and plastic bags ActaVeterinaria Scandinavica 32, 455^461 Cabrita E., Robles V., Alvarez R & HerraŁez M.P (2001) Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization Aquaculture 201, 301^304 Carosfeld J., Godinho H.P., Zaniboni-Filho E & Harvey B.J (2003) Cryopreservation of sperm in Brazilian migratory ¢sh conservation Journal of Fish Biology 63, 472^489 Ciereszko A., Drabrowski K., Lin F., Chris S.A & Toth G.P (1999) E¡ects of extenders and time of storage before freezing on motility and fertilization of cryopreserved Muskellunge spermatozoa Transactions of the American Fisheries Society 128, 542^548 Cruz V.L.B (2001) Criopreservac° aìo seŒmen de curimbataŁ (Prochilodus lineatus) Masters thesis in Vertebrate Zoology, Pontif|¤ cia Universidade Cato¤lica de Minas Gerais, Belo Horizonte, MG, Brazil, 59pp Cruz-Casallas P.E (2001) Te¤cnicas de laboratorio para la evaluacio¤n de la calidad seminal en peces Orinoquia 5, 155^163 Cruz-Casallas P.E., Velasco-Santamar|¤ a Y & Medina-Robles V.M (2006) Determinacio¤n del espermatocrito y efecto r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 Aquaculture Research, 2011, 42, 738^745 Cryopreservation of Piaractus brachypomus sperm J A Ramirez-Merlano et al del volumen de la dosis seminante sobre la fertilidad en yamu¤ (Brycon amazonicus) Revista Colombiana de Ciencias Pecuarias 19,140^145 Fogli Da Silveira W., Kavamoto E.T & Narahara M.Y (1985) Avaliac°aìo da qualidade e criopreservac°aìo em forma de ‘pellets’do se¤men bagre, Rhamdia hilarii (Valenciennes, 1840) Boletin Instituto de Pesca 12,7^11 Fresneda A., Lenis G., Agudelo E & Olivera-AŁngel M (2004) Espermiacio¤n inducida y Crioconservacio¤n de Cachama blanca (Piaractus brachypomus) Revista Colombiana de Ciencias Pecuarias Suplemento 17, 46^52 HernaŁndez A., Munìoz D., Ferraz De Lima J.A., De Santis D F.R., VaŁsquez-Torres W., GonzaŁlez R., Morales R., Alcantara F., Luna TM & Kossowski C (1992) Estado actual del cultivo de Colossoma y Piaractus en Brasil, Colombia, PanamaŁ, Peru¤ yVenezuela Segunda Reunio¤n Internacional del grupo de trabajo te¤cnico de Colossoma y Piaractus Bolet|¤ n Red deAcuicultura 6, 3^27 Labbe C., Crowe L.M & Crowe J.H (1997) Stability of the lipid component of Trout sperm plasma membrane during freeze-thawing Cryobiology 34,176^182 Lahnsteiner F., Patzner R.A & Weismann T (1992) Energy metabolism in spermatozoa of the grayling (Thymallus thymallus) In: Proceedings of the Fourth International Symposium of the Reproductive Physiology of Fish (ed by A.P Scott, J.P.K Sumpter, D.E Kime & M.S Rofle), pp 279 Sheffield, UK: FishSymp Lahnsteiner F.,Weismann T & Patzner R.A (1997) Methanol as cryoprotectant and the suitability of 1.2 mL and 5.0 mL straws for cryopreservation of semen from salmonid ¢shes Aquaculture Research 28, 471^479 Lahnsteiner F., Berger B., Horvath A., Urbanyi B & Weismann T (2000) Cryopreservation of spermatozoa in Ciprinid ¢shes Theriogenology 54, 1477^1498 Linhart O., Rodina M & Cosson J (2000) Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos Cryobiology 41, 241^250 Nascimento A.F., Maria A.N., Pessoa N.O., Carvhalo M.A.M & Viveiros A.T.M (2010) Out-of-season sperm cryopreserved in di¡erent media of the Amazonian freshwater ¢sh piraitinga (Piaractus brachypomus) Animal Reproduction Science 118, 324^329 Navarro O.J., Velasco-Santamar|¤ a Y.M & Cruz-Casallas P.E (2004) Evaluacio¤n de cinco protectores para la crioconservacio¤n de semen de cachama blanca (Piaractus brachypomus) Revista Colombiana de Ciencias Pecuarias Supl 17, 53^59 Ogier De Baulny L.E., Vern Y., Kerboeuf D & Mais G (1997) Flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved Rainbow trout (Oncorhynchus mykiss) spermatozoa Cryobiology 34, 141^149 Piironen J (1994) Composition and cryopreservation of sperm from some Finnish freshwater teleost ¢sh Finnish Fisheries Research 15, 27^48 Rakitin A., Moira M., Ferguson E & Trippel A (1999) Spermatocrit and spermatozoa density in Atlantic cod (Gadus morhua): correlation and variation during the spawning season Aquaculture 170, 349^358 Rana K (1995) Cryopreservation of aquatic gametes and embryos: recent advances and applications In: Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish (ed by F.W Goetz & P Thomas), pp 85–89 Austin, TX, USA: University of Texas at Austin Richardson G.F., Wilson C.E., Crim L.W & Yao X.Z (1999) Cryopreservation of yellowtail £ounder (Pleuronectes ferrugineus) semen in large straws Aquaculture 174, 89–94 Richardson G.F., Miller T.L & McNiven M.A (2000) Cryopreservation of Arctic charr Salvelinus alpinus semen in various extenders and three sizes of straw Aquaculture Research 31, 307^315 Rurangwa E., Kime D.E., Ollevier F & Nash J.P (2004) The measurement of sperm motility and factors a¡ecting sperm quality in cultured ¢sh Aquaculture 234, 1^28 Sarvi K., Niksirat H., Mojazi Amiri B., Mirtorabi S.M., Ra¢ee G.R & Bakhtiyari M (2006) Cryopreservation of semen from the endangered Caspian brown trout (Salmo trutta caspius) Aquaculture 256, 564^569 Steinberg H.A., Hedder R., BaulainY & HoltzW (1995) Cryopreservation of rainbow trout (Oncorhynchus mykiss) semen in straws In: Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish (ed by F.W Goetz & P Thomas), pp 146 Austin, TX, USA: University of Texas at Austin Tiersch T.R.,Williamson J.H., Carmichael G.J & Gorman O.T (1998) Cryopreservation of sperm of the endangered razorback sucker Transactions of the American Fisheries Society 127, 95^104 Velasco-Santamar|¤ aY.M., Medina-Robles V.M & Cruz-Casallas P.E (2006) Cryopreservation of yamu¤ (Brycon amazonicus) sperm for large scale fertilization Aquaculture 256, 264^271 Vincent C., Pruliere G., Pajot-Augy E., Campion E & Douzou P (1998) Biophysical chemical aspects of cellular cryobehavior Biophysical Chemistry 29, 161^169 Viveiros A.T.M., Orfaìo L.H., Maria A.N & Allaman I.B (2009) A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen Animal Reproduction Science 112, 293^300 Wamecke D & Pluta H.J (2003) Motility and fertilizing capacity of frozen/thawed common carp (Cyprinus carpio L.) sperm using dimethyl-acetamide as the main cryoprotectant Aquaculture 187, 361^375 Wheeler P & Thorgaard G (1991) Cryopreservation of rainbow trout semen in large straws Aquaculture 93, 95^100 Yao Z., Crim L.W., Richardson G.F & Emerson C.J (2000) Motility, fertility and ultrastructural changes o¡ ocean put (Macrozoarces americanus L.) sperm after cryopreservation Aquaculture 181, 361^375 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 738^745 745 Aquaculture Research, 2011, 42, 878^886 (a) 24 Water parameters a¡ect silver cat¢sh anaesthesia D P Gomes et al (b) 24 a 18 * a a ab 12 ab b 0 b b 20 40 PvCO2(mm Hg) Pv O2(mm Hg) a pH * a a a a 12 ab ab b b b 0 20 40 Eugenol (mg L–1) (c) 10 18 a b b a a a a b b 15 °C 23 °C 30 °C 0 20 40 Eugenol (mg L–1) Figure PvO2 (a), PvCO2 (b) and blood pH (c) in silver cat¢sh maintained at di¡erent water temperatures after being anaesthetized (stage IV) with eugenol, except for those exposed to 20 mg L À of eugenol at 30 1C, which did not anaesthetize and remained in stage II for 20 All values are mean Æ SE Di¡erent lowercase letters indicate signi¢cant di¡erences between the water temperatures in the same eugenol concentration (Po0.05) ÃSigni¢cant di¡erence between the eugenol concentrations in the same water temperature (Po0.05) 1867) (Walsh & Pease 2002) exposed to clove oil, as well as to rainbow trout exposed to AQUI-S (an isoeugenol-based anaesthetic) (Stehly & Gingerich 1999), when these species were acclimated to higher temperatures Induction and recovery times were generally shorter in Atlantic cod, Gadus morhua (Linnaeus 1758), exposed to benzocaine, metacaine (MS-222), metomidate and 2-phenoxyethanol at 16 1C as compared with 1C (Zahl et al 2009) The temperature^ related e¡ects of an anaesthetic could be explained by the dependence of metabolic rate on temperature The faster induction and recovery times at higher water temperatures may be related to an increased oxygen demand due to increased basal metabolism and lower oxygen solubility, leading to enhanced respiration and blood £ow, and thus accelerated physiologic processes involving absorption and elimination of the anaesthetics (Zahl et al 2009) Rainbow trout exposed for 10 to AQUI-S showed higher isoeugenol levels in ¢llets obtained at higher temperatures (Meinertz, Greseth, Schreier, Bernardy & Gingerich 2006), and silver perch, Bidyanus bidyanus (Mitchell 1838), exposed to 50 mg L À of clove oil also presented higher eugenol levels in ¢llets obtained at higher temperatures (Kildea, Allan & Kearney 2004) In addition, silver perch were also able to clear their £esh of eugenol more rapidly at higher temperatures (Kildea et al 2004), which would help to explain the shorter recovery times of silver cat¢sh acclimated to higher temperatures Earlier reports indicate that ¢sh size does not have a clear unidirectional e¡ect on the time required to induce anaesthesia (Houston & Woods 1972; Akhlaghi & Mirab-Brojerdi 1999; Prince & Powell 2000; Walsh & Pease 2002; Woody et al 2002; Tsantilas, Galatos, Athanassopoulou, Prassinos & Kousoulaki 2006), and considerable variation exists both within a species and, especially, between species White¢sh of larger size had shorter anaesthesia induction times with 40 mg L À of clove oil than smaller ¢sh, but the opposite trend was observed in rainbow trout (Hoskonen & Pirhonen 2004) It was observed in the present study that at most temperatures, the sedation and anaesthesia induction times were higher in larger silver cat¢sh exposed to eugenol than in smaller specimens This is in agreement with the fact that larger ¢sh have a smaller gill surface area in relation to body weight, and consequently a smaller area for anaesthetic di¡usion (Zahl et al 2009) In addition, larger silver cat¢sh probably have lower metabolic rates (Bolner & Baldisserotto 2007), which may also contribute to a slower rate of anaesthetic absorption r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 878^886 883 Water parameters a¡ect silver cat¢sh anaesthesia D P Gomes et al Values of PvO2 and PvCO2 in silver cat¢sh varied according to water temperature and ranged from approximately 5.0^20.0 and 6.0^13.0 mmHg respectively Dourado, Salminus brasiliensis (Cuvier 1816), exhibited similar PvO2 values (Souza, Soncini, Glass, Sanches & Rantin 2001), but these values are highly dependent on the species in question, and whether the blood is arterial or venous Overall mean PvO2 and PvCO2 values in tambaqui, Colossoma macropomum (Cuvier 1816), exposed to di¡erent water pHs (3.0, 4.0, 5.0 and 6.5 at 28^30 1C) were approximately 40.0 and 5.0 mmHg respectively Values of PvO2 and PvCO2 (4.93 and 12.73 mmHg respectively) of silver cat¢sh exposed to the highest tested eugenol concentration (40 mg L À 1; 23 1C) were similar to those reported by Sladky et al (2001) for red pacu, Piaractus brachypomus (Cuvier 1818), acclimated to a temperature of 21.5 1C and exposed to 50 mg L À of clove oil (3.50 and 8.90 mmHg respectively) Blood pH values of silver cat¢sh were similar to those reported for tambaqui (Wood, Wilson, Gonzalez, Patrick, Bergman, Narahara & Val 1998) and red pacu (Sladky et al 2001) Silver cat¢sh maintained at 23 1C (both control ¢sh and those exposed to 40 mg L À of eugenol) presented signi¢cantly higher PvCO2 values as compared with those kept at 15 1C This PvCO2 at 23 1C would lead to respiratory acidosis, which would explain the signi¢cantly lower blood pH These ¢ndings are in agreement with Sladky et al (2001) In conclusion, water quality and ¢sh size a¡ect the anaesthetic e⁄cacy of eugenol in silver cat¢sh, particularly at the lower concentrations tested Sedation of this species can be induced with as little as 20 mg L À of eugenol, but anaesthesia requires a concentration of at least 40 mg L À to compensate for the in£uence of ¢sh size and water quality Blood gas tensions and pHs were a¡ected by eugenol anaesthesia, but only in ¢sh acclimated to a temperature of 30 1C Acknowledgments B Baldisserotto and A G Becker received research and PhD fellowships, respectively, from CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Cient|¤ ¢co, Brazil) References Akhlaghi M & Mirab-Brojerdi M (1999) Anaesthetic e¡ect of clove tree and LC50 determination in rainbow trout 884 Aquaculture Research, 2011, 42, 878^886 (Oncorhynchus mykiss) Journal of the Faculty of Veterinary Medicine 54, 52^57 Anderson W.G., McKinley R.S & Collavechia M (1997) The use of clove oil as an anaesthetic for rainbow trout and its e¡ects on swimming performance North American Journal of Fisheries Management 17, 301^307 Baldisserotto B (2004) Silver cat¢sh culture.World Aquaculture 35, 65^67 Black E.C & Conner A.R (1964) E¡ects of MS-222 on glycogen and lactate levels in rainbow trout (Salmo gairdneri) Journal of the Fisheries Research Board of Canada 21, 1539^ 1542 Bolner K.C.S & Baldisserotto B (2007) Water pH and urinary excretion in silver cat¢sh Rhamdia quelen Journal of Fish Biology 70, 50^64 Boyd C.E & Tucker C.S (1992) Water Quality and Pond Soil Analysis for Aquaculture Alabama Agricultural Experiment Station, Auburn University, Auburn, AL, USA Chippari-Gomes A.R., Gomes L.C & Baldisserotto B (1999) Lethal temperatures for silver cat¢sh, Rhamdia quelen, ¢ngerlings Journal of Applied Aquaculture 9, 11^21 Cho G.K & Heath D.D (2000) Comparison of trica|¤ ne methanesulphonate (MS 222) and clove oil anaesthesis e¡ects on the physiology of juvenile Chinook salmon Oncorhynchus tabawytscha (Walbaum) Aquaculture Research 31, 537^546 Copatti C.E., Coldebella I.J., Radˇnz Neto J., Garcia L.O., Da Rocha M.C & Baldisserotto B (2005) E¡ect of dietary calcium on growth and survival of silver cat¢sh ¢ngerlings, Rhamdia quelen (Heptapteridae), exposed to di¡erent water pH Aquaculture Nutrition 11, 345^350 EndoT., Ogishima K.,Tanaka H & Ohshima S (1972) Studies on the anaesthetic e¡ect of eugenol in some freshwater ¢shes Bulletin of the Japanese Society of Scienti¢c Fisheries 38,761^767 Garcia L.O., Copatti C.E.,Wachholz F., Pereira Filho W & Baldisserotto B (2008) Freshwater temperature in the state of Rio Grande Sul, southern Brazil, and its implication for ¢sh culture Neotropical Ichthyology 6, 275^281 Gue¤nette S.A., Uhland F.C., He¤lie P., Beaudry F & Vachon P (2007) Pharmacokinetics of eugenol in rainbow trout (Oncorhynchus mykiss) Aquaculture 266, 262^265 Hanley C.S., Clyde V.L.,Wallace R.S., Paul-Murphy J., Patterson T.A., Keuler N.S & Sladky K.K (2010) E¡ects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca £avescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio) Journal of the American Veterinary Medical Association 236,1104^1108 Hikasa Y., Takase K., Ogasawara T & Ogasawara S (1986) Anaesthesia and recovery with tricaine methanesulfonate, eugenol and tripental sodium in the carp, Cyprinus carpio JapaneseJournal of Veterinary Science 48, 341^351 Hoskonen P & Pirhonen J (2004) Temperature e¡ects on anaesthesia with clove oil in six temperature-zone ¢shes Journal of Fish Biology 64,1136^1142 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 878^886 Aquaculture Research, 2011, 42, 878^886 Water parameters a¡ect silver cat¢sh anaesthesia D P Gomes et al Houston A.H & Woods R.J (1972) Blood concentrations of tricaine methane sulfonate in brook trout, Salvelinus fontinalis, during anaesthetization, branchial irrigation, and recovery Journal of the Fisheries Research Board of Canada 29,1344^1346 Howell B.J., Baumgardner F.W., Bondi K & Rahn H (1970) Acid-base balance in cold-blooded vertebrates as a function of body temperature American Journal of Physiology 218, 600^606 Inoue L.A.K.A., Santos Neto C & Moraes G (2003) Clove oil as anaesthetic for juveniles of matrinxaì Brycon cephalus (Gunther, 1869) CieŒncia Rural 33, 943^947 Iversen M., Finstad B., McKinlay R.S & Eliassen R.A (2003) The e⁄cacy of metomidate, clove oil, AQUI-STM and Bens zoak as anaesthetics in Atlantic salmon (Salmo solar L.) smolts, and their potential stress ^ reducing capacity Aquaculture 221, 549^566 Keene J.L.I., Noakes D.L.G., Moocia R.D & Soto C.G (1998) The e⁄cacy of clove oil as an anesthetic for rainbow trout Oncorhynchus mykiss (Walbaum) Aquaculture Research 29, 89^101 Kildea M.A., Allan G.L & Kearney R.E (2004) Accumulation and clearance of the anaesthetics clove oil and AQUI-S (TM) from the edible tissue of silver perch (Bidyanus bidyanus) Aquaculture 232, 265^277 Lazzari R., Radˇnz-Neto J., Emanuelli T., Pedron F.A., Costa M.L., Losekann M.E., Correia V & Bochi V.C (2006) Diferentes fontes prote¤icas para a alimentac°aìo jundiaŁ (Rhamdia quelen) CieŒncia Rural 36, 240^246 Limsuwan C., Grizzle J.M & Plumb J.A (1983) Etomidate as an anesthetic for ¢sh ^ its toxicity and e⁄cacy Transactions of the American Fisheries Society 112, 544^550 McFarland W.N (1959) A study of the e¡ects of anaesthetics on the behavior and physiology of ¢shes Publications of the Institute of Marine Sciences 6, 22^25 Meinertz J.R., Greseth S.L., Schreier T.M., Bernardy J.A & GingerichW.H (2006) Isoeugenol concentrations in rainbow trout (Oncorhynchus mykiss) skin-on ¢llet tissue after exposure to AQUI-S (TM) at di¡erent temperatures, durations, and concentrations Aquaculture 254, 347^354 Munday P.L & Wilson S.K (1997) Comparative e⁄cacy of clove oil and other chemicals in anesthetization of Pomacentrus amboinensis, a coral reef ¢sh Journal of Fish Biology 51, 931^938 Mylonas C.C., Cardinaletti G., Sigelaki I & Polzonetti-Magni A (2005) Comparative e⁄cacy of clove oil and 2-phenoxyethanol as anesthetics in the aquaculture of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) at di¡erent temperatures Aquaculture 246, 467^481 Peake S (1998) Sodium bicarbonate and clove oil as potential anaesthetics of non-salmonid ¢shes North American Journal of Fisheries Management 18, 919^924 Pereira-da-Silva E.M., Oliveira R.H.F., Ribeiro M.A.R & Coppola M.P (2009) Efeito aneste¤sico o¤leo de cravo em alevinos de lambari CieŒncia Rural 39,1851^1856 Piedras S.R.N., Moraes P.R.R & Pouey J.L.O.F (2004) Crescimento de juvenis de jundiaŁ (Rhamdia quelen), de acordo com a temperatura da aŁgua Boletim Instituto de Pesca 30, 177^182 Piper R.G., McElwain I.B., Orme L.E., McCraren J.P., Fowler L.G & Leonard J.R (1982) Fish Hatchery Management United States Department of the Interior Fish and Wildlife Service,Washington, DC, USA, 517pp Pirhonen J & Schreck C.B (2003) E¡ects of anaesthesia with MS-222, clove oil and CO2 on feed intake and plasma cortisol in steelhead trout (Oncorhynchus mykiss) Aquaculture 220, 507^514 Prince A & Powell C (2000) Clove oil as an anaesthetic of invasive ¢eld procedures on adult rainbow trout North AmericanJournal of Fisheries Management 20, 1029^1032 Ross L.G & Ross B (2008) Anaesthetic and SedativeTechniques for Aquatic Animals, 2nd edn Blackwell, London, UK, 159pp Schoettger R.A & Julin M (1967) E⁄cacy of MS-222 as an anesthetic on four salmonids Investigation of Fisheries Contribution, US Department of Interior, 13,1^15 Silva L.V.F., Golombieski J.I & Baldisserotto B (2003) Incubation of silver cat¢sh, Rhamdia quelen (Pimelodidae), eggs at di¡erent calcium and magnesium concentrations Aquaculture 228, 279^287 Silva L.V.F., Golombieski J.I & Baldisserotto B (2005) Growth and survival of silver cat¢sh larvae, Rhamdia quelen (Heptapteridae), at di¡erent calcium and magnesium concentrations Neotropical Ichthyology 3, 299^304 Sladky K., Swanson C., Stoskopf M., Loomis M & Lewbart G (2001) Comparative e⁄cacy of tricaine methanesulfonate and clove oil for use as anesthetic in red pacu (Piaractus brachypomus) American Journal of Veterinary Research 62, 337^342 Small B.C (2004) E¡ect of isoeugenol sedation on plasma cortisol, glucose and lactate dynamics in channel cat¢sh Ictalurus punctatus exposed to three stressors Aquaculture 238, 469^481 Soto C & Burhanuddin S (1995) Clove oil as a ¢sh anaesthetic for measuring length a weight of rabit¢sh (Siganus lineatus) Aquaculture 136,149^152 Souza R.H.D., Soncini R., Glass M.L., Sanches J.R & Rantin F.T (2001) Ventilation, gill perfusion and blood gases in dourado, Salminus maxillosus Valenciennes (Teleostei, Characidae), exposed to graded hypoxia Journal of Comparative Physiology B 171, 483^489 Stehly G.R & Gingerich W.H (1999) Evaluation of AQUI-S (e⁄cacy and minimum toxic concentration) as ¢sh anaesthetic-sedative for public aquaculture in the United States Aquaculture Research 30, 365^372 Sylvester J.R (1975) Factors in£uencing the e⁄cacy of MS-222 to striped mullet (Mugil cephalus) Aquaculture 6, 163^169 Sylvester J.R & Holland L.E (1982) In£uence of temperature, water hardness, and stocking density on MS-222 response in three species of ¢sh Progressive Fish-Culture 44,138^141 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 878^886 885 Water parameters a¡ect silver cat¢sh anaesthesia D P Gomes et al Tort L., Puigcerver M., Crespo S & Padros F (2002) Cortisol and hematological response in sea bream and trout subjected to the anesthetics clove oil and 2-phenoxyethanol Aquaculture Research 33, 907^910 Townsend C.R & Baldisserotto B (2001) Survival of silver cat¢sh ¢ngerlings exposed to acute changes of water pH and hardness Aquaculture International 9, 413^419 Townsend C.R., Silva L.V.F & Baldisserotto B (2003) Growth and survival of Rhamdia quelen (Siluriformes, Pimelodidae) larvae exposed to di¡erent levels of water hardness Aquaculture 215, 103^108 Tsantilas H., Galatos A.D., Athanassopoulou F., Prassinos N.N & Kousoulaki K (2006) E⁄cacy of 2-phenoxyethanol as an anaesthetic for two size classes of white sea bream, Diphodus sargus L., and sharp snout sea bream, Diplodus puntazzo C Aquaculture 253, 64^70 Tyler P & Hawkins A.D (1981) Vivisection, anaesthetics and minor surgery In: Aquarium Systems (ed by A.D Hawkins), pp 247–278 Academic Press, New York, NY, USA Wagner E., Arndt R & Hilton B (2002) Physiological stress responses, egg survival and sperm motility for rainbow trout broodstock anaesthetized with clove oil, tricaine methanesulfonate or carbon dioxide Aquaculture 211, 353^366 886 Aquaculture Research, 2011, 42, 878^886 Walsh C.T & Pease B.C (2002) The use of clove oil as an anaesthetic for the long ¢nned eel, Anguilla reinhardtii (Steindachner) Aquaculture Research 33, 1^9 Wedemeyer G (1970) Stress of anesthesia with MS-222 and benzocaine in rainbow trout (Salmo gairdneri) Journal of the Fisheries Research Board of Canada 27, 909^914 Wood C.M., Wilson R.W., Gonzalez R.J., Patrick M.L., Bergman H.L., Narahara A & Val A.L (1998) Responses of an Amazonian teleosts, the tambaqui (Colossoma macropomum), to low pH in extremely soft water Physiological Zoology 71, 658^670 Woody C.A., Nelson J & Ramstad K (2002) Clove oil as an anaesthetic for adult sockey salmon: ¢eld trials Journal of Fish Biology 60, 340^347 Zahl I.H., Kiessling A., Samuelsen O.B & Hansen M.K (2009) Anaesthesia of Atlantic cod (Gadus morhua) ^ e¡ect of pre-anaesthetic sedation, and importance of body weight, temperature and stress Aquaculture 295, 52^59 Zaions M.I & Baldisserotto B (2000) Na1 and K1 body levels and survival of ¢ngerlings of Rhamdia quelen (Siluriformes, Pimelodidae) exposed to acute changes of water pH CieŒncia Rural 30, 1041^1045 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 878^886 Aquaculture Research, 2011, 42, 887^891 doi:10.1111/j.1365-2109.2010.02727.x SHORT COMMUNICATION Cellulase-producing Bacillus strains isolated from the intestine of Amazon basin fish Samanta Balsini Peixoto, Florencia Cladera-Olivera, Daniel Joner Daroit & Adriano Brandelli Laborato¤rio de Bioqu|¤ mica e Microbiologia Aplicada, Instituto de CieŒncia e Tecnologia de Alimentos, Universidade Federal Rio Grande Sul, Porto Alegre, RS, Brazil Correspondence: A Brandelli, Laborato¤rio de Bioqu|¤ mica e Microbiologia Aplicada, Instituto de CieŒncia e Tecnologia de Alimentos, Universidade Federal Rio Grande Sul, Av Bento Gonc°alves 9500, 91501-970 Porto Alegre, RS, Brazil E-mail: abrand@ufrgs.br The intensi¢cation of aquaculture practices has generated a demand for alternative feed ingredients In this sense, plant biomass is an inexpensive and highly available nutrient source mainly composed of cellulose, a recalcitrant polymer consisting of glucose units linked by b-1,4-glucosidic bonds (Lynd,Weimer, van Zyl & Pretorius 2002) As ¢sh usually not produce cellulolytic enzymes, the cellulase activity observed in ¢sh is mainly produced by the intestinal microbiota (Saha & Ray 1998) Various cellulolytic bacteria have been isolated from ¢sh intestines (Bairagi, Ghosh, Sen & Ray 2002; Saha, Roy, Sen & Ray 2006; Kar & Ghosh 2008; Mondal, Roy, Sen & Ray 2008), which may contribute to the digestion of plant materials The proposed mechanism of enzymatic cellulolysis, based on fungal cellulase systems, is essentially composed of endo-b-1,4-glucanase, exo-b-1,4-glucanase (cellobiohydrolase) and b-glucosidase (cellobiase) activities The endo- and exo-glucanases act synergistically to produce mainly cellobiose, which is cleaved by b-glucosidases (Singh & Hayashi 1995; Lynd et al 2002) The South American warm water teleosts pacu (Piaractus mesopotamicus Holmberg 1887) and piaucom-pinta (Leporinus friderici Bloch 1794) possess herbivorous/omnivorous feeding habits (Albrecht, Ferreira & Caramaschi 2001; Abimorad, Carneiro & Urbinati 2007) Because of the ingestion of cellulosic materials, herbivorous/omnivorous ¢sh often harbour cellulase-producing intestinal microorganisms As the intestinal microbiota of Amazon basin ¢sh is poorly studied, and microbial diversity is a major re- r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd source for novel products and processes in aquaculture and biotechnology, the present study evaluated the cellulolytic ability of bacterial strains isolated from the intestine of Amazonian ¢sh Bacterial strains, P6 and P11, were isolated previously from the midgut of P mesopotamicus and L friderici, respectively, on Luria^Bertani (LB) agar plates (10 g L À lacto-peptone, g L À yeast extract, 10 g L À NaCl and 15 g L À agar) for 24 h at 30 1C The strains were identi¢ed as Bacillus species based on 16S rDNA sequencing, clustering with Bacillus subtilis and Bacillus velesensis respectively (Giongo, Lucas, Casarin, Heeb & Brandelli 2007) Strains were maintained on LB agar plates at 1C These bacteria were kindly provided by Dr Spartaco Astol¢-Filho (UFAM, Manaus, Brazil) Bacterial growth was investigated on LB agar plates at di¡erent temperatures (15^55 1C) for 24 h In submerged cultivations, LB broths with di¡erent pH (5.0^10.0, adjusted with either M NaOH or M HCl) were inoculated, incubated at 30 or 37 1C and growth was determined by optical density at 600 nm Alternatively, bacteria were cultivated in 250 mL Erlenmeyer £asks containing 50 mL of Bushnell^ Haas medium (0.2 g L À MgSO4, 0.02 g L À CaCl2, 1g L À KH2PO4, 1g L À K2HPO4, 1g L À NH4NO3 and 0.05 g L À FeCl3, pH 7.0) plus10 g L À of di¡erent carbon sources (Table 1), and incubated at 30 1C for 24 h with shaking (125 rpm) At de¢ned periods, aliquots of culture were centrifuged (10 000 g for 10 min), the supernatant was ¢ltered (0.22 mm membrane) and used as a crude extracellular enzyme (Giongo et al 2007) The pellet was washed with 887 Cellulase production by ¢sh Bacillus strains S B Peixoto et al Table Cellulase (CMCase) activity of Bacillus subtilis P6 and Bacillus velesensis P11 growing on di¡erent carbon sourcesà Activity (U mL À 1)w Carbon source Control (LB medium) Glucose Galactose Mannose Arabinose Maltose Sucrose Lactose Cellobiose Carboxymethyl cellulose Cellulose Starch a-Methyl-Dglucopyranose Bacillus subtilis Bacillus velesensis P6 P11 72 75 79 78 72 72 86 79 93 82 86 72 75 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 5 5 76 83 76 83 76 76 83 84 99 79 87 76 76 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 5 4 ÃStrains P6 and P11 were grown in a mineral medium containing 10 g L À carbon source wValues are the means of three independent experiments phosphate-bu¡ered saline, recentrifuged and (i) used directly in order to investigate whole cells enzyme activities or (ii) resuspended in bu¡er, followed by cell lysis through sonication (three cycles of 10 s at 20 000 Hz) After centrifugation (10 000 g for 10 min), enzyme activities were determined in cell debris and cell lysate Cellulase activity (CMCase) was determined using the Congo red staining method (Carder 1986) using agar plates with 0.1g L À carboxymethylcellulose Enzyme samples (50 mL) were placed in mm wells on the CMC-agar plates and, after incubation (16 h at 30 1C), plates were £ooded with a 0.1g L À Congo red solution After 30 min, plates were rinsed with distilled water, followed by two washes with 1mol L À NaCl The diameters of the hydrolysis zones (mm) were measured and CMCase units (U mL À 1) were determined according to a standard curve (Carder 1986) developed with various concentrations of commercial cellulose (Fluka) b-glucosidase and exo-b-1,4-glucanase activities were monitored in the culture supernatants, whole cell, cell debris and cell lysate with the chromogenic substrates p-nitrophenyl-b-D-glucopyranoside and p-nitrophenyl-b-D-cellobioside respectively (Geimba, Ri¡el, Agostini & Brandelli 1999) In assays with whole cells or cell debris, the reaction mixtures were centrifuged (10 000 g for min) before absorbance readings 888 Aquaculture Research, 2011, 42, 887^891 Agroindustrial wastes were tested as substrates for cellulase production by P6 and P11.Wheat straw was subjected to three di¡erent pretreatments: (i) shearing into small fragments (approximately mm) using a laboratory blender; (ii) shearing plus chemical treatment with 0.8 mol L À NaOH for h at 80 1C; and (iii) milling (0.5 mm particle size) Rice straws/ rice hulls were only subjected to milling Mineral medium containing 10 g pretreated substrate L À was autoclaved (121 1C, 105 kPa for 15 min), inoculated and incubated at 30 1C, 125 rpm After 48 h, the media were centrifuged, and the supernatants subjected to the CMCase activity assay Bacillus spp are commonly isolated from ¢sh intestines (Ghosh, Sen & Ray 2002a; Kim, Brunt & Austin 2007; RingÖ, Sperstad, Kraugerud & Krogdahl 2008) Bacillus subtilis P6 and B velesensis P11 grew on CMC-agar plates, indicating the presence of carboxymethylcellulolytic bacteria in the intestines of pacu and piau-com-pinta respectively Microbial cellulase, through the breakdown of ingested cellulosic materials, might be signi¢cant for the nutrition of omnivorous/herbivorous ¢sh (Ghosh, Sen & Ray 2002b; Saha et al 2006) The function of ¢sh intestinal microbiota is emphasized as its composition seems to be a¡ected by food habits and dietary changes (Mondal et al 2008; RingÖ et al 2008) In a previous study, culturable cellulolytic microorganisms were not detected in the intestines of carnivorous ¢sh but microbial cellulase activity was detected in herbivorous/omnivorous ¢sh (Bairagi et al 2002) Nevertheless, it should be emphasized that the culture-based methods used in that study might have failed to detect cellulolytic microorganisms present in low numbers and, additionally, non-culturable cellulolytic bacteria could have been present in the ¢sh intestines, contributing to cellulolytic activities Strains P6 and P11 grew at 20^45 1C on LB-agar plates, with an optimum at 37 1C (result not shown) In submerged cultivations, the growth of both strains was higher at 37 1C than that at 30 1C, reaching the stationary growth phase after 16 h (Fig 1a) For instance, P mesopotamicus is found in temperatures from 15 to 35 1C (Milstein, Zoran, Peretz & Joseph 2000), and the temperatures used in feeding trials with this species and also Leporinus spp usually range from 25 to 29 1C (Wilhelm Filho, Torres, Zaniboni-Filho & Pedrosa 2005; Abimorad et al 2007) Bacillus subtilis P6 and B velesensis P11 grew similarly at pH 6.0^8.0, poor growth occurred at pH 9.0 and no growth was observed at pH 5.0 and 10.0 (data not shown) These values relate well with the pH r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 887^891 Aquaculture Research, 2011, 42, 887^891 Cellulase production by ¢sh Bacillus strains S B Peixoto et al 120 Residual activity (%) (a) OD 600nm 100 80 60 40 20 0 10 20 30 Time (h) 40 50 Cellulase activity (U/ml) 10 pH Figure E¡ect of pH on the cellulase produced by strains P6 (white symbols) and P11 (black symbols) on LB broth Data are the means of three independent experiments Curve estimation was carried out by polynomial regression using MICROSOFT EXCEL software 90 (b) 80 70 60 50 40 30 20 10 0 10 20 30 Time (h) 40 50 Figure Growth (a) and cellulase production (b) by Bacillus strains on LB broth Optical density and cellulase activity were monitored during the cultivation of strains P6 (white symbols) and P11 (black symbols) at 30 1C (circles) or 37 1C (squares) Data are the means of three independent experiments Curve estimation was carried out by polynomial regression using MICROSOFT EXCEL software found in ¢sh intestines (7.0^9.0) (Payne 1978; Garc|¤ aCarrenìo, Albuquerque-Cavalcanti, del Toro & Zaniboni-Filho 2002; RingÖ, Olsen, Mayhew & Myklebust 2003) Previously, cellulolytic Bacillus circulans and Bacillus megaterium, isolated from tilapia and grass carp, respectively, grew within broad temperature (15^42 1C) and pH (5.0^11.0) ranges (Saha et al 2006) On LB broth, strains P6 and P11 showed maximal CMCase activity at 24 h (Fig.1b), coinciding with the stationary growth phase (Fig.1a) Although higher growth occurred at 37 1C, the maximum cellulase production was observed at 30 1C (Fig 1b) Extracellular cellobiohydrolase activities of strains P6 and P11 were 0.9 and 2.1U mL À 1, respectively, and b-glucosidase was not detected In bacteria from the aquatic environment, cell-bound enzymes may be advantageous, avoiding enzyme loss and facilitat- ing the uptake of hydrolysis products (Eveleigh, Bok, El-Dorry, El-Gogary, Elliston, Goyal, Waldron, Wright & Wu 1995) However, only low to undetectable cellobiohydrolase and b-glucosidase activities were observed in whole cells, cell debris and cell lysate This suggests that endo-b-1,4-glucanase (CMCase) is the major enzyme e¡ecting cellulose hydrolysis in P6 and P11 Cellulolytic bacteria seem to produce mainly endo-glucanases Among 34 Bacillus strains isolated from soil, CMCase activity was produced by all isolates, 28 isolates produced b-glucosidase and exo-glucanases were not produced (Pajni, Dhillon, Vadehra & Sharma 1989) The absence of b-glucosidase activity is also observed in many bacteria where cellobiose and cellodextrins enter the cell and are metabolized by phosphorylases (Singh & Hayashi 1995; Lynd et al 2002) CMCases from P6 and P11 were active over broad pH ranges, showing optimal activity at pH and 5, respectively, maintaining about 90% of their maximal activity between pH and (Fig 2), suggesting their potential activity in the gut of ¢sh During the cultivation on various carbon sources, the highest cellulase activity was produced with cellobiose, followed by cellulose (Table 1) Both strains produced constitutive levels of cellulase (Fig 1b) and the addition of carbon sources tended to enhance CMCase production (Table 1) Disaccharides such as cellobiose and lactose often stimulate cellulase production (Singh & Hayashi 1995; Geimba et al 1999), while glucose typically inhibits cellulase production through catabolite repression (Lynd et al 2002) Therefore, the CMCase activity observed in the r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 887^891 889 Cellulase production by ¢sh Bacillus strains S B Peixoto et al presence of glucose indicates that a di¡erent mechanism might regulate its production by P6 and P11 Similar results were reported for Bacillus sp KSM-635 (Ito 1997), and the constitutive production of cellulases is also reported in other bacteria, such as Paenibacillus curdlanolyticus strain B-6 (Waeonukul, Kyu, Sakka & Ratanakhanokchai 2008) and Bacillus species (Zvauya & Zvidzai 1995) Although cellulase production by the majority of fungal species is usually induced by cellulose or its derivatives, the regulation of cellulase biosynthesis among bacteria is not governed by such a uniform pattern (Robson & Chambliss 1989; Suto & Tomita 2001) In aquaculture, attention is being focused on the utilization of enzyme-producing bacterial isolates as probiotics in formulating aquafeeds Intestinal Bacillus isolates are shown to improve growth performance and feed utilization in ¢sh, and stimulate the ¢sh immune responses (BalcaŁzar, Decamp, Vendrell, De Blas & Ruiz-Zarzuela 2006; Merri¢eld, Dimitroglou, Foey, Davies, Baker, BÖgwald, Castex & RingÖ 2010) The addition of cellulase to ¢sh diets might positively a¡ect growth, feed utilization and nutrient digestibility, potentially reducing feed costs (Oliveira, Logato, Freitas, Rodrigues, Fialho & Diodatti 2007; Ayhan, Diler, Arabaci & Sevgili 2008) The successful utilization of cellulosic materials for ¢sh feed depends on the economic production of cellulases (Saha et al 2006) High productivity of cellulases may be achieved by using pretreated lignocellulosic biomass as inexpensive growth substrates (Krishna 1999; Romero, Aguado, GonzaŁlez & Ladero 1999) Therefore, CMCase production by strains P6 and P11 was evaluated during cultivation on milled wheat straws, rice straws, rice hulls and milled/alkali-treated wheat straws Higher CMCase production by P11 occurred with milled wheat straw and the alkali pretreatment did not stimulate increased enzyme e⁄cacy (Table 2) Rice straw was a better substrate than rice hulls for CMCase production, and similar enzymatic activities were observed for both P6 and P11 (Table 2) These results emphasize the importance of choosing an appropriate inducing substrate for a high enzyme yield Krishna (1999) showed that autoclaving banana fruit stalk favoured cellulase production, was more economical and produced less wastewater than chemical treatments The cellulolytic potential of B subtilis P6 and B velesensis P11was investigated Bacterial growth, cellulase production (mainly endoglucanases) and cellulase activity were observed at pH values found 890 Aquaculture Research, 2011, 42, 887^891 Table Production of cellulase (CMCase) by Bacillus subtilis P6 and Bacillus velesensis P11 during growth on agricultural residuesà Cellulase activity (U mL À 1) Substrate Sheared wheat straws Alkali-treated wheat straws Milled wheat straws Milled rice straws Milled rice hulls Bacillus subtilis Bacillus velesensis P6 P11 48 Æ 5A 45 Æ 6A 50 Æ 5A 25 Æ 3B 60 Æ 19A 65 Æ 12A 18 Æ 5B 160 Æ 23C 59 Æ 8A 12 Æ 4D ÃStrains P6 and P11 were grown in a mineral medium containing 10 g L À of each crop residue After 48 h of submerged cultivations, the enzyme activity was determined in the culture supernatants Values are the means of three independent experiments Superscript letters indicate signi¢cant di¡erences within the same column at Po0.05 (Student’s t-test) in ¢sh intestine, and agricultural residues supported cellulase production As discussed, the characterization of celullolytic microorganisms and microbial cellulases from ¢sh intestines addresses important subjects in aquaculture Acknowledgments This work was supported by Conselho Nacional de Desenvolvimento Cient|¤ ¢co e Tecnolo¤gico (CNPq, Brazil) funding References Abimorad E.G., Carneiro D.J & Urbinati E.C (2007) Growth and metabolism of pacu (Piaractus mesopotamicus Holmberg 1887) juveniles fed diets containing di¡erent protein, lipid and carbohydrate levels Aquaculture Research 38, 36^44 Albrecht M.P., Ferreira M.F.N & Caramaschi E.P (2001) Anatomical features and histology of the digestive tract of two related neotropical omnivorous ¢shes (Characiformes; Anostomidae) Journal of Fish Biology 58, 419^430 Ayhan V., Diler I., Arabaci M & Sevgili H (2008) Enzyme supplementation to soybean based diet in gilthead sea bream (Sparus aurata): e¡ects on growth parameters and nitrogen and phosporus excretion Kafkas Universitesi Veteriner Fakultesi Dergisi 14, 161^168 Bairagi A., Ghosh K.S., Sen S.K & Ray A.K (2002) Enzyme producing bacterial £ora isolated from ¢sh digestive tracts Aquaculture International 10,109^121 BalcaŁzar J.L., Decamp O., Vendrell D., De Blas I & Ruiz-Zarzuela M (2006) Health and nutritional properties of probiotics in ¢sh and shell¢sh Microbial Ecology in Health and Disease 18, 65^70 r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 887^891 Aquaculture Research, 2011, 42, 887^891 Carder J.H (1986) Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate di¡usion assay Analytical Biochemistry 153,75^79 Eveleigh D.E., Bok J.D., El-Dorry H., El-Gogary S., Elliston K., Goyal A.,Waldron C.,Wright R & WuY.M (1995) Cellulase lessons revealed through the microbe’s perspective Applied Biochemistry and Biotechnology 51/52, 169^177 Garc|¤ a-Carrenìo F.L., Albuquerque-Cavalcanti C., del Toro M.A.N & Zaniboni-Filho E (2002) Digestive proteinases of Brycon orbignyanus (Characidae, Teleostei): characteristics and e¡ects of protein quality Comparative Biochemistry and Physiology Part B 132, 343^352 Geimba M.P., Ri¡el A., Agostini V & Brandelli A (1999) Characterization of cellulose-hydrolyzing enzymes from the fungus Bipolaris sorokiniana Journal of the Science of Food and Agriculture 79,1849^1854 Ghosh K., Sen S.K & Ray A.K (2002a) Characterization of bacilli isolated from the gut of rohu, Labeo rohita, ¢ngerlings and its signi¢cance in digestion Journal of Applied Aquaculture 12, 33^42 Ghosh K., Sen S.K & RayA.K (2002b) Growth and survival of rohu, Labeo rohita (Hamilton) spawn fed diets supplemented with ¢sh intestinal micro£ora Acta Ichthyologica et Piscatoria 32, 83^92 Giongo J.L., Lucas F.S., Casarin F., Heeb P & Brandelli A (2007) Keratinolytic proteases of Bacillus species isolated from the Amazon basin showing remarkable de-hairing activity World Journal of Microbiology and Biotechnology 23, 375^382 Ito S (1997) Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents Extremophiles 1, 61^66 Kar N & Ghosh K (2008) Enzyme producing bacteria in the gastrointestinal tracts of Labeo rohita (Hamilton) and Channa punctatus (Bloch) Turkish Journal of Fisheries and Aquatic Sciences 8,115^120 Kim D.-H., Brunt J & Austin B (2007) Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss) Journal of Applied Microbiology 102,1654^1664 Krishna C (1999) Production of bacterial cellulase by solid state bioprocessing of banana wastes BioresourceTechnology 69, 231^239 Lynd L.R., Weimer P.J., van Zyl W.H & Pretorius I.S (2002) Microbial cellulose utilization: fundamentals and biotechnology Microbiology and Molecular Biology Reviews 66, 506^577 Merri¢eld D.L., Dimitroglou A., Foey A., Davies S.J., Baker R.T.M., BÖgwald J., Castex M & RingÖ E (2010) The current status and future focus of probiotic and prebiotic applications for salmonids Aquaculture 302, 1^18 Milstein A., Zoran M., Peretz Y & Joseph D (2000) Low temperature tolerance of pacu, Piaractus mesopotamicus Environmental Biology of Fishes 58, 455^460 Mondal S., Roy T., Sen S.K & RayA.K (2008) Distribution of enzyme-producing bacteria in the digestive tracts of some freshwater ¢sh Acta Ichthyologica et Piscatoria 38,1^8 Cellulase production by ¢sh Bacillus strains S B Peixoto et al Oliveira G.R., Logato P.V.R., Freitas R.T.F., Rodrigues P.B., Fialho E.T & Diodatti F.C (2007) Nutrient digestibility in diets with enzyme complex for Nile tilapia Brazilian Journal of Animal Science 36(Suppl.),1945^1952 Pajni S., Dhillon N.,VadehraV.D & Sharma P (1989) Carboxymethyl cellulase, b-glucosidase and xylanase production by Bacillus isolates from soil International Biodeterioration 25, 1^5 Payne A.I (1978) Gut pH and digestive strategies in estuarine grey mullet (Mugilidae) and tilapia (Cichlidae) Journal of Fish Biology 13, 627^629 RingÖ E., Olsen R.E., Mayhew T.M & Myklebust R (2003) Electron microscopy of the intestinal micro£ora of ¢sh Aquaculture 227, 395^415 RingÖ E., Sperstad S., Kraugerud O.F & Krogdahl — (2008) Use of16S rRNA gene sequencing analysis to characterize culturable intestinal bacteria in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy Aquaculture Research 39, 1087^1100 Robson L.M & Chambliss G.H (1989) Cellulases of bacterial origin Enzyme and MicrobialTechnology 11, 626^644 Romero M.D., Aguado J., GonzaŁlez L & Ladero M (1999) Cellulase production by Neurospora crassa on wheat straw Enzyme and MicrobialTechnology 25, 244^250 Saha A.K & RayA.K (1998) Cellulase activity in rohu ¢ngerlings Aquaculture International 6, 281^291 Saha S., Roy R.N., Sen S.K & Ray A.K (2006) Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes) Aquaculture Research 37, 380^388 Singh A & Hayashi K (1995) Microbial cellulases: protein architecture, molecular properties, and biosynthesis Advances in Applied Microbiology 40,1^44 Suto M & Tomita F (2001) Induction and catabolite repression mechanisms of cellulase in fungi Journal of Bioscience and Bioengineering 92, 305^311 Waeonukul R., Kyu K.L., Sakka K & Ratanakhanokchai K (2008) E¡ect of carbon sources on the induction of xylanolytic-cellulolytic multienzyme complexes in Paenibacillus curdlanolyticus strain B-6 Bioscience, Biotechnology, and Biochemistry 72, 321^328 Wilhelm Filho D., Torres M.A., Zaniboni-Filho E & Pedrosa R.C (2005) E¡ect of di¡erent oxygen tensions on weight gain, feed conversion, and antioxidant status in piapara, Leporinus elongatus (Valenciennes, 1847) Aquaculture 244, 349^357 Zvauya R & Zvidzai C.J (1995) Constitutive production of endoglucanase by a Bacillus sp isolated from a Zimbabwean hot spring.World Journal of Microbiology and Biotechnology 11, 658^660 Keywords: intestinal microbiota, Bacillus, cellulase, endoglucanase, Amazon basin r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 887^891 891 Aquaculture Research, 2011, 42, 892^897 doi:10.1111/j.1365-2109.2011.02824.x SHORT COMMMUNICATION Endogenous feeding and morphological changes in hatchery-reared larval palm ruff Seriolella violacea (Pisces: Centrolophidae) under starvation Claudia A Bustos1 & Alfonso Silva2 Programa de Doctorado en Acuicultura, Universidad Cato¤lica del Norte campus GuayacaŁn, Coquimbo, Chile Laboratorio de Cultivo de Peces, Facultad de Ciencias del Mar, Universidad Cato¤lica del Norte campus GuayacaŁn, Coquimbo, Chile Correspondence: C A Bustos, Programa de Doctorado en Acuicultura, Universidad Cato¤lica del Norte campus GuayacaŁn, Larrondo1281, Coquimbo, Chile E-mail: clausbustos@gmail.com There is an increasing e¡ort towards diversi¢cation of potential marine species for ¢sh culture around the world (Aristizabal, SuaŁrez, Vega & Bargas 2009; Cavalin & Weirich 2009; Leu, Liou, Wang, Yang & Meng 2009) However, to avoid negative impacts from the introduction of exotic species, the investigation of indigenous species becomes important and worthwhile (Ross, Mart|¤ nez Palacios & Morales 2008) Species such as southern hake Merluccius australis (Bustos & Landaeta 2005; Bustos, Landaeta, BaySchmith, Lewis & Moraga 2007), amberjack Seriola lalandi (Moran, Smith, Gara & Poortenaar 2007) and palm ru¡ Seriolella violacea Guichenot, 1848 have been considered to be excellent candidates for mariculture in Chile Particularly, the palm ru¡, S violacea, is a high-value marine ¢n¢sh that inhabits subtropical waters, and it is distributed between 11S and 341S in the eastern Paci¢c Ocean (Iannacone 2003) Successful maintenance of wild-caught broodstock, natural spawning, larviculture and pilot production of juveniles have been achieved in recent years at Fish Culture Laboratory, Universidad Cato¤lica del Norte, Chile Nevertheless, it is necessary to improve the knowledge of the biology of its early life stages to optimize their larviculture During larviculture, although plenty of live food is supplied to the culture tank, ¢sh may still be su¡ering from starvation due to poor vision and mouth gape limitation (Planas & Cunha 1999; Nakagawa & Miyashita 2008) After yolk resorption, energy reserves support the larvae for a limited period; the utilization 892 of some substances as energy sources by larvae faced with starvation promotes a series of changes in morphological characteristics, such as a decrease in their body height and degenerated gut height (Theilacker & Watanabe 1989; Yufera, Pascual, Polo & Sarasquete 1993), inducing poor performance in feeding and an adverse e¡ect on growth (Blaxter & Ehrlich1974) Because of their ease of observation and measurement both in the laboratory and in the ¢eld, the general collapse of gut and myotome development could be sensitive indicators for assessing larval starvation, especially at an early larval stage (e.g irreversible starvation age) (Yufera et al.1993; Dou, Masuda,Tanaka & Tsukamoto 2002) Internally, starvation usually changes the shape of enterocyte cells in the intestine of several ¢sh species, reducing the height of the midgut mucosal cells (Ehrlich, Blaxter & Pemberton 1976; Yufera et al 1993; Chen, Qin, Carragher, Clarke, Kumar & Hutchinson 2007) Furthermore, the changes in the digestive tract, liver and pancreas may a¡ect the synthesis of the digestive enzymes and functions of the digestive system (Gawlicka, Parent, Horn, Ross, Opstad & Torrissen 2000; Bolasina, Pe¤rez & Yamashita 2006) Information on starvation resistance and time to start external feeding is necessary to reduce the costs related to live feeds of larval palm ru¡ and to reduce larval mortality in ongoing e¡orts to develop a commercial mass culture system for this species The objectives of the work are to establish for the ¢rst time, under laboratory-controlled conditions, larval r 2011 Blackwell Publishing Ltd Aquaculture Research, 2011, 42, 892^897 Starvation e¡ects on larval Seriolella violacea C A Bustos & A Silva performance during endogenous feeding and the effect of early starvation on the morphological features and survival of larval S violacea Fertilized eggs were collected from captive adult S violacea that spontaneously spawned during August and September 2008 in two12000 L broodstock tanks under ambient conditions (14 Æ 1C, 34 ppt) at Fish Culture Laboratory, from Universidad Cato¤lica del Norte, Coquimbo, Chile Eggs were then stocked at 500 eggs L À in one 500 L cylinder conical tank supplied with ¢ltered seawater and gentle aeration Temperature in the incubator tank ranged from 141 to 16 1C and a light regime of 12 L:12 D was provided throughout the embryo development and until hatching occurred Hatching occurred 72^74 h after fertilization Newly hatched larvae were then transferred into two cylindrical tanks (100 L), with sterilized and ¢ltered seawater (1 mm), at an initial density of 50 larvae L À During the endogenous feeding, ten larval S violacea were extracted daily from one of the tanks Measurements of the larval length (LL), yolksac length and height were performed on freshly anaesthetized larvae with MS-222 under a stereomicroscope (Fig.1a).Yolk-sac volume was estimated considering the yolk-sac as an ellipsoid (V 4/3pab2), where a is half of the yolk-sac length and b is half of the yolk-sac height, Bustos et al 2007) Then, an exponential negative curve was ¢tted to calculate the yolk-sac absorption rate of larval S violacea Two days post hatching (2 DPH), larvae were stocked in two 60 L cylinder conical tanks with central drains of ¢ltered seawater (1 mm) at 16 1C and a Figure Larval Serioella violacea and morphological characteristics measured in larvae (a) larva of 4.1mm NL and days post hatching (DPH), (b) larva of 5.0 mm NL and DPH, (c) larva of 7.3 mm NL and 14 DPH NL, notochord length; HL, head length; ED, eye diameter; BD, body depth; MH, myotome height standpipe ¢tted with a 150 mm mesh Larvae in the latter tank were not fed and those in the other tank were fed enriched rotifers (Brachionus plicatilis) twice a day at a density of 10 rotifers mL À Rotifers were cultured using a batch culture system, fed a dry diet of bakers yeast combined with concentrated microalgae Nannochloropsis oculata and Isochrysis galbana In addition, rotifers were enriched with AlgaMac 3050 (Aquafauna Bio-Marine, Hawthorne, CA, USA) The morphology of 10^20 larvae from both nonfed and fed groups was measured daily Morphological measurements were performed on freshly anaesthetized larvae (MS-222) using a calibrated ocular micrometer ¢tted to a stereomicroscope Five parts of the body of the larvae were measured: notochord length (NL, from the tip of the mouth to the tip of the notochord), eye diameter (ED), body depth (BD, depth immediately posterior to the pectoral bud), head length (HL, from the tip of the snout to the opercular margin) and myotome height (MH, height immediately posterior to the anus) (Fig.1b) Comparisons were made for each measurement, at a daily scale, between fed and unfed larvae using Mann^Whitney U-tests The daily mortality rate from yolk absorption time was de¢ned as the percentage of the dead ¢sh to the survivors at the beginning of each day Instantaneous mortality rate (Z) was used to examine mortality among trials (fed vs non-fed) and was de¢ned as: Z (ln N0^ln Nt)t À 1, where N0 and Nt are the numbers of initial and surviving larvae, respectively, and t is the duration of the developmental stage in days Newly hatched larvae [mean Æ standard deviation (SD), 2.97 Æ 0.18 mm NL] had a yolk-sac 0.34 Æ 0.11mm3 in volume (range: 0.19^0.57 mm3) with a lipid droplet of 0.25 Æ 0.04 mm diameter (range: 0.15^ 0.31mm) The intercept of the growth linear model (i.e., the size at hatching) was ¢xed at 2.97 mm The estimated linear model was LL 0.316 A12.97, where LL is the larval length (mm) and A is the age (days) Therefore, larval growth in length during the endogenous period was fast, with an estimated linear rate of 0.316 mm day À (Fig 2a) The yolk-sac was consumed quickly during the ¢rst day and decreased exponentially after hatching, being almost completely absorbed on 4^5 days The estimated exponential model was YSV 0.279e À 0.573 A, where YSV is the yolk-sac volume (mm3) and A is the age (days) (Fig 2b) The lipid droplet was completely absorbed at 10 DPH, and larvae survived until 14 DPH without food (Fig.1c) Starved larvae showed a reduction in growth in notochordal length between and14 DPH compared r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 892^897 893 Starvation e¡ects on larval Seriolella violacea C A Bustos & A Silva Aquaculture Research, 2011, 42, 892^897 Figure (a) Larval growth of Seriolella violacea during endogenous feeding at 16 1C; (b) Larval rate of yolk-sac consumption during endogenous feeding in a warehouse reared at 16 1C LL, larval length; A, age;YSV, yolk-sac volume with normally fed larvae (Fig 3a) Signi¢cant di¡erences (Po0.01) were detected between fed and unfed larvae ¢rst in the MH (9 DPH), BD (11DPH) and NL (12 DPH) (Fig 3e, c and a) respectively ED and HL were less sensitive to unfed conditions, because they showed signi¢cant di¡erences only at the end of the experiment (14 DPH), when all the larvae died (Fig 3a and d) Therefore, starved S violacea larvae were smaller and more slender than normal fed larvae Unfed larvae also showed higher mortality rates (0.419 Æ 0.367 day À 1) than larvae fed with rotifers (0.320 Æ 0.324 day À 1), particularly up to DPH; at DPH unfed larvae showed a reduction in mortality (Fig 3f), coinciding with the reduction in MH (Fig 3e) Because mortality estimation data did not show normality (Shapiro^Wilks test, W 0.78, Po0.001), comparisons were made using the Mann^ Whitney U-test A non-parametric test showed significant di¡erences in mortality estimations between fed and unfed larvae (U 2702, P 0.046) Several aspects of the early life history of S violacea emerged from our study First, during endogenous feeding, larvae grows fast in length (0.316 mm day À 1), but when the yolk-sac is exhausted and exogenous feeding starts, larvae reduce their growth in length and increase the BD Second, at 16 1C, starved larval S violacea can survive at least more week after yolk absorption Third, MH is the ¢rst morphological feature a¡ected during starvation, and is probably the source of energy for the survival of larval S violacea when food is not available 894 Larval growth during early development, particularly before ¢rst feeding, is slow in several taxa (Paralabrax maculatofasciatus, o0.05 mm day À 1, Penìa & Dumas 2005; Merluccius australis, 0.03^0.04 mm day À 1, Bustos et al 2007) Other species, particularly with small larvae at hatching (i.e., Sparus aurata, $ 0.1mm day À 1, Yufera et al 1993), show a rapid early development at similar water temperatures Estimations of larval growth in length were large for S violacea at 16 1C and may be a strategy to increase chances of ¢rst feeding after yolk-sac absorption, which compensates the high mortality estimates (Fig 3f) during early ontogeny The lipid droplet was completely absorbed by fed and unfed larvae at 10 DPH Probably a combination of fatty acids from the lipid droplet and proteins from musculature kept unfed larvae alive until14 DPH On the contrary,Yufera et al (1993) found that unfed larval S aurata were unable to completely absorb the lipid droplet, and it remains in dying larvae Although simple, morphological measurements provide a rapid, real and signi¢cant representation of the changes caused by the lack of food in the early life stages of ¢sh, there is also a disadvantage in the morphological method due to the lack of validation of the criteria derived from laboratory studies for use with natural populations The starvation due to the non-availability of feed or delayed initial feeding can have an adverse e¡ect on the growth of larval ¢sh, and it is very important to understand the appropriate time to introduce the ¢rst feeding (Gawlicka et al 2000; Dou, Masuda, Tanaka & Tsukamoto 2005; r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 892^897 Aquaculture Research, 2011, 42, 892^897 Starvation e¡ects on larval Seriolella violacea C A Bustos & A Silva Figure Temporal variability in the morphometric measurements of fed and unfed larval Seriorella violacea reared at 16 1C Asterisks indicate from which day signi¢cant di¡erences (Po0.01) between fed and unfed groups were detected (a) Notochord length (NL), (b) eye diameter (ED), (c) body depth (BD), (d) head length (HL), (e) myotome height (MH) and (f) daily mortality Bars indicate one standard deviation (SD) Kailasam, Thirunavukkarasu, Selvaraj & Stalin 2007) In the case of palm ru¡ larvae, we observed that DPH is the appropriate time to introduce the ¢rst feeding and that starved individuals showed reduced growth in length and their trunk musculature was a¡ected Similarly, Dou et al (2002) reported for Japanese £ounder larvae Paralichtys olivaceus that MH and BD decreased signi¢cantly between unfed and fed larvae The utilization of body tissues as an energy source produces considerable alterations in the digestive tract and adjacent glands in starved larvae of marine species such as larval sardine Sardinops sagax (Uriarte & Balbont|¤ n 1987), S aurata (Yufera et al 1993) and Seriola lalandi (Chen et al 2007), among others Starvation usually results in shrinkage of enterocytes and a reduction in the height of enterocyte cells both in the midgut and in the hindgut (Theilacker & Watanabe 1989; Bisbal & Bengtson 1995), lack of supranuclear vacuoles in the hindgut (Oozeki, Ishii & Hirano 1989; Crespo, Marin de Mateo, Santamaria, Sala, Grau & Pastor 2001), degeneration of the cellular structure both in the liver and in the pancreas (O’Connell 1976; Kjorsvik, van der Meeren, Kryvi, Arn¢nnson & Kvenseth 1991) and disorder of trunk musculature (Bisbal & Bengtson 1995; Gisbert, r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 892^897 895 Starvation e¡ects on larval Seriolella violacea C A Bustos & A Silva Conklin & Piedrahita 2004) Coincidently, the ¢rst effect observed in larval S violacea occurred in the musculature or MH, considering all the observations made in the study, because probably in other tissues an early damage occurred In fed larvae, there was a continuous increase in MH, but in unfed larvae the data were signi¢cantly lower (Fig 3e) A similar pattern has been observed in unfed larval Sparus aurata (Yufera et al.1993) and Paralicthys olivaceus (Dou et al 2002) Typical damages in starved larvae are severe muscle ¢bre separation and lack of intermuscular tissue, which directly a¡ect MH and BD Additionally, the reduction in the musculature due to starvation after ¢rst feeding may a¡ect the swimming capabilities of ¢sh larvae, both for escape predators and for pursuit preys (Yin & Blaxter 1987; Skiftesvik 1990), reason for which in a culture it is predictable that these larvae are destined to die Knowledge of the early life history of S violacea is scarce and this ¢rst e¡ort will help to understand the larval dynamics under hatchery conditions Biochemical and histological examination of the larvae during starvation was needed to validate the practicability and applicability of the morphological indicators suggested by this study Future investigations are necessary to answer questions about the variability among batches during the reproductive season, reduce mortality and optimize its larviculture Acknowledgments The authors thanks two anonymous referees for suggestions on an early version of this manuscript, and Mauricio F Landaeta and Marcia Oliva for their help in the laboratory and data analysis This investigation was funded by project FONDEF DO2I1161 ‘Research and development of a base technology for culture and production of the palm ru¡ (Seriolella violacea)’ adjudicated to Dr A Silva During the writing of the manuscript, CAB was partially funded by a CONICYT doctoral scholarship References Aristizabal E., SuaŁrez J.,Vega A & Bargas R (2009) Egg and larval quality assessment in the Argentinean red porgy (Pagrus pagrus) Aquaculture 287, 329^334 Bisbal G.A & Bengtson D.A (1995) E¡ects of delayed feeding on survival and growth of summer £ounder Paralichthys dentatus larvae Marine Ecology Progress Series 121, 301^306 Blaxter J.H.S & Ehrlich K.F (1974) Changes in behaviour during starvation of herring and plaice larvae In: The 896 Aquaculture Research, 2011, 42, 892^897 Early Life History of Fish (ed by J.H.S Blaxter), pp 575^ 588 Springer-Verlag, Berlin, Germany Bolasina S., Pe¤rez A & Yamashita Y (2006) Digestive enzimes activity during ontogenetic development and e¡ect of starvation in Japanese £ounder, Paralichthys olivaceus Aquaculture 252, 503^515 Bustos C.A & Landaeta M.F (2005) Development of eggs and early larvae of the southern hake Merluccius australis, reared under laboratory conditions Gayana 69, 402^408 (in Spanish) Bustos C.A., Landaeta M.F., Bay-Schmith E., Lewis R & Moraga X (2007) E¡ects of temperature and lipid droplet adherence on mortality of hatchery-reared southern hake Merluccius australis larvae Aquaculture 270, 535^540 Cavalin F.G & Weirich C.R (2009) Larval performance of aquacultured Florida pompano (Trachinotus carolinus) fed rotifers (Brachionus plicatilis) enriched with selected commercial diets Aquaculture 292, 67^73 Chen B.N., Qin J.G., Carragher J.F., Clarke S.M., Kumar M.S & HutchinsonW.G (2007) Deleterious e¡ects of food restrictions in yellowtail king¢sh Seriola lalandi during early development Aquaculture 271, 326^335 Crespo S., Marin de Mateo M., Santamaria C.A., Sala R., Grau A & Pastor E (2001) Histopathological observations during larval rearing of common dentex Dentex dentex L (Sparidae) Aquaculture 192, 121^132 Dou S.Z., Masuda R.,Tanaka M & Tsukamoto K (2002) Feeding resumption, morphological changes and mortality during starvation in Japanese £ounder larvae Journal of Fish Biology 60,1363^1380 Dou S.Z., Masuda R., Tanaka M & Tsukamoto K (2005) Effects of temperature and delayed initial feeding on the survival and growth of Japanese £ounder larvae Journal of Fish Biology 66, 362^377 Ehrlich K.F., Blaxter J.H.S & Pemberton R (1976) Morphological and histological changes during the growth and starvation of herring and plaice larvae Marine Biology 35, 105^118 Gawlicka A., Parent B., Horn M.H., Ross N., Opstad I & Torrissen O.J (2000) Activity of digestive enzymes in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus): indication of readiness for ¢rst feeding Aquaculture 184, 303^314 Gisbert E., Conklin D.B & Piedrahita R.H (2004) E¡ects of delayed ¢rst feeding on the nutritional condition and mortality of California halibut larvae Journal of Fish Biology 64, 116^132 Iannacone J (2003) Three metazoan parasites of palm ru¡ Seriolella violacea Guichenot (Pisces, Centrolophidae), Callao, Peru Revista Brasileira de Zoologia 20, 257^260 (in Spanish) Kailasam M.,Thirunavukkarasu A.R., Selvaraj S & Stalin P (2007) E¡ect of delayed initial feeding on growth and survival of Asian sea bass Lates calcarifer (Bloch) larvae Aquaculture 271, 298^306 Kjorsvik E., van der Meeren T., Kryvi H., Arn¢nnson J & Kvenseth P.G (1991) Early development of the digestive r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 892^897 Aquaculture Research, 2011, 42, 892^897 Starvation e¡ects on larval Seriolella violacea C A Bustos & A Silva tract of cod larvae, Gadus morhua L., during start-feeding and starvation Journal of Fish Biology 38, 1^15 Leu M.Y., Liou C.H.,Wang W.H.,Yang S.D & Meng P.J (2009) Natural spawning, early development and ¢rst feeding of the semicircle angel¢sh [Pomacanthus semicirculatus (Cuvier, 1831)] in captivity Aquaculture Research 40, 1019^1030 Moran D., Smith C.K., Gara B & Poortenaar C.W (2007) Reproductive behaviour and early development in yellowtail king¢sh (Seriola lalandi Valenciennes 1833) Aquaculture 262, 95^104 NakagawaY & Miyashita S (2008) Prey selectivity of nakedhead large-eye bream (Gymnocranius griseus,Temminck & Schlegel 1844) larvae on natural plankton at ¢rst feeding Aquaculture Research 39, 217^220 O’Connell C (1976) Histological criteria for diagnosing the starving condition in early post yolk sac larvae of the northern anchovy, Engraulis mordax Girard Journal of Experimental Marine Biology and Ecology 25, 285^312 Oozeki Y., Ishii T & Hirano R (1989) Histological study of the e¡ects of starvation on reared and wild-caught larval stone £ounder, Kareius bicoloratus Marine Biology100, 269^275 Penìa R & Dumas S (2005) E¡ect of delayed ¢rst feeding on development and feeding ability of Paralabrax maculatofasciatus larvae Journal of Fish Biology 67, 640^651 Planas M & Cunha I (1999) Larviculture of marine ¢sh: problems and perspectives Aquaculture 177, 171^190 Ross L.G., Mart|¤ nez Palacios C.A & Morales E.J (2008) Developing native ¢sh species for aquaculture: the interacting demands of biodiversity, sustainable aquaculture and livelihoods Aquaculture Research 39, 675^683 Skiftesvik A.B (1990) Changes in behaviour at onset of exogenous feeding in marine ¢sh larvae Canadian Journal of Fisheries and Aquatic Science 49, 1570^1572 Theilacker G.H & Watanabe Y (1989) Midgut cell height de¢nes nutritional status of laboratory raised larval northern anchovy, Engraulis mordax Fishery Bulletin 87, 457^469 Uriarte I & Balbontin F (1987) Characterization of the starving condition of the sardine Sardinops sagax musica, larvae (Pisces, Clupeiformes) by morphometric and histological criteria Revista de Biolog|¤ a Marina 23,77^106 Yin M.C & Blaxter J.H.S (1987) Escape speeds of marine ¢sh larvae during early development and starvation Marine Biology 96, 459^468 Yufera M., Pascual E., PoloA & Sarasquete M.C (1993) E¡ect of starvation on the feeding ability of gilthead seabream (Sparus aurata L) larvae at ¢rst feeding Journal of Experimental Marine Biology and Ecology 169, 259^272 Keywords: yolk, lipid droplet, morphometrics, Chile r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 892^897 897 [...]... Marine Science and Technology to CAS r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 746^753 751 Sex determination in Odontesthes hybrids J Inazawa et al Aquaculture Research, 2011, 42, 746^753 References Chevassus B (1983) Hybridization in ¢sh Aquaculture 33, 245^262 Cnaani A & Levavi-Sivan B (2009) Sexual development in ¢sh, practical applications for aquaculture Sexual Development 3, 164^175... (1996) Sex di¡erentiation and hormonal feminization in pejerrey Odontesthes bonariensis Aquaculture 139, 31^45 Strˇssmann C.A., Saito T., Usui M.,Yamada H & Takashima F (1997) Thermal thresholds and critical period of ther- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 746^753 Aquaculture Research, 2011, 42, 746^753 molabile sex determination in two atherinid ¢shes, Odontesthes bonariensis... Michigan, 596pp Goddard S (1996) Feed Management in IntensiveAquaculture Chapman & Hall, NewYork, 194 pp Greenberg A.E., Clesceri L.S & Eaton A.D (1992) Standard Methods for the Examination of Water and Wastewater, 18thh ed APHA,Washington, DC, USA 762 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 Aquaculture Research, 2011, 42, 754^763 Growth of Patagonian pejerrey in net cages J... (Provincias de Neuque¤ n y R|¤ o Negro) Informacio¤n BaŁsica Secretar|¤ a de Agricultura, Ganader|¤ a, Pesca yAlimentos (SAGPyA), 52pp r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 763 Aquaculture Research, 2011, 42, 764^775 doi:10.1111/j.1365-2109 .2011. 02803.x Hatching rate and larval growth variations in Pseudoplatystoma punctifer : maternal and paternal effects Jesu¤s Nu¤nìez1,2,... genotype from the ¢rst Obo,  Oha< cross Gonads of sub-adults from this cross were also examined by histology as described above r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 746^753 747 Sex determination in Odontesthes hybrids J Inazawa et al Aquaculture Research, 2011, 42, 746^753 Results DNA extraction and polymerase chain reaction (PCR) analysis of genotypic sex A sex-linked SNP marker (Koshimizu... number of individuals bearing the X and Y chromosome of O hatcheri respectively Note: not all samples available were sex genotyped ND, not determined 748 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 746^753 Aquaculture Research, 2011, 42, 746^753 Sex determination in Odontesthes hybrids J Inazawa et al Table 3 Sex ratios, allocation of Odontesthes hatcheri X and Y chromosomes and survival... weighing 4.14 Æ 1.3 g (mean Æ SD) (lot 1) was transferred to one rearing cage on March 2006 A second lot of 4500 juveniles r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 755 Growth of Patagonian pejerrey in net cages J P Hualde et al Aquaculture Research, 2011, 42, 754^763 weighing 2.4 Æ 1.3 g (lot 2) was transferred to the other cage on May 2006 Fish were transported in a 500 L tank... – – – – – – – – – – – – 93.0 452.0 136.0 136.0 184.0 19.2 18.8 6.6 79.0 440.0 95.0 169.0 215.0 17.9 24.6 1.6 ^, not indicated in the commercial formula r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 Aquaculture Research, 2011, 42, 754^763 Growth of Patagonian pejerrey in net cages J P Hualde et al (see Table 3) for determination of the amount of experimental feed and natural prey... 80 Summer (2) Fall (3) Winter (1) Number of samplings per season is shown between parentheses UA, unidenti¢ed arthropods r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 757 Growth of Patagonian pejerrey in net cages J P Hualde et al Aquaculture Research, 2011, 42, 754^763 ing, gonads dark and reduced The weight increment in male and female gonads was evaluated through the gonadosomatic... in the Ramos Mex|¤ a reservoir during the experiment 758 Figure 2 Growth of two groups of Patagonian pejerrey in net cages in the Ramos Mex|¤ a reservoir r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 754^763 Aquaculture Research, 2011, 42, 754^763 Growth of Patagonian pejerrey in net cages J P Hualde et al feed supplied to lot1was replaced for the experimental pejerrey feed, mortality was