Aquaculture Research, 2009, 41, 129^134 doi:10.1111/j.1365-2109.2009.02314.x

Spawning induction of pejerrey bonariensis in captivity using sustained-release gonadotropin releasing hormone agonist implants

LeandroA. Miranda & Gustavo M. Somoza Laboratorio de Ictio¢siolog|¤a yAcuicultura, Instituto de Investigaciones Biotecnolo¤gicas-Instituto Tecnolo¤gico de Chascomu¤s (IIB-INTECH) (CONICET-UNSAM), Buenos Aires, Argentina

Correspondence: L A Miranda, Laboratorio de Ictio¢siolog|¤a y Acuicultura, Instituto de Investigaciones Biotecnolo¤gicas-Instituto Tec- nolo¤gico de Chascomu¤s (IIB-INTECH) (CONICET-UNSAM), Camino de Circunvalacio¤n Laguna Km, 6 (B7130IWA) Chascomu¤s, Buenos Aires, Argentina. E-mail: [email protected]

Abstract nos Aires, Argentina. Its typical habitats are ‘lagu- nas’, the most common lentic water bodies of the The aim of this study was to induce and synchronize Argentinean , which frequently have salinity spawning of pejerrey Odontesthes bonariensis (Valen- levels from 1 to 24 g L 1 (Toresani, Lo¤pez & Go¤mez ciennes, 1835), using gonadotropin releasing hor- 1994). It has been reported recently that pejerrey mone agonist (GnRHa) implants. In the ¢rst has better survival and growth performance in experiment, the ovarian condition was assessed by brackish water, and the positive e¡ects of salinity on ovarian biopsies and the measurement of the genital ionic and osmoregulatory balance and mitigation of pore width (GPW). Females having the leading clutch stress response were also demonstrated (Somoza, of oocytes with a diameter of around 800^900 mm Miranda, Berasain, Colautti, Remes-Lenicov & and a GPW between 4.5 and 5.5 mm were treated Strˇssmann 2008). with GnRHa implants. Eighty per cent of females Because of the economic movement generated by spawned between 2 and 9 days after treatment, 12 pejerreyas a game ¢sh and also because of the quality days earlier than 20% of the ¢sh in the control group of its £esh, this species has been introduced in other that presented signs of spawning activity. In order to provinces of Argentina as well as in other countries avoid any possible ovarian injury and/or stress by the (Somoza et al. 2008). For this reason, it has been con- catheterization procedure, in a second experiment, fe- sidered that pejerrey can be an alterna- males were selected only by visual inspection of the tive for ¢sh production in the Argentinean pampas, abdomen and GPW measurement. As in experiment where more than 2 million square hectometre of 1,80%of femalesspawnedbetween 2 and8 daysafter water bodies have been described (Reartes 1995; Lo¤- treatment, 8 days earlier than 30% of the ¢sh that pez, Baigu¤n, Iwaskiw, Del¢no & Pad|¤n 2001). In spite spawned in the control group. In both experiments, of this, and although some experimental trials have fertilization and hatching success were similar be- been performed, pejerrey culture has not been fully tween control and GnRHa-treated groups. These re- developed in Argentina (Miranda, Berasain, Velasco, sults clearly demonstrated that GnRHa implantation Shirojo & Somoza 2006; Somoza, Miranda, Guilgur & can advance and synchronize ovulation and spawn- Strobl-Mazzulla 2006; Somoza et al. 2008). ing in pejerrey without a¡ecting egg quality. The pejerrey is a multiple spawnner with a marked seasonal reproductive cycle under natural condi- Keywords: GnRHa implants, induced spawning, tions, with a major spawning period during spring pejerrey, reproduction and a minor one in autumn, with water temperatures between 18 and 20 1C (Calvo & Morriconi 1972; Introduction Strˇssmann 1989). The spawning events and the The pejerrey Odontesthes bonariensis is an inland length of the intervals between successive spawn- water atherinopsid ¢sh native to the Province of Bue- ings di¡ered considerably between and within

r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd 129 Induced spawning of pejerrey by GnRHa implants L A Miranda & G M Somoza Aquaculture Research, 2009, 41, 129^134 females. However, the number of eggs of the ¢rst In pejerrey,it has already been reported that it was spawning of a single female was estimated to be possible to induce spermiation by environmental and o4000 eggs in experimental ¢sh with a standard hormonal treatments without a¡ecting sperm con- length of 25^26cm (Strˇssmann 1989). Pejerrey’s centration and motility (Miranda, Escaray, Bustin- eggs are spherical, demersal and they have a dia- gorry & Somoza 2001; Miranda, CassaraŁ , & Somoza meter of 1.6 mm approximately.They also have a ser- 2005). These treatments included the increase of the ies of ¢laments that allow them to attach to di¡erent light phase duration, hCG, heterologous pituitary ex- substrates (GonzaŁ lez Regalado & Mastrarrigo1954). tracts and GnRHa injection. However, no attempts It has also been reported that the pejerrey repro- have been reported to induce spawning in pejerrey ductive period can be extended in captivity by ma- females. nipulating the photoperiod and temperature The aim of the present study was to optimize pejer- (Strˇssmann1989; Miranda et al. 2006). For example, rey reproduction in captivity using sustained-release it was possible to obtain reproductive activity almost GnRHa delivery systems to induce and synchronize throughout the year if the water temperature was ovulation and spawning, and thus provide basic in- maintained around17 1C (Toda,Tonami,Yasuda & Su- formation to establish a spawning protocol to obtain zuki 1995; Miranda et al. 2006). However, due to the eggs and larvae in a massive form. marked asynchrony and the relatively low fecundity of this species, a large number of mature ¢sh would be necessary to obtain enough eggs for the operation Materials and methods of a hatchery (Somoza et al. 2008). Experiment 1 Di¡erent hormones have been tested in many ¢sh species to induce ovulation and spawning in captiv- Sixty captive-reared adult pejerrey with external ity, including pituitary extracts, piscine gonadotro- signs of active reproductive status (spermiating pins and human chorionic gonadotropin (hCG). males and females with a prominent abdominal re- Furthermore, superactive agonists of gonadotropin gion) were selected from a broodstock kept at the out- releasing hormones (GnRHa) have been used suc- doors aquatic facilities of Instituto de Investigaciones cessfully to induce spawning in many species Biotecnolo¤gicas-Instituto Tecnolo¤gico de Chascomu¤s, (Mylonas & Zohar 2001) including, for example Chascomu¤s, Argentina. Females were anaesthetized di¡erent salmonids (Mylonas, Hinshaw & Sullivan with benzocaine, and the following measurements 1992), Sparus auratus (Linnaeus, 1758, Zohar 1989), were taken: standard length, total weight and the Pseudopleuronectes americanus (Walbaum, 1792, Har- genital pore width (GPW,Table 1 and Fig. 1) using a min & Crim 1992), Morone saxatilis (Walbaum, 1792, digital calibre (0.1mm). The ovarian biopsies were s Mylonas,Tabata, Langer, & Zohar1995) and Limanda performed using a 2 mm internal diameter Silastic ferruginea (Storer, 1839, Larsson, Mylonas, Zohar, & catheter (Dow Corning, Midland, MI, USA) intro- Crim 1997). However, due to the relatively short duced into the genital pore. For each female, the dia- half-life of GnRHa, some ¢sh require multiple GnRHa meters of at least 10 of the largest oocytes were injections for an e¡ective treatment (Mylonas, Sigela- measured using a stereoscope. Only those females ki, Divanach, Mananos, Carillo & Afonso-Polyviou with oocytes with diameters between 800 and 2003). For this reason, the use of sustained-release, 900 mm were selected for this experiment (Fig.1).This GnRHa delivery systems (implants, microspheres, criterion was used by Strˇssmann (1989) because it osmotic pumps, etc.) to stimulate long-term release represents oocytes at the end of vitellogenesis/early of luteinizing hormone, which is the gonadotropin ¢nal oocyte maturation (FOM). Ten selected females responsible for maturation, has been proven to were intraperitoneally implanted with commercial be e¡ective in inducing ovulation and spawning in pellets containing 75 mg of sGnRHa (salmon type di¡erent species (Mylonas & Zohar 2001; Mylonas, GnRHa, OvaplantTM, Syndel, Vancouver, Canada). Papandroulakis, Smboukis, & Divanach 2004; These females were placed together with 10 un- Agulleiro, Anguis, Canavate, Mart|¤nez-Rodr|¤guez, treated males in a 3000-L indoor tank (implanted Mylonas & CerdaŁ 2006; Corriero, Medina, Mylonas, group). Another10 females were subjected to a sham Abascal, De£orio, Arago¤n, Bridges, Santamaria, operation and placed in a similar tank with 10 un- Heinisch, Vassallo-Agius, Belmonte, Fauvel, Garcia, treated males (control group). The ¢sh were kept un- Gordin & De Metrio 2007; Ibarra-Castro & Duncan der simulated natural light conditions (14 h light:10h 2007). dark) with water temperature maintained at

r 2009 The Authors 130 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 129^134 Aquaculture Research, 2009, 41, 129^134 Induced spawning of pejerrey by GnRHa implants L A Miranda & G M Somoza

Table 1 Pre-experimental data (means SE) of pejerrey before treatment with GnRHa implants

N SL (cm) W (g) GPW (mm) OD (lm)

I1 10 36.1 1 463.4 37.5 5.1 0.06 880 8

C1 10 35.8 0.7 448.7 21.7 4.9 0.09 890 11

I2 10 35.4 0.2 477.1 6.3 4.9 009 NM

C2 10 36.8 0.8 468.9 15.9 4.8 008 NM

SL, standard length; W, weight; GPW, genital pore width; OD, oo- cyte diameter; N, number of females; NM, not measured. I1 and

C1 (implanted and control females in experiment 1), I2 and C2 (implanted and control females in experiment 2).

Figure 2 Photograph of late vitellogenic oocytes from pejerrey Odontesthes bonariensis collected by ovarian biopsy.Scale bar 510 0 mm.

Spawning and egg quality

Every morning after implantation and for 20 days, each tank was checked for the presence of spawned eggs. They were collected using nets located at the out£ow of the tanks. The relative fecundity was cal- culated by dividing the total number of eggs collected in each group by the total weight of presumed spawned females. When the experiment concluded, it was possible to infer the number of spawned fe- Figure 1 Photograph showing the genital pore width males by observing the dilated genital pore with (GPW) of pejerrey female Odontesthes bonariensis. signs of blood and with a £accid abdominal region. Every day, the eggs were counted using a volumetric 18.1 0.6 1C using an open-£ow system with brack- measure (1mL ffi 250 eggs) and samples of 100 eggs ish water (15g L 1 salinity as NaCl) at an in£ow rate were observed under a stereoscope in order to deter- of about 6 L min 1.The were fed three times mine the fertilization success. The eggs were incu- a day with arti¢cial pelleted food. The experiment bated in £ow-through freshwater incubators until was performed during October^November 2007. hatching. Each batch of eggs collected was incubated in a se- parate glass jar until hatching in a £owing-water sys- Experiment 2 tem at temperatures between 18 and 19 1C and at a Taking into account the results obtained in the ¢rst salinity of 5 g L 1. In all cases, dead eggs or larvae experiment and in order to avoid any possible ovar- were removed and counted in order to determine ian injury and/or stress by the catheterization proce- hatching success. Fertilization and hatching success dure, a second trial was performed using only the results of the implanted or the control group were GPW measure (Fig. 2) as the criterion of gonad ma- analysed using one-wayanalysis of variance at a level turation. Only those females with a GPW between of signi¢cance of Po0.05. 4.5 and 5.5 mm were used in this experiment. Similar to experiment1, selected females were anaesthetized, measured, implanted or sham operated and placed together with spermiating males as described above Results (Table 1). The ¢sh were kept under simulated natural Experiment 1 light conditions (15h light:9 h dark) with the water temperature at 19.4 0.4 1C. The experiment was The ¢rst batch of spawned eggs was found 2 days performed during January^February 2008. after GnRHa implantation (Fig. 3). During the ¢rst 10

r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 129^134 131 Induced spawning of pejerrey by GnRHa implants L A Miranda & G M Somoza Aquaculture Research, 2009, 41, 129^134

days, 41250 eggs were collected from eight out of the only 30% of the females showed signs of spawning, 10 females of the implanted group. The relative fe- producing 21500 eggs with a relative fecundity of cundity of this group was calculated as 15281eggs kg 1. There were no signi¢cant di¡er- 11114 eggs kg 1. During the same period, no spawn- ences between treatments in fertilization and hatch- ing was observed in the control group. However, ing success (Table 2). 10750 eggs were collected after day 14, with only two out of 10 females showing signs of spawning. The relative fecundity in the control group was Discussion 11984 eggs kg 1. Females implanted with GnRHa spawned 12 days earlier than control females (Table The establishment of pejerrey aquaculture requires a 2, Fig. 3). No signi¢cant di¡erences were found in massive production of larvae, and the e⁄cient pro- fertilization and hatching success between treat- duction of seed would allow reducing the cost of ments (Table 2). hatchery operations, thus increasing pro¢tability during grow-out. As a ¢rst step, it was necessary to maintain a pejerrey broodstock in captivity and then control their reproductive activity (Miranda et al. Experiment 2 2006). So far, pejerrey-fertilized eggs have been ob- Gonadotropin releasing hormone agonist-implanted tained with large numbers of broodstock because of females began to spawn 2 days after they were im- the low female fecundity (up to a few thousand egg- planted, while in the control group spawning started s individual^1spawning act^1) and spawning asyn- after 10 days (Fig. 4). In the implanted group, the chrony among females (Strˇssmann 1989; Miranda spawning period lasted for 8 days, with 80% of fe- et al. 2006; Somoza et al. 2008). males showing signs of spawning (Table 2). A total The present results have clearly demonstrated that of 60 000 eggs were collected and the relative fe- GnRHa implantation can be used to advance and cundity was 15707 eggs kg 1. In the control group, synchronize ovulation and spawning in pejerrey.

Figure 3 Daily egg production by pejerrey Odontesths bo- Figure 4 Daily egg production by pejerrey Odontesths bo- nariensis females implanted with a gonadotropin releas- nariensis females implanted with a gonadotropin releas- ing hormone agonist delivery system (Ovaplant) or sham ing hormone agonist delivery system (Ovaplant) or sham operated. Experiment1. operated. Experiment 2.

Table 2 Spawning performance and egg quality from pejerrey treated with GnRHa implants

I1 C1 I2 C2

First spawn (days) 2 14 2 10 Fecundity (eggs) 41 250 10 750 60 000 21 500 Presumed spawned females (%) 80 20 80 30 Relative fecundity (eggs kg 1 spawned female) 11 114 11 984 15 707 15 281 Fertilization (%) 57.71 25 (8) 54.5 3.6 (3) 46.43 28 (8) 48.5 19 (3) Hatching (%) 42 1.3 (8) 46 3.2 (3) 39 2.6 (8) 41 3.4 (3)

I1 and C1 (implanted and control females in experiment 1), I2 and C2 (implanted and control females in experiment 2). No statistical di¡erences were observed in fertilization and hatching percentages (Po0.05). Experimental N in brackets.

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Although the dose used was high, and lower doses panikos & Dunham 2007). Moreover, the e⁄cacy of might have been e¡ective, the treatment was selected GnRH analogues has also been shown to be in£u- because the implants are easily available commercial enced by water temperature (Harmin & Crim 1992; products. The same dose was used to induce spawn- King & Pankhurst 2004). ing in the spotted rose snapper Lutjanus guttatus,and It is important to note that in both experiments, it proved to be the best compared with lower and fertilization and hatching success were similar higher doses (Ibarra-Castro & Duncan 2007). among the control and implanted groups, stressing It is well known that treatment with a GnRHa the fact that this treatment does not a¡ect egg quality. sustained-release delivery system has been used This is in agreement with previous results on di¡er- successfully to induce FOM, ovulation and spawn- ent species (Zohar & Mylonas 2001). ing, as well as spermiation in many di¡erent teleost In summary,the present results demonstrated that species (Mylonas & Zohar 2001; Mylonas et al.2004; it is possible to induce spawning of pejerrey using Agulleiro et al. 2006; Corriero et al. 2007; Ibarra- GnRHa implants without a¡ecting egg quality. To Castro & Duncan 2007). However, a key issue for the best of our knowledge, this is the ¢rst report on the successful induction of spawning in cultured the use of GnRHa on an Atheriniform species. ¢sh is the accurate diagnosis of the gonadal stage Although much work is necessary in order to support before hormonal treatment. Thus, in any induced a true and sustainable aquaculture industry for pe- spawning protocol, the hormone should be admi- jerrey, the development of induced maturation and nistered when the ¢sh have completed vitellogen- synchronization of spawning methods are a valuable esis (Mylonas, Woods III & Zohar 1997; Corriero tool to optimize seed production, which would make et al. 2007). As has already been reported, pejerrey hatching operations more e⁄cient. females are characterized by a leading clutch of oo- cytes between 800 and 900 mm at the end of vitello- genesis or beginning of FOM (Strˇssmann1989). For Acknowledgments this reason, females with these characteristics were selected in the present study. However, because This work was supported by grants provided to Gus- ovarian biopsies can produce ovarian injury and/or tavo Somoza (from CONICET, PIP #5425 and AMP- stress, and a well-trained sta¡ is necessary to per- CyT PICTR 528). We are also indebted to Jim Powell form this task, the GPW was used as the criterion (Syndel, Canada) for the donation of some of the im- for readiness for hormonal treatment. The present plants used in this study. results showed that GnRHa implantation not only induced successful spawning in pejerrey females se- lected based on ovarian biopsy (Experiment 1) but References also in females selected usingonly the GPW (Experi- Agulleiro M.J., AnguisV., Canavate J.P., Mart|¤nez-Rodr|¤guez ment 2). G., Mylonas C.C. & Cerda J. (2006) Induction of spawning It is important to note that after experimental time, Ł of captivereared Senegal sole (Solea senegalensis) using dif- new batches of eggs were found at day 35 in the tank ferent delivery systems for gonadotropin-releasing hor- of the implanted group and at day 48 in the control mone agonist. Aquaculture 257,511^524. group (Experiment 1). This ¢nding could represent a Calvo J. & Morriconi E.R. (1972) Feno¤menos reproductivos en second spawning event. However, because of the ex- el pejerrey Basilichthys bonariensis. III Estudio de la fecun- perimental conditions, it was not possible to assure didad, e¤poca y nu¤mero de desoves. Anales de la Sociedad that some females had spawned twice. Cient|¤¢ca Argetina 93,75^84. Other di¡erences were found between the two ex- Corriero A., Medina A., Mylonas C.C., Abascal F.J., De£orio periments. For example, in the second experiment the M., Arago¤n L., Bridges C.R., Santamaria N., Heinisch G., relative fecundity was higher. This may have been Vassallo-Agius R., Belmonte A., Fauvel C., Garcia A., Gordin H. & De Metrio G. (2007) Histological study of the due to di¡erences in the experimental temperature, e¡ects of treatment with gonadotropin-releasing hor- which was somewhat higher in the second trial. It is mone agonist (GnRHa) on the reproductive maturation known that each ¢sh species has an optimal tem- of captive-reared Atlantic blue¢n tuna (Thunnus thynnus perature range for spawning and when an is L.). Aquaculture 272, 675^ 686. induced to spawn within that temperature range, GonzaŁ lez Regalado T. & MastrarrigoV.(1954) Piscicultura. El small di¡erences in temperature may delay or accel- pejerrey. Ministerio de Agricultura y Ganader|¤a, Buenos erate ovulation (Phelps, Hastey, Pendetar, Linley, Pa- Aires, Publicacio¤nMiscelaŁ nea no. 268,53pp (in Spanish). r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 129^134 133 Induced spawning of pejerrey by GnRHa implants L A Miranda & G M Somoza Aquaculture Research, 2009, 41, 129^134

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