BERNARD-ANTONIN DUPONT CYR
COMPRESSION DU CYCLE REPRODUCTEUR DU LOUP DE MER (ANARHICHAS LUPUS ET A. MINOR)
Mémoire présenté à la Faculté des études supérieures et postdoctorales de l’Université Laval dans le cadre du programme de Maîtrise en Sciences Animales pour l’obtention du grade de Maître ès sciences (M.Sc.)
DÉPARTEMENT DE SCIENCES ANIMALES FACULTÉ DE L’AGRICULTURE ET DE L’ALIMENTATION UNIVERSITÉ LAVAL QUÉBEC
2012
© Bernard-Antonin Dupont Cyr, 2012
Résumé Le développement aquacole du loup de mer (Anarhichas minor et A. lupus) est limité par la disponibilité en juvénile de qualité et par les difficultés d'instaurer une production annuelle constante. L'objectif de cette étude est d’utiliser la photopériode pour comprimer le cycle reproducteur du loup de mer (loup atlantique (A. lupus), loup tacheté de souche Québécoise (A. minor QC) et de loup tacheté de souche norvégienne (A. minor NW)). Deux photopériodes ont été testées : une naturelle simulée de 12 mois (SNP) et une comprimée sur 8 mois (CP). L'expérience s’est déroulée de février 2006 à décembre 2007. Des échantillons de sang ainsi que des mesures de croissance (masse et longueur) ont été prélevés chaque mois. La maturation a été suivie mensuellement par les teneurs des stéroïdes sexuels (11-kétotestostérone et 17-β-oestradiol) dosés avec des ELISA compétitifs. En 2007, la fraie du groupe CP a été devancé de 3 mois chez les A. lupus, de 5 mois chez les A. minor QC et de 6 mois chez les A. minor NW comparativement au groupe SNP. La réalisation de ce projet a permis d’instaurer une ponte hors saison au printemps et l’observation d'un dimorphisme sexuel de croissance avantageant les mâles (A. lupus 21.9%, A. minor QC 26.6% et A. minor NW 17.2%) pour les deux espèces en présence.
ii
Abstract Wolffishes (Anarhichas minor and A. lupus) aquaculture development is limited by the supply of quality juvenile and difficulties to establish a year-round juvenile production. The aim of this study was to compress reproduction cycle of two wolffish species (Common wolfish (A. lupus), Spotted wolffish Canadian strain (A. minor QC) and spotted wolffish Norwegian strain (A. minor NW)) by photoperiod manipulation. Two photoperiod treatments were tested: a 12 months simulated natural photoperiod (SNP) and an 8 months compressed photoperiod (CP). Experiments were conducted from February 2006 to December 2007. Blood samples and growth measurement (body mass and total length) were collected monthly. Sexual maturation was assessed monthly by plasma steroids levels (11-ketotestosterone and 17-β-oestradiol) and analyzed by competitive ELISA. In 2007, CP broodstock A. lupus spawned 3 months earlier, A. minor QC spawned 5 months earlier and A. minor NW spawned 6 months earlier compared to SNP. This project enabled the establishment of an off-season spawning in the spring and suggests the presence of a sexrelated growth dimorphism benefiting males (A. lupus 21.9%, A. minor QC 26.6% et A. minor NW 17.2%).
Avant-Propos Ce projet a été réalisé dans le cadre d'un partenariat entre le Centre Aquacole Marin de Grande-Rivière (opéré par le Ministère de l'Agriculture, de l'Alimentation et des Pêches du Québec), l'Institut Maurice Lamontagne (ministère des Pêches et Océans Canada) et l'Université Laval. Il a été subventionné par les programmes PCRDA et AquaNet. Les travaux se sont échelonnés de 2006 à 2009. Plusieurs personnes ont participé de près ou de loin à la réalisation de ce projet et je veux, ici, les remercier toutes sans exception. Je veux tout d’abord remercier le Dr Nathalie R. Le François et le Dr Robert Roy pour leur encouragement soutenu, leurs conseils éclairés, et leur présence rassurante tout au long du projet. Merci de la confiance que vous m’avez témoignée et de m'avoir donné la chance de vivre une si belle aventure. Le séjour que j’ai passé à Grande-Rivière fut toujours très plaisant et des plus instructifs. Grâce à vous, j’ai eu la chance de côtoyer des équipes de recherche dynamiques dans des ambiances de travail des plus agréables, toujours avec le souci du travail bien fait et le goût de la réussite au final. Je veux aussi remercier l'équipe des Sciences Animales de l’Université Laval, et particulièrement le Dr Grant Vandenberg pour son accueil au sein des équipes de son département. Mon passage à l'Université Laval m’a donné l’occasion d’élargir mes horizons, de découvrir plusieurs autres domaines de recherche connexes fort intéressants et d’œuvrer dans des environnements différents que j’ai également trouvés très stimulants et enrichissants. Je veux remercier tous les autres membres de l'équipe qui ont participé au projet : Dany Ouellet, Sarah Tremblay et Tony Grenier; mes remerciements à Arianne Savoie, sans qui Grande-Rivière aurait été beaucoup moins drôle; Maître Savoune, vous êtes irremplaçable; merci beaucoup à mon mentor de laboratoire Domynick Maltais, vous avez été indispensable. Je veux aussi remercier mes parents, Sylvie Dupont et Alain Cyr, qui m'ont toujours encouragé et supporté dans mes projets les plus audacieux. Ensemble vous avez
iv grandement contribué à la personne que je suis et l’aboutissement de ce travail est le fruit de vos efforts et de vos sacrifices. En espérant que cela puisse enfin me permet de gagner mon autonomie complète. Je souhaite enfin remercier de tout cœur Lucie Meynier qui, par sa patience et sa délicatesse légendaire, m’a toujours soutenu dans la poursuite de mes rêves et de mes objectifs de carrière, même lorsque cela m’amenait à m’éloigner pour des périodes prolongées. La réalisation de ce projet a demandé beaucoup de sacrifices de sa part et entraîné le report de plusieurs chantiers qui pourront dorénavant prendre forme. Merci Lucie pour ta compréhension et tes encouragements. Je te promets que je vais finir par finir le plafond de la cuisine! Cette maîtrise fut pour moi une très belle aventure. Je suis maintenant prêt pour la suite et j'ai encore plus de motivation et d’idées qu'il y a trois ans lorsque j'ai signé avec le loup. Un merci spécial aux loups de mer sans lesquels ce projet n’aurait pas pu se réaliser. Cette espèce m’a permis de découvrir un aspect de la biologie que je connaissais moins et de piquer ma curiosité pour toujours. Le loup est une espèce incroyable et j’espère que mon aventure avec elle ne fait que commencer; comme dit si bien le proverbe, « In wolffish we should all trust ». Sur ce, je vous remercie encore et je vous souhaite une bonne lecture.
Bernard-Antonin Dupont Cyr
Table des matières Résumé ....................................................................................................................................i Abstract..................................................................................................................................ii Avant-Propos ........................................................................................................................iii Table des matiè res.................................................................................................................v Liste des tableaux ................................................................................................................vii Liste des figures ..................................................................................................................viii Introduction ...........................................................................................................................1 Biologie du loup de mer......................................................................................................3 La reproduction ...................................................................................................................3 Les gamètes mâles ..........................................................................................................7 Les gamètes femelles ......................................................................................................8 La reproduction des téléostéens en aquaculture................................................................11 Le système endocrinien de la reproduction.......................................................................12 Horloge biologique des téléostéens et photopériode ........................................................14 Mise en contexte ...............................................................................................................17 Chapitre 1 ............................................................................................................................19 Régulation de la maturation sexuelle et de la période du frai par la manipulation de la photopériode chez le loup de mer (Anarhichas lupus et A. minor) : teneurs en stéroïdes sexuels (11-kétotestostérone et 17-β-oestradiol) Photoperiod regulates the timing of sexual maturation, spawning and sex steroid profiles in wolffishes (Anarhichas minor and A. lupus). Résumé..............................................................................................................................21 Abstract .............................................................................................................................22 Introduction .......................................................................................................................23 Materials and methods ......................................................................................................24 Fish and experimental conditions .................................................................................24 Sampling .......................................................................................................................26 Steroid analyses.............................................................................................................26 Statistics ........................................................................................................................27 Results ...............................................................................................................................28 Sexual maturation .........................................................................................................28 Steroid profiles ..............................................................................................................29 Discussion .........................................................................................................................31 Acknowledgements ...........................................................................................................36 Tables and captions ...........................................................................................................37
vi Chapitre 2 ............................................................................................................................43 Observation d’une différence de croissance lié au sexe chez le loup de mer (Anarhichas lupus et A. minor). Characterization of the growth rate of adult wolffishes (Anarhichas minor and A. lupus) unravels a marked sexual dimorphism. Résumé..............................................................................................................................45 Abstract .............................................................................................................................46 Introduction .......................................................................................................................47 Materials and methods ......................................................................................................50 Broodstock origin..........................................................................................................50 Rearing condition ..........................................................................................................51 Steroid analysis .............................................................................................................52 Growth measurements...................................................................................................52 Statistical analysis .........................................................................................................53 Results ...............................................................................................................................54 Body mass and total length ...........................................................................................54 Common wolffish (A. lupus) ........................................................................................54 Spotted wolffish (A. minor-QC and NW).....................................................................54 Specific growth rate and condition factor .....................................................................55 Food conversion efficiency ...........................................................................................56 Discussion .........................................................................................................................56 Acknowledgements ...........................................................................................................58 Tables and captions ...........................................................................................................60 Discussion générale .............................................................................................................66 Compression du cycle reproducteur..................................................................................66 Croissance .........................................................................................................................70 Condition d’élevage ..........................................................................................................73 Conclusion générale ..........................................................................................................75 Bibliographie .......................................................................................................................77
Liste des tableaux Table 1 : Proportion (%) of maturation of common wolffish (A. lupus), and Canadian (A. minor QC) and Norwegian (A. minor NW) strains of spotted wolffish, exposed to the different photoperiod regimes. ......................................................................................37 Table 2: Proportions (%) of ovulation and oocyte atresia in maturing female common wolffish (A. lupus), and Canadian (A. minor QC) and Norwegian (A. minor NW) strains of spotted wolffish exposed to the different photoperiod regimes. Different superscript letters indicate significant differences between groups. .............................38
Liste des figures Figure 1: Séquence du comportement reproducteur du loup de mer atlantique (Anarhichas lupus) (Johannessen et al., 1993). ...................................................................................4 Figure 2: A) Schémas de la régulation hormonale de la croissance des oocytes (Gonadotropin = GTH-I.). B) Schémas de la vitellogènèse (Gonadotropin = GTH-II.) (Nagahama, 1994). ........................................................................................................14 Figure 3: Photoperiod regimes of the different treatment groups from April 2006 to December 2007 2006: SNP, (Grande-Rivière; 48° 24′ 00″ North, 64° 30′ 00″ West) simulated natural photoperiod; CP, photoperiod compressed to 8 months. .................39 Figure 4 : Timing of cumulative ovulation in female A) A.lupus, B) A.minor QC and C) A.minor NW. .................................................................................................................40 Figure 5 : Temporal changes in plasma concentrations of 17β-estradiol (E2) in female A) A. lupus, B) A. minor QC and C) A. minor NW exposed to different photoperiod regimes; CP = compressed 8 month photoperiod and SNP = simulated natural photoperiod; Values represent mean ± S.E.M. (SNP n = 11, 7 and 7 for A. lupus, A. minor QC and A. minor NW, respectively; CP n = 10, 8 and 7 for A. lupus, A. minor QC and A. minor NW respectively). Different letters indicate significant differences in mean levels of E2 for the same date between photoperiod treatments. S.E.M. values (vertical lines) may be obscured by the symbol. ...................................................................................41 Figure 6 : Temporal changes in plasma concentrations of 11-ketotestosterone (11-KT) in male A) A. lupus, B) A. minor QC and C) A. minor NW exposed to different photoperiod regimes; CP = compressed 8 month photoperiod, SNP = simulated natural photoperiod; Values represent mean ± S.E.M. (SNP: n = 10, 7 and 7 for A. lupus, A. minor QC and A. minor NW, respectively; CP: n = 10, 8 and 6 for A. lupus, A. minor QC and A. minor NW, respectively). Different letters indicate a significant difference in mean 11-KT levels for the same date between photoperiod treatments. S.E.M. values (vertical lines) may be obscured by the symbol. ...............................................42 Figure 7 : Photoperiod regimes of the different treatment groups in the period of April 2006 to December 2007 2006: SNP, (Grande-Rivière; 48° 24′ 00″ North, 64° 30′ 00″ West) simulated natural photoperiod; CP, photoperiod compressed to 8 months. .................60 Figure 8 : A) Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A.lupus reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA followed by TuckeyHSD, p < 0.05). N = 3 rearing tanks for each mean value, with 7-9 females and 16-18 males per tanks.............................................................................................61 Figure 9 : Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A.minor-QC reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA
ix followed by TuckeyHSD, p < 0.05). N =2 rearing tanks for each mean value, with 6-7 females and 8-9 males per tanks. ..................................................................................62 Figure 10 : A) Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A.minor-NW reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA followed by TuckeyHSD, p < 0.05) N = 2 rearing tank for each mean value, with 910 females and 14-15 males per tanks. .........................................................................63 Figure 11 : Broodstock SGRW (A) A.lupus, B) A.minor-QC, C) A.minor-NW), SGRl (D) A.lupus, E) A.minor-QC, F) A.minor-NW) and condition factor (k) (G) A.lupus, H) A.minor-QC, I) A.minor-NW) reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod according to female pre-maturation (Imm), maturation (Mat), spawning (Spaw), post-spawning (Post) and male (Male). Results are given in mean ± S.E. Different lettes show significant difference beetween each period or group. .................................................64 Figure 12 : Food conversion efficiency (FCE) for wolffishes reared under two photoperiods regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod Results are given in mean ± S.E. Different letters show significant difference beetween each photoperiod treatment and group. .......................................65
Introduction Depuis plusieurs années, les stocks sauvages de poissons marins sont en baisse et plusieurs recherches dressent un portrait peu optimiste de leurs perspectives de rétablissement (Stokastad, 2006; Worm et al., 2006). Ainsi pour préserver les vestiges des grands stocks sauvages et répondre à l’augmentation de la demande de produits marins de haute qualité, il devient impératif de développer une alternative durable et diversifiée aux pêcheries traditionnelles (Gulbrandsen, 2009). Dans plusieurs pays vivant principalement des pêches traditionnelles, comme la Norvège et l’Islande, le développement de l’aquaculture est perçu comme une solution durable aux problèmes socio-économiques reliés à l’effondrement des grands bancs. Depuis la dernière décennie, cette industrie connait une forte croissance dans l’Atlantique Nord (Foss et al., 2004). La majorité des installations se sont spécialisées dans l’élevage des poissons pélagiques tel que les salmonidés (e.g. saumon de l’Atlantique (Salmo salar), omble de fontaine (Salvelinus fontinalis), omble chevalier (Salvelinus alpinus), truite arcen-ciel (Oncorhynchus mykiss), etc.) qui contrairement à celui des poissons benthiques demande relativement peu de surface. Au Canada, la mariculture (élevage en milieu marin) est une activité économique très peu développée et la majorité des installations sont spécialisées dans la culture des salmonidés ou la conchyliculture. Au Québec, les caractéristiques géographiques et climatiques de la région
du
Fleuve
et
du
Golfe
du
Saint-Laurent
ainsi
qu’une
règlementation
environnementale rigoureuse sur les installations classiques en mer ou sur terre, freinent considérablement le développement de cette industrie. Bref, le contexte d’implantation d’une entreprise spécialisée en aquaculture représente un défi significatif mais non insurmontable.
Un choix judicieux de l’espèce est primordial afin de maximiser l’atteinte
des objectifs et de réduire au maximum les coûts d’exploitation. Le loup tacheté (Anarhichas minor) et le loup atlantique (A. lupus) ont été identifiés comme d’excellents candidats pour le développement d’une mariculture en eau froide en Norvège, en Islande et au Canada (Tilseth, 1990; Moksness et Pavlov, 1996; Falk-Petersen et al., 1999; Foss et al., 2004; Le François et al., 2002, 2010). Ces espèces démersales, non
2 grégaires de l’Atlantique Nord sont aussi protégées par la loi canadienne sur les espèces en péril
(www.cosewic.gc.ca)
ce
qui
limite
l‘implantation
d‘une
pêche
commerciale
significative. D’où l’intérêt pour le développement de la mariculture de ces espèces au Québec et dans l’est du Canada. En plus de la qualité de leur chair (Pavlov et Moksness, 1993), le loup tacheté et le loup atlantique possèdent plusieurs qualités biologiques intéressantes : un taux de croissance élevé en eaux froides ( 0.05). E2 levels were low or non-detectable in all A. lupus photoperiod groups during February, March and April 2006 (Figure 5A). E2 levels started to rise in May of that year and peak levels appeared in June - July, with a mean of 4.93 ± 0.59 ng·ml-1 . E2 levels
30 peaked immediately prior to or during the ovulation period, then decreased and returned to basal levels. In 2007, the photoperiod treatment had a significant impact on the profiles of E2 (p < 0.001; Figure 5). There was a shift in the annual profiles of E2 in the CP treatments compared to the SNP ones, for all groups (A. lupus, A. minor QC and A. minor NW). The E2 profiles of the CP groups followed the same pattern, with basal levels from January to March, peak levels in June and decreases to basal levels in August - September. Peak levels of E2 in the CP groups were 14.93 ± 1.74, 9.69 ± 0.75 and 9.51 ± 1.37 ng·ml-1 for the A. lupus, A. minor QC and A. minor NW fish, respectively (Figure 5). Peak levels of E2 in A. lupus fish were lower in 2006 than in 2007. In the SNP groups, peak levels of E2 appeared later and lasted for a longer period, compared to fish in the compressed photoperiod, ranging from 11.04 ± 2.50 ng·ml-1 in Norwegian A. minor, to 17.07 ± 1.56 in A. lupus (Figure 5). In 2006, there was no significant effect of the CP treatment on the annual pattern of 11-KT in A. lupus (p < 0.001, Figure 6A). For both photoperiod treatments, levels of 11-KT were low or non-detectable during the first months of the study. In June, 11-KT levels increased, reaching values in July of 6.56 ± 1.63 ng·ml-1 and 4.14 ± 0.39 ng·ml-1 , for the SNP and CP groups, respectively. 11-KT levels remained elevated for 3 months, before decreasing to basal levels in October In 2007, differences in the profiles of 11-KT between CP and SNP treatments were found for A. lupus, and both A. minor strains. In the A. lupus CP group, mean 11-KT increased in March, reached a maximum of 7.38 ± 0.72 ng·ml-1 in May, then returned to basal levels by August. In contrast, August was the month of the highest mean 11-KT, 10.66 ± 1.17 ng·ml1
, in the corresponding SNP group. For two the A. minor QC and NW 11-KT profiles were
different between CP and SNP, but similar between each strain (Figure 6B and Figure 6C). Maximum levels of 11-KT in both A. minor strains were lower in the CP treatments compared to the SNP groups (Figure 6B and Figure 6C). Mean levels of 11-KT in the CP
31 treatments were highest in May, 4.49 ± 0.67 ng·ml-1 and 3.47 ± 0.67 ng·ml-1 for male A. minor QC and NW, respectively.
Discussion The present results clearly indicate that the timing of spawning of Atlantic (A. lupus) and spotted wolffish (A. minor QC and NW) can be modified by seasonal photoperiodic compression and suggest that photoperiod could be a major cue in the onset of sexual maturation in captive wolffish (A. minor and A. lupus). The homogeneity in profiles of steroids (E2 and 11-KT) and the significant difference in timing of ovulation indicate that compression of seasonal photoperiod may be effective in the management of maturation and for the obtainment of an out-season spawning period similarly to many other species (Prat et al., 1999; Bromage et al., 2001; Howell et al., 2003). The results are in agreement with Foss et al., (2004) who briefly reported a successful compression of the reproduction cycle in a commercial setting of spotted wolffish by exposing broodstock to a photoperiod of nine months. Wolffish responses to photoperiod manipulation are in agreement with other work on other commercial species (Duston and Bromage, 1986; Hansen et al., 1992; Björnsson et al., 1994; Davis et al., 1999; Prat et al., 1999; Bromage et al., 2001; Howell et al., 2003; Norberg et al., 2004). In this study, the 2007 spawning period of wolffish in the CP treatments was 3 - 6 months earlier than that in the SNP groups featuring both species. A. lupus spawning season seems to vary depending on the origin of the stock. Previous in vitro studies reported that spawning started in June - July to September in Russian stocks (Scott and Scott, 1988; Pavlov and Radzikhovskaya, 1991; Dzerzhinskij and Pavlov 1992). Johannessen et al., (1993) reported that Norwegian stock spawned from mid-October to mid-February and Tveiten et al., (2001) reported a spawning period from November to January. In addition to these observations, the absence of significant differences between the timing of ovulation in A. minor QC and NW strains suggest that the reproductive cycle has an endogenous circannual oscillation that may be affected by photoperiod. This suggests that the life cycle of the wolffish is very flexible and they could be adaptable to a photoperiodic treatment. The results of this study support this hypothesis and also suggest a
32 useful method to manipulate the reproduction cycle of A. lupus, A. minor QC and A. minor NW. Photoperiod acts on melatonin circadian secretion, which has a major impact on the hypothalamic pituitary-gonadal axis (Porter et al., 2000a, b; Bromage et al., 2001). Therefore, the onset of longer day lengths during the summer solstice is perceived as a stimulus to initiate the development of reproduction; the onset of shorter day lengths (winter solstice) would be a signal for triggering the later stages of gametogenesis and ovulation (Bromage et al., 1984; Takashima and Yamada, 1984; Bromage et al., 2001). According to the natural photoperiodic regime of this study, 48 ° 24 '00 "N, 64 ° 30' 00" W, sexual maturation of wolffish (A. minor and A. lupus) was stimulated by increasing the length of the days associated with the summer solstice, with spawning taking place 1 - 2 month before the winter solstice. This is in good agreement with the spawning pattern of wild wolffish at the same latitude (Brander, 1993). Our results also suggest that reproduction of common and spotted wolffish is not annual and fish may skip one or two years of spawning. Johannessen et al., (1993) have observed the same phenomenon and suggested that maturation in captivity may be curtailed by the complexity of the reproduction cycle, in addition to the possible partial inadequacy of the rearing environment (including nutrition) for optimal reproduction. This could partly explain the less than 100% rate of maturation observed in both species and strains. Compression of the reproductive cycle also appears to reduce the percentage of maturation in the early years of treatment exposure (Duston and Bromage, 1988, Blythe et al., 1994; Björnsson et al., 1998). Acclimatization to photoperiod compression can take up two years before observing an effect on reproduction cycle (Björnsson et al., 1998; Martin-Robichaud and Berlinsky, 2004). According to Foss et al., (2004), wolffish acclimatization period would be 18 months. The results obtained in this study are consistent with this observation and the effect of photoperiod was more pronounced during the second year of experimentation, after 18 to 20 months of exposure.
33 Results suggested no diminution in eggs diameters, ovulation rate or atresia related to photoperiod. Compression of the reproductive cycle by photoperiod may decrease the diameter of eggs and ovulation rates in females (Blythe et al., 1994; Morehead et al., 2000; Foss et al., 2004). The effects of photoperiod on the quality of sexual products are not easy to clarify. They usually involve the modification of several parameters throughout oogenesis (Frantzen et al., 2004; Bobe and Labe, 2010). Compression of the reproductive cycle could not only affect the duration between periods of spawning, but also all of the endogenous parameters of the animal. For example, the compression of the reproduction cycle could reduce the feeding period and recovery period between each spawning. This could lead to weakened fish that have not the necessary adequate reserves to achieve an additional successful gametogenesis. In this study, no impact on eggs diameters, ovulation rate, maturation rate or atresia could be attributed to photoperiod. Maturation rates were slightly lower in the A. minor QC and NW compared to A. lupus. The success of sexual maturation depends on an individual reaching a threshold optimal metabolism during a spawning period (Duston and Bromage, 1988; Duston and Saunders, 1992; Thorpe, 1994). In salmonids, the age, size, growth rate, metabolism and stage of gonadal development are key factors for successful maturation in response to photoperiod treatment (Rowe et al., 1991; Berglund, 1995). According to the results, the age and the size of broodstock would be the factors affecting the maturation rate. In wolffish, there is a proportional relationship between the size of the fish and the quality of sexual products (Templeman, 1986 a, b). Previous fields studies proposed that spotted wolffish matured at a length of 76.9 ± 5.0 cm and/or at the age of 8.5 ± 0.6 years olds depending of the area (Barsukov, 1959; Østvedt, 1963; Templeman, 1986b; Gunnarsson et al., 2008). While wild common wolffish matured at a length of 56.7 ± 6.3 cm and/or at the age of 7.9 ± 1.5 years of age (Templeman, 1986a; Hansen et al., 1992; Pavlov and Novikov, 1993; Gunnarsson et al., 2006. According to this information, the size of the broodstock used in this study was slightly below the natural maturation mass found for this species. In this study the A. minor QC and NW weighed approximately 1kg in February 2006 and reached 3 kg in December 2007 while A. lupus weighed 1kg in February and reach 1.3 kg in December 2007. However, the primary hypothesis would be most likely related to the difference of age
34 between both species and the status of first time spawner of A. minor QC and NW. A. minor of this study were 3+ years old, and at their first maturation period in 2006 in comparison to the A. lupus were 6+ years old and not at their first spawning period. This assumption was support by the work of Karlsen et al., (1995) who reported that maturation rate at first spawning of wolffish in captivity is usually less than 80%. Tveiten and Johnsen (1999) and Tveiten et al., (2001) reported that the timing of wolffish maturation is sensitive to temperature without being considered the main factor. Therefore, in order to isolate the effect of photoperiodic treatment on sexual maturation, wolffish in this study were maintained at a constant temperature of 8 °C. However, it should be noted that the optimal temperature for reproduction of wolffish is 6 °C (Moksness and Pavlov, 1996; Hansen and Falk-Petersen, 2001; Tveiten et al., 2001; Tveiten and Johnsen, 2001; Imsland et al., 2006; Sund and Falk-Petersen, 2005). However, the CAMGR water cooling capacity was insufficient to maintain 6 °C all year round so a constant temperature of 8 °C was adopted. The shift in the 11-KT profiles between the CP and SNP groups clearly demonstrates an effect of photoperiod on the reproductive cycle of males in both species. 11-KT is the principal androgen in many teleost’s and is involved in spermatogenesis and in the development of secondary sexual characters (Borg, 1994; Shulz and Miura, 2002). The plasma profiles of sex steroids of the wolffish (A. lupus, A. minor QC and A. minor NW) in the SNP treatments are similar to those observed in teleosts with synchronous annual maturation (Pankhurst, 1998). The increase in plasma 11-KT levels observed during the spawning period is consistent with results of the literature on other species (Billard et al., 1982; Fostier et al., 1983; Borg, 1994; Bromage et al., 2001). A large proportion of wolffish (A. lupus and A. minor) were spermiating throughout the year. This preliminary observation would support the hypothesis that male wolffish are spermiant throughout the year to attract females, with small ejaculations of milt containing small amounts of pheromones (Johannessen et al., 1993; Pavlov et al., 1997; Foss et al., 2004). Similar patterns are reported in other cultured species including sea bass
35 (Dicentrarchus labrax) (Prat et al., 1990) and Senegalese sole (Solea senegalensis) (GarciaLòpez et al., 2007). The year 2006 was the first spawning season for the spotted wolffish populations included in our study. The 11-KT cycles in the male A. minor QC and A. minor NW groups under CP treatment were still not clearly defined. The low levels observed suggest the initiation of the endocrine system associated with puberty (Holland et al., 2000). For fish culture, it is often observed that more than one cycle is needed before obtaining maximum levels of steroids (Holland et al., 2000, Norberg et al., 2004; Martin-Robichaud et al., 2004). The low levels observed could be attributed to the status of a young adult fish, since high quality gametes and a high fertilization rates were reported for this group after the experiment (Pers. Comm. Le François, 2008). In 2007, peak levels of E2 in all of the SNP treatment females were observed in the months of August to October, compared to June in the CP treatment groups. E2 levels in the A. minor CP groups remained low, in comparison to the SNP groups. E2 induces the synthesis by the liver of the yolk precursor protein, VTG (Idler and Ng Bunn, 1983; Specker and Sullivan, 1994). Nagahama (1994) reported that E2 and VTG are positively correlated. The shifted E2 profile in the CP treatments may indicate that VTG synthesis may also be affected by photoperiod manipulation. The CP treatment also resulted in a shift of ovulation, compared to the SNP. However, the relatively lower levels of E2 in the CP groups could influence gametogenesis and possibly lead to an incomplete development of gametes and lower fertilization rates. Significant mortality in A. lupus occurred in February to April 2006. The behaviour of A. lupus of this study appeared abnormal compared with other CAMGR stocks and no similar observation has been reported in the literature. It is possible that the light intensity was inappropriate for the biology of the species. Light intensity is an important parameter affecting the growth and reproduction cycle of several teleost livestock (Downing et Litvak, 2002; Hole and Pittman, 1995). The aggressive behaviour of these wolffish disappeared completely after the light intensity was modified, suggesting that the animals were probably
36 undergoing a chronic stress during the period from February 2006 to April 2006 (Stratholt et al., 1997; Contreras-Sanchez et al., 1998). This is the first time that light intensity is suggested as a factor that may lead to behaviours such as cannibalism and aggression in A. lupus. This component should be the subject of further research to validate the best breeding conditions for captive A. lupus broodstocks. In conclusion, this study has demonstrated that photoperiod manipulation could be a powerful tool for controlling reproduction in wolffish. Consequently, application of photoperiod manipulations should prove to be an effective approach in ongoing efforts to establish a year-round production of gametes and increased the availability of eggs and larvae for future wolffish cultivation initiatives. We note that, following this experiment, the photoperiod treatment was maintained at Mérinov inc., to obtain out-of-season spawning in May and two spawnings per year at this research facility.
Acknowledgements The present study was funded by Fisheries and Oceans Canada through the ACRDP program and Aquanet initiative, with the collaboration of the Ministère de l'agriculture, des pêcheries et de l'alimentation du Québec (MAPAQ). The authors thank Domynick Maltais, Dany Ouellet, Arianne Savoie and Tony Grenier for their technical support.
37
Tables and captions Table 1 : Proportion (%) of maturation of common wolffish (A. lupus), and Canadian (A. minor QC) and Norwegian (A. minor NW) strains of spotted wolffish, exposed to the different photoperiod regimes. % maturation
Species
Sex
Photoperiod treatment
2006
2007
n (total)
A. Lupus
Male
CP
92.0
98.0
50
SNP
96.0
100.0
47
CP
82.6
87.0
23
SNP
100.0
81.0
21
CP
72.7
81.8
11
SNP
80.0
100.0
10
CP
73.3
73.3
15
SNP
68.4
52.6
19
CP
50.0
65.0
20
SNP
52.6
68.4
20
CP
66.7
79.2
24
SNP
96.4
75.0
28
Female
A. minor QC
Male
Female
A. minor NW
Male
Female
38
Table 2: Proportions (%) of ovulation and oocyte atresia in maturing female common wolffish (A. lupus), and Canadian (A. minor QC) and Norwegian (A. minor NW) strains of spotted wolffish exposed to the different photoperiod regimes. Different superscript letters indicate significant differences between groups.
Species
A. lupus
A. minor QC
A. minor NW
Photoperiod treatment
% ovulation
% atresia
Egg diameter (mm ± S.E.M)
2006
2007
2006
2007
2006
2007
CP
94.7
90.0
0.0
0.0
4.4 ± 0.1c
3.7 ± 0.2ab
SNP
95.2
94.1
0.0
0.0
3.9 ± 0.1bc
3.3 ± 0.2a
CP
54.5
90.9
18.2
0.0
4.8 ± 0.2b
3.6 ± 0.2a
SNP
100.0
90.0
0.0
0.0
4.6 ± 0.2b
4.9 ± 0.1b
CP
18.8
52.6
81.3
26.3
4.7 ± 0.4a
3.7 ± 0.4b
SNP
37.0
38.1
22.2
14.3
4.2 ± 0.2a
3.6 ± 0.3b
39
Figure 3: Photoperiod regimes of the different treatment groups from April 2006 to December 2007 2006: SNP, (Grande-Rivière; 48° 24′ 00″ North, 64° 30′ 00″ West) simulated natural photoperiod; CP, photoperiod compressed to 8 months.
40
Figure 4 : Timing of cumulative ovulation in female A) A. lupus, B) A. minor QC and C) A. minor NW.
41
Figure 5 : Temporal changes in plasma concentrations of 17β-estradiol (E2) in female A) A. lupus, B) A. minor QC and C) A. minor NW exposed to different photoperiod regimes; CP = compressed 8 month photoperiod and SNP = simulated natural photoperiod; Values represent mean ± S.E.M. (SNP n = 11, 7 and 7 for A. lupus, A. minor QC and A. minor NW, respectively; CP n = 10, 8 and 7 for A. lupus, A. minor QC and A. minor NW respectively). Different letters indicate significant differences in mean levels of E2 for the same date between photoperiod treatments. S.E.M. values (vertical lines) may be obscured by the symbol.
42
Figure 6 : Temporal changes in plasma concentrations of 11-ketotestosterone (11-KT) in male A) A. lupus, B) A. minor QC and C) A. minor NW exposed to different photoperiod regimes; CP = compressed 8 month photoperiod, SNP = simulated natural photoperiod; Values represent mean ± S.E.M. (SNP: n = 10, 7 and 7 for A. lupus, A. minor QC and A. minor NW, respectively; CP: n = 10, 8 and 6 for A. lupus, A. minor QC and A. minor NW, respectively). Different letters indicate a significant difference in mean 11-KT levels for the same date between photoperiod treatments. S.E.M. values (vertical lines) may be obscured by the symbol.
43
Chapitre 2
Observation d’une différence de croissance lié au sexe chez le loup de mer (Anarhichas lupus et A. minor).
44
Characterization of the growth rate of adult wolffishes (Anarhichas minor and A. lupus) unravels a marked sexual dimorphism.
Bernard-Antonin Dupont Cyr1 , Robert L. Roy2, Grant W.Vandenberg1 and Nathalie R. Le François1,3,4* . 1. Faculté des Sciences de l'Agriculture et de l'Alimentation, Département des Sciences Animales, Université Laval, Pavillon Paul-Comtois 2425 rue de l'agriculture Local 1122, Québec (Québec), Canada, G1V 0A6. 2. Institut Maurice-Lamontagne, Pêches et Océans Canada, 850 route de la Mer, Mont-Joli (Québec), Canada, G0J 2L0. 3. Biodôme de Montréal, 4777 Avenue Pierre-De Coubertin, Montréal (Québec), Canada, H1V 1B3. 4. Département de biologie, chimie et géographie, Université du Québec à Rimouski, 300, Allée des Ursulines, Rimouski, QC G5L 3A1 *Corresponding author:
[email protected] Keyword: Sexual dimorphism; Anarhichas spp.; Growth; broodstock.
45
Résumé Le but de cette étude est de documenter les patrons de croissance du loup atlantique (6+; A. lupus) et de loup tacheté (3+; A. minor de souche canadienne (QC) et Norvégienne (NW)) de 1 à 3.5 Kg en fonction du sexe et d’un traitement photopériodique. Durant la période de février 2006 à juillet 2007, les différents groupes (A. lupus, A. minor-QC et NW) ont été soumis à deux régime photopériodique (naturelle simulée et compressée sur huit mois). La croissance (masse totale humide et longueur totale) a été suivie mensuellement sur l’ensemble des individus. Les résultats suggèrent l’établissement d’un dimorphisme de la croissance relié au sexe avantageant les mâles (A. lupus 21.9%, A. minor-QC 26.6% et A. minor-NW 17.2%) qui serait directement relié à la maturation sexuelle lorsqu’elle se déroule en conditions d’élevage en captivité et ce chez les deux espèces. Ce dimorphisme n’a jamais été observé lors d’études en milieu naturel dirigées sur ces espèces. En contexte d’élevage aquacole, cette observation pourrait être attribuable à l’absence d’investissement parental du mâle en captivité (garde des masses d’œufs fécondés et cessation de l’alimentation).
Dans
un
contexte
d’aquaculture
commerciale,
la
présence
d’un
dimorphisme sexuel de croissance suggère l’évaluation de méthodes de contrôle du sexe si la taille ciblée est supérieure à 1.5 Kg.
46
Abstract Sex-related growth patterns of common (6+ Anarhichas lupus) and spotted wolffishes (3+ A. minor; Canadian (QC) and Norwegian (NW) strain) broodstock of average weight from 1 to 3.5 Kg were monitored. During the period February 2006 to July 2007 the fish were subjected to two photoperiods (simulated natural and compressed to eight months) (three tanks of 25 A. lupus by photoperiod, two tanks of 15 A. minor-QC and 25 A. minor-NW by photoperiod). Growth (total wet weight and total length) was monitored monthly. Results suggest the establishment of sex-related growth dimorphism benefiting males (A. lupus 21.9%, A. minor-QC 26.6% and A. minor-NW 17.2%) possibly linked to sexual maturation when it takes place under captivity and this for both species under study. This dimorphism never been clearly observed in wild populations of wolffish. In an aquaculture situation, these observations seems related to the absence, in captivity, of male egg-guarding behaviour and thus sustain their growth compared to the females who continue their gonadal development and undergo the associated growth reduction. In commercial settings, the presence of a sex-related growth dimorphism strongly suggests the application of sex control techniques for commercial target sizes greater than 1.5 kg.
47
Introduction Growth dimorphism related to sex is a complex phenomenon widely observed in fish. Two major mechanisms are involved in the emergence of growth dimorphism related to sex. The first mechanism is generally expressed during early life stages and is linked to the particularity of the species to promote growth of one gender in particular such as is seen in Poecilia reticulate (guppy: Goodrich et al., 1934), Oreochromis niloticus (Nile tilapia: Toguyeni et al., 2007) and in Pseudobagrus ichikawai (bagrid catfish: Wanatabe, 1994). It is explained by genetic differences between sexes, environmental factors (temperature, photoperiod, food availability) and/or intrinsic characteristics (food conversion) benefiting a gender over the other. The second mechanism implies different morphological and physiological modifications between sexes appearing at the onset of sexual maturation. The latter is generally observed when age of maturation and/or reproduction investment differs between sexes (Craig, 1977; Rijnsdorp and Ibelings, 1989; Thorpe, 1994). Individuals of both sexes must reach a specific size and/or body condition to successfully complete maturation and breeding (Policansky, 1983; Dutil, 1986; Jobling, 1995; Kadri et al., 1996). Sexual maturation (gametogenesis, vitellogenesis, specific reproduction behaviour, etc.) requires the mobilization of energy and some body constituents (lipid, protein, glycogen, carotenoid, etc.) that could differ between sexes and may result in modifications of the growth pattern (Kadri et al., 1996; Adams and Thorpe, 1989; Rowe and Thorpe, 1990 a, b; Rowe et al., 1991; Thorpe, 1994; Jobling, 1995; Adams and Hunthingford, 1997). In addition to the intrinsic characteristics of a species, the growth rates for the same cohort are also variable and generally explained by several factors such as the genetics of broodstock (Forsberg, 1996; Imsland et al., 1997), social status (Jobling and Koskela, 1996) and antagonistic behaviour (McCarthy et al., 1992; Imsland et al., 1998). If not properly addressed, the presence of early maturation and growth dimorphism in aquaculture stocks and populations can significantly impair profitability through reduced growth rate and flesh quality and can also lead to immuno-depression that may increase susceptibility to diseases and increased mortality.
48 Many aquaculture species are well known to exhibit sexual growth dimorphism. Early sexual maturation and/or sex-related growth dimorphism is a problem in intensive farming of several specie such as Rainbow trout (Oncorhynchus mykiss; Bonnet et al., 1999), Atlantic salmon (Salmo salar; Thorpe et al., 1994), Artic charr (Salvelinus alpinus; Adam and Huntingford, 1997), Brook charr (Salvelinus fontinalis)(Le François et al., 1999), tilapia (Oreochromis niloticus; Mair et al., 1995, 1997), European sea bass (Dicentrarchus labrax; Saillant et al., 2001; Zanuy et al., 2001), Atlantic cod (Gadus morhua; Karlsen et al.,1995), Atlantic halibut (Hippoglossus hippoglossus; Norberg et al., 2001). The wolffishes have been identified as good candidate for the diversification of cold-water mariculture (Tilseth, 1990; Falk-Petersen et al., 1999; Foss et al., 2004; Le François et al., 2002, 2010). Since 1998, a large-scale multi-disciplinary research program has been active in Québec, Canada to diversify the aquaculture sector and develop wolffish aquaculture in Eastern Canada (Le François et al., 2010). Research on Canadian spotted wolffish populations has focused principally on early stages and involved immature fish (from hatching to 200g). The work of Foss et al., (2004) and Le François et al., (2002, 2010) reviewed the on-growing parameters and the details of wolffishes aquaculture. Wolffish are known to exhibit high growth rates in captivity and we can rely on a well-known characterized reproductive cycle (Johannessen et al., 1993; Ringø and Lorentsen, 1987; Tveiten and Johnsen, 1999; Kime and Tveiten, 2002) that can be effectively controlled by photoperiod modulation (Dupont Cyr et al., to be submitted Chapter 1 of this memoir). In the wild, wolffish sexual maturation occurs at the age of 4-5 years at a minimal size of around 0.5-1kg and 3-4 kg (A. lupus and A. minor) (Moksness, 1994). Most female begin maturing at lengths of 50-70 cm and 75-80 cm and higher (Templeman, 1986a, b; Gunnarsson et al., 2006, 2008) for Common and spotted wolffish respectively. Wolffish are internal fertilizers (copulation occurs) and annual single-batch spawners. Courting phase begin 3-5 months before the spawning period (Johannessen et al., 1993). The male finds a nest (Keats et al., 1985) and attracts a female by releasing a small quantity of milt (Johannessen et al., 1993). After couple formation, couples remain together for the entire oocyte maturation period (3-5 months). The spawning period is variable, depending on the location, but generally takes place in late fall - early winter (Templeman, 1986a,b). A few
49 weeks before ovulation, the female will cease feeding. Once spawning is completed the eggs will be deposited in an egg mass and the male will protect the fertilized eggs until hatching (Keat et al, 1985; Ringø and Lorentsen, 1987). During most of the incubation period, which typically lasts 900-1000 degree day (5-6 months at 3-4 ºC) in both species, males and females will lose part of their dentition and will not be feeding very actively (males slightly more actively feeding than females) (Templeman 1986a, b; Ringø and Lorentsen, 1987). After spawning, the female will rapidly leave the fertilized egg mass and the nest to feed actively (Ringø and Lorentsen, 1987). Due to the complex courtship and the apparent difficulty to
create an optimal environment to
trigger natural spawning
behaviours, male and female gametes are extracted from the fish manually and fertilization in vitro are necessary (Foss et al., 2004; Moksness et al., 2004; Le François et al., 2010). In wolffish aquaculture-related studies, no sex-related growth dimorphism was ever reported for common and spotted wolffishes (Moksness, 1994; Falk-Petersen et al., 1999; Foss et al., 2004). More specifically, Johannessen et al., (1993) reported no sex-related size differences prior to reproductive maturation for the common wolffish. However, based on Canadian fisheries reports, Templeman (1986 a, b) suggested that males of Anarhichas minor and A. lupus had a tendency to weighed more than female at a given length at size over 50 and 60 cm respectively. Johannessen et al., (1993) reported no sex-related size differences prior to reproductive maturation for the common wolffish but described in great details the apparition of secondary sex characteristics (potbelly shape, papilla on urogenital pore, courting and reproductive behavior etc.). Later, Liao and Lucas (2000) and Gunnarsson at al. (2006, 2008) suggested no sex-related difference in weight and/or length for common wolffish population of the North Sea and common and spotted wolffish in Icelandic waters respectively In 2006, first domesticated F1 spotted wolffish became mature. The availability of both first-time breeders of A. minor and a mature captive population of A. lupus, enabled the initiation of research activities aimed at the control of maturation by photoperiod manipulations and the characterization of male and female growth patterns beyond 1 Kg in captivity. Early stages and juvenile growth performances of both wolffish species (A. minor
50 and A. lupus) has been the object of several aquaculture related studies (A. lupus: Stefanussen et al., 1993; Le François et al., 2004; Lamarre et al., 2004; A. minor: Savoie et al., 2006, 2008; Imsland et al., 2006, 2007; Tremblay-Bourgeois et al., 1010). Adult stage and reproduction have been the object of fewer research efforts (see Le François et al, 2010) and to our knowledge no study on sex-related differences of growth in captivity at the adult stage is reported.
The objectives of this work are multiple: 1) characterize the
growth rate of adult common and spotted wolffish, 2) compare the growth of adult spotted wolffish of Canadian and Norwegian origin and 3) examine the expression of a sexual dimorphism in growth in adult A. minor and A. lupus.
Materials and methods Broodstock origin The study was conducted at the Centre Aquacole Marin de Grande-Rivière (CAMGR, operated by the Ministère de l’Agriculture et de l’Alimentation du Québec), during the period of April 2006 to July 2007 on Canadian and Norwegian spotted wolffish of 2003year class and Common wolfish of 2000-year class. The Canadian stock of spotted wolffish
(A. minor-QC) originated from F1 of a wild
captive broodstock and was a mix of two females with multiple males. The Norwegian stock (A. minor-NW) was obtained from fertilized eggs from five females with multiple male from Troms Steinbit A/S (Rubbestad, Norway), a commercial operation.
Common
wolffish (A. lupus) stock was obtained from wild-fertilized eggs. All experimental fish were reared under ambient water temperature and photoperiod conditions prior to the experiments that started in February 2006. A. lupus could be qualified as a mature broodstock population (spawned in 2004 and 2005) and A. minor (NW and QC) first time spawners. No previous monitoring had been conducted to identify the sexes and no maturation sign were observed in A. minor. Initially, sex was identified by regular oocyte maturation monitoring using a Sonosite 180 plus ultrasound equipped with L38/10-5 transducer (SonoSite Canada Inc., Markham, ON, Canada) and later confirmed with analyse of plasma sexual steroids analysis (see Dupont Cyr et al., chapter 1 of this memoir).
51
Rearing condition Photoperiod and temperature acclimation began on February 16 th 2006. Fishes were individually tagged with an internal microchip (AVID Identification Systems Inc. Folson, LA, USA) in the dorsal muscle at the base of the dorsal fin and with an external tag model Rototag (Dalton I.D., Henley on Thames, Oxon, UK) through the base of the dorsal fin. In February 2006, A. minor were distributed randomly into fours raceways (500L). Each raceway contained 25 A. minor NW (wet weight 1121.8 ± 41.4g (S.E.); total length 44.6 ± 0.4cm) and 15 A. minor QC (wet weight 1075.8 ± 46.8g; total length 43.8 ± 0.5cm). Raceways were separated in two sections to separate each spotted wolffish strain (A. minor NW and A. minor QC). Spotted male-female ratio was 1.5:1. In April 2006, A. lupus was distributed randomly into six Swedish tanks (400L) with 25 A. lupus (wet weight 962.1 ± 24.7g; total length 46.1 ± 0.3cm) and a ratio male-female 1.7:1. Tanks were supplied with flowing seawater (flow: 8L/min, temperature: 7.5 - 8˚C), oxygen saturation was maintained at approximately 90% and salinity were approximately 30 g L-1 . Rearing tanks were held under two photoperiod regimes (Figure 7) (3 tanks per photoperiod for A. lupus wolffish and 2 raceways per photoperiod for A. minor); a natural simulated photoperiod (SNP)(48˚N on a 12 month period) and a compressed photoperiod (CP)(48˚ N compressed on an 8 month period). A Lightproof canopy enclosed each photoperiodic group, and four 100W bulbs positioned inside the tents provided light. On April 2006, light intensity was initially fixed at 50 lx at the water surface for both species; in June it was slightly reduced to 25 lux to control intra-specific competition and antagonistic behaviour in the A. lupus stock. This was applied to both species. Seasonal changes and daily cycles with simulated dawn and dusk were controlled with a photoperiodic controller (Sunmatch; Aquabiotech Inc., Coaticook, QC, Canada). Fishes were hand-fed 3 times per week to satiety. A. minor QC and A. minor NW were fed with Corey Aqua Clear 10 mm pellets (Corey Feed Mills Ltd, Fredericton, NB, Canada)
52 and A. lupus were fed with Inver Lanky Breed (INVE Aquaculture Inc., Salt Lake City, Utah, USA) 8mm pellets. Steroid analysis Steroid analyses were conducted at the Maurice Lamontagne Institute (MLI) (Fisheries and Oceans, Mont-Joli, Québec, Canada). Samples were transported from CAMGR to MLI in a dry shipper (liquid nitrogen) and stored at -80˚C. Plasma concentrations of 17-β-oestradiol (E2) and 11-ketotestosterone (11-KT) were measured by enzyme linked immunoassays (ELISA kits, Cayman Chemical, Ann Arbor, MI, USA) according to Roy et al., 2009. Frozen samples were thawed on ice and centrifuged at 11500 rpm. 11-KT samples were diluted in ELISA kit buffer, heated at 80°C for 30 min and then centrifuged at 11500 rpm. E2 samples were diluted in distilled water then spiked with H3-E2 (2500 cpm, GE healthcare, Montreal, QC, Canada) and vortexed with ether. The aqueous phase was frozen in liquid nitrogen whereas the organic phase was transferred to a coated glass tube. This step was repeated 3 times. Coatings were performed with Sigmacote (Sigma Aldrich, StLouis, MO, USA). Glass tubes were rinsed with Sigmacote, dried over night and rinsed with water. The organic phase was evaporated under a stream of nitrogen at room temperature and then reconstituted in ELISA buffer. The inter- and intra-assay coefficients of variation for the E2 assay were 13,7% (n = 20) and the KT assays were 7,8% (n = 21) (pools of samples with 50% binding). 11-KT was measured in male plasma and E2 was measured in female plasma only. Fish that died during the experiment were excluded from subsequent data analyses. Growth measurements Sampling was carried out every first week of every month between April 2006 and July 2007. Fish were anaesthetized with 80 ppm of benzocaine (4-Aminobenzoic acid ethyl ester; Sigma-Aldrich, Oakville, ON, Canada) and weighed to the nearest 0.2g (W) and total length (L) to the nearest 0.1cm were measured. Weight and length data were used to calculate specific growth rates (SGR) for individual fish, according to the formula: SGRW = 100*[ln(Wf) - ln(Wi)] / (tf - ti)
53 SGRL = 100*[ln(Lf) - ln(Li)] / (tf - ti) where Wf is the final wet weight, Wi is the initial wet weight, Lf is the final total length, Li is the initial total length and (tf - ti) is the number of day between each period. Sexual dimorphism (SD) was evaluated according to Saillant et al. (2001) with the formula: SDW = 100(Wmale - Wfemale) / Wmale SDL = 100(Lmale - Lfemale) / Lmale where Wmale and Wfemale are the respective mean weights of male and female at a specific sampling period and Lmale and Lfemale are the respective mean total length of male and female at a specific sampling period. The condition factor (CF) was calculated from the formula: CF = 100(W / L3 ) where W is the wet weight (g) and L is the corresponding total length (cm). Food conversion efficiency (FCE) was calculated as (Kinghorn, 1983): FCE = (W2 – W1 ) / C where C is the feed intake cover by the period of W 2 and W1 . Statistical analysis For all data homoscedasticity and normality were tested. When rejected, a logarithmic transformation was performed (Sokal and Rolf, 1995). A three way nested ANOVA (Searle et al. 1992), where the replicates (rearing tanks) were nested within photoperiod treatment (i.e., CP or SNP photoperiod), was applied to calculate the effect of different photoperiod and gender on weight, and length with a Bonferronni correction. SDW and SDL were tested with a Kruskal-Wallis Rank Sum Test. Significant ANOVA were followed by
54 TukeyHSD multiple comparison (Quinn and Keough, 2004) to located differences among the treatments. Significant difference were accepted when p < 0.05. All statistical tests were performed using R (R Development Core Team, 2011).
Results Body mass and total length Result suggest a statistical difference in body mass and total length between sexes in both species (A. lupus and A. minor) and strain (QC and NW). Suggesting the presence of a sexrelated growth dimorphism. Common wolffish (A. lupus) Mean body mass of both sexes of A. lupus was found significantly different for the entire sampling period (April 2006 - July 2007: 16 months), at the exception of data acquired in May and June 2006 (Figure 8A). Growth in length also suggests an overall statistical difference between sexes (Figure 8B). These observations strongly suggest that for repeated spawners such as 6+ A. lupus, sex-related growth differences were already established prior to the growth trial. Body mass SDW was of 15.0% in April 2006, increasing trough the spawning period to a level of 25.8% in November 2006 and decreased to 17.3% before the onset of the 2007 spawning period (Figure 8C). SDL was established at 5.9% in April 2006 and gently increased throughout the experiment to reach 7.0%in July 2007. For female and male the overall body mass gains of 25.1% and 30.8% and overall total length gains of 7.3% and 10.0% were recorded respectively. Spotted wolffish (A. minor-QC and NW) Body mass and total length of both sexes of A. minor-QC (Figure 9A, B) and A. minor-NW (Figure 10A, B) were not significantly different during the period prior to spawning events. Body mass was significantly different between sexes on July 2006 for A. minor-QC and September 2006 for A. minor-NW respectively. Thereafter, mean body mass of both sexes was at all time significantly different. In addition, total length was significantly different between sexes after the months of September 2006 (A. minor-QC; Figure 9B) and
55 November (A. minor-NW; Figure 10B). Growth differences suggest the establishment of a sex-related growth dimorphism occurring at first maturation. At the beginning of the growth trial (April 2006), mean body mass and mean total length percentage of difference between sexes for A. minor-QC were 14.3% and 2.8% respectively. After 16 month this value increased to 27.3% and 7.9%. The overall body mass gain was of 56.7% and 62.9% for female and male respectively. Whereas overall total length increases was of 18,6% and 23.1% for female and male respectively. For A. minor-NW, mean body mass and mean total length percentage of difference between sexes were 2.8% and 10.9% respectively on April 2006 and were 5.2% and 17.1% after 16 months (July 2007). The overall body mass increases of 59.4% and 62.1% for female and male respectively. Whereas overall total length increases of 21.2% and 23.1% for female and male respectively. There was no significant difference observed between each strain (QC and NW) for A. minor. Specific growth rate and condition factor Female SGRw of both species suggest a synchronization with the unfolding of the reproduction events. In comparison with female, male SGRW, SGRL and CF of both species and strains were similar for all the experiment period (p > 0.050). Therefore, female SGRW, SGRL and CF patterns of will be discussed according to three common phases i.e. immature, mature, spawning and post-spawning. Immature period: from the first day of the growth trial to the first observation of developing oocytes reaching 1mm, mature period: the period between oocyte at 1mm to the last sampling prior to ovulation, spawning period: the period before and after the ovulation and finally the post-spawning period: covered the period after the ovulation to the beginning of the next reproduction cycle. SGRW was significantly lower for A. lupus (p < 0.000) compared with both strain of A. minor (NW and QC). For all groups (A. lupus, A. minor NW and QC), SGRW , SGRL and CF were significantly lower for female (p < 0.000) at all times in particular during the spawning period. As expected, sexual maturation effect on SGRL was less pronounced than on SGRW (p = 0.102). A. lupus CF was significantly lower than A. minor of both strains.
56 Food conversion efficiency Overall FCE was significantly different between spotted and common wolffish (2 ways ANOVA, p < 0.000). Overall FCE covering the experiment period were significantly higher for spotted wolffish compared with common wolffish. For both species and strain, results did not proposed a FCE seasonal variation and did not proposed an effect of photoperiod treatment.
Discussion Our results suggest the presence of a sex-related growth dimorphism for wolffishes of both species (A. lupus and A. minor): adult males display higher growth rate and final body mass than adult females. To our knowledge this is the first time that a size dimorphism between sexes is clearly demonstrated for this family of fish in captivity and these results are reinforced by indirect field observations and conclusion proposed by Templeman in 1986 (a, b). Our results strongly suggest that sex-related growth dimorphism for wolffishes is directly linked to the maturation. In this study, two distinct groups of wolffishes were followed: a group of well-established spawners born in captivity and of similar life history (6 +, A. lupus) and a group of first time spawners (3 +, A. minor). A. lupus males were significantly larger then female for all the period covered by the experiment compared to A. minor were no difference in body mass were observed prior-maturation. These differences between each spawning status (mature, immature) strongly indicate that sexual maturation is the main factor explaining the presence of a sex-related growth dimorphism in wolffishes. We however propose that in captivity two main phenomenons could be acting in synergy for the emergence of a sex-related growth dimorphism: physiological and behavioural dissimilarities. In gonochoric species (species with separate sexes), physiological investment in oogenesis and spermatogenesis can widely vary between sexes (Malison et al., 1988). In wolffishes, ovaries represented 0.7 - 28% of the body mass of the female compared to testes which are
57 1.3% (Templeman, 1986a). Based on these results, Templeman (1986a) suggested the likelihood of a sex-related growth dimorphism benefiting males because of the disparity in energy allocation between sexes for gonadal growth. Our results support this assumption. The absence of significant gender difference observed in A. minor prior-maturation suggests that the physiological differences between sexes during gametogenesis would be a key factor for the expression of sex-related growth dimorphism in wolffishes. The second parameter involved is based on behavioural differences between wild and captive wolffishes. Keats et al. (1985) previously described dynamic parental care in wild A. lupus and observed feed intake reduction as gonadal growth progressed in both sexes as well as during the incubation of the egg masses by the male for a prolonged period. Wolffishes are also reported to lose their teeth few weeks before ovulation (Barsukov, 1959; Le François and Archer, 2005). Jönsson (1982) observed that during the incubation period, A. lupus wild adult male change from well nourished to an emaciated condition. In captivity, the male wolffishes of both species do not invest much energy into the reproduction events:
semen is collected from the fish manually, used to fertilize the eggs,
which are then incubated in incubators units without expression of their egg-guarding behaviour.
Whereas, females in captivity still experience energetic expenditures associated
with ovarian maturation. This difference could be a decisive factor in the detection of sexrelated growth dimorphism in captivity compare to wild observations. This shared lost of body mass during the reproduction/incubation events in the wild environment could explain the absence of clear sex-related growth dimorphism in previous studies in the wild. The absence of fasting in the captive male and his passive behaviour during reproduction seems to confer a male growth benefit compared to females in both species. Our results clearly suggest that the presence of growth dimorphism related to sex is in favour of males due to the alleviation of the reproductive behaviour in captive conditions linked to egg incubation, egg-guarding behaviour and to the lower energetic investment of males during spermatogenesis compared to oogenesis.
58 Photoperiod treatment had no impact on growth for A. lupus and A. minor. The effect of photoperiod on growth has been extensively studied in several species such as barramundi (Lates calcarifer) (Barlow et al., 1995) and Atlantic salmon (Salmo salar) (Hansen et al., 1992). A large number of studies describe the impact of photoperiod on juvenile growth, reduce larval period and/or changed the pattern of sexual maturation (Rowe and Thorpe, 1990a; Thorpe et al., 1994; Duston and Saunders, 1997). For this study, the photoperiod was changed to compress the reproductive cycle and not to maximize growth. Our data on A. minor enabled us to observe and describe the establishment of a sexual dimorphism over a long period after first maturation and to appreciate growth rate variations. The presence in the initial experimental design of a well-established sex-related difference in A. lupus group reinforced the hypothesis of sexual maturation as a breaking point in growth performances between the sexes in both species. Until now, no detailed growth study featuring adult fish had been proposed for the broodstock of these species in captivity. According to the result, the maturation of wolffishes of both species begins at the age of 3 - 4 years old when they reach 900 – 1000g. Sexual maturation begins sooner in captivity than in the wild. In a commercial aquaculture production context, our results suggest that the development of a monosex male production is desirable if commercial size is beneath 1.5 Kg.
Over that size selection of male fish
could enable to obtain higher average growth rate (male are 17.3%, 27.3% and 17.1% heavier than female for A. lupus, A. minor QC and NW respectively) and steady growth (male SGR are not affected by maturation) and possible induce higher productivity in the order of 10-15% higher, reduction of variation in harvest size and reduction of aggressive interactions. Techniques for production of monosex males are widely adopted by the aquaculture industry given their considerable benefits (Beardmore et al., 2000) it could be adapted to wolffishes.
Acknowledgements The present study was financed by the ACRDP and Aquanet (Department of Fisheries and Oceans) and the Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec.
59 Authors want to express their gratitude to the CAMGR staff as well as Tony Grenier, Arianne Savoie and Dany Ouellet (UQAR) for their technical support.
60
Tables and captions
Figure 7 : Photoperiod regimes of the different treatment groups in the period of April 2006 to December 2007 2006: SNP, (Grande-Rivière; 48° 24′ 00″ North, 64° 30′ 00″ West) simulated natural photoperiod; CP, photoperiod compressed to 8 months.
61
Figure 8 : A) Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A. lupus reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA followed by TukeyHSD, p < 0.05). N = 3 rearing tanks for each mean value, with 7-9 females and 16-18 males per tanks.
62
Figure 9 : Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A. minor QC reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA followed by TukeyHSD, p < 0.05). N =2 rearing tanks for each mean value, with 6-7 females and 8-9 males per tanks.
63
Figure 10 : A) Body mass (g), B) total length (cm) and C) % of sexual dimorphism of broodstock A. minor NW reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod. Results are given in mean ± S.E. There was no statistical different observed for the photoperiod or for the interaction between photoperiod and sexes at a given date. Sexes statistical differences at a given date was indicate with the * symbol (Three-way nested ANOVA followed by TukeyHSD, p < 0.05) N = 2 rearing tank for each mean value, with 9-10 females and 1415 males per tanks.
a
a
a
Spaw
Post
-0.05
Mat
Male
2.0
G) A.lupus
c
a
bc
ab
Mat
Spaw
Post
Male
0.0
a
Imm
0.6 0.4
Male
b abc
a abc
abc abc
bc bc
Mat
Spaw
Post
Male
0.0
0.2
Post
F) A.minor NW
0.05
SGRl (%) ab
bc
ab bc
Imm
Mat
Spaw
Post
Male
H) A.minor QC CP SNP
Condition factor
Condition factor
a
0.5
1.0 0.5
a CP SNP
1.5
CP SNP
Spaw
CP SNP
Imm
I) A.minor NW CP SNP
0.5
Imm
Mat
0.00 c
-0.05
a
2.0
a
a
0.10
E) A.minor QC
1.5
a
a
CP SNP
Imm
0.10 a
b
-0.2
Male
0.05
SGRl (%) a
a
-0.4 -0.6
Post
1.0
b
a
-0.8
Spaw
0.15
Mat
0.00
0.05
SGRl
a
0.0
Imm
0.00 -0.05
a
-0.4 -0.6
Male
CP SNP
1.5
2.0
b
CP SNP
-0.8
Post
0.15
Spaw
0.10
0.15
Mat
D) A.lupus
ab
Condition Condition factor factor (k)
a
-0.2
0.0 -0.2
SGRw
-0.4 -0.6 -0.8
CP SNP
Imm
0.0
a
0.2
a
C) A.minor NW
1.0
a
b
B) A.minor QC
a
a
b
bc
c
Imm
Mat
Spaw
Post
Male
0.0
0.6
a
0.2
a
0.4
A) A.lupus
0.4
0.6
64
a
a
Imm
a
ab
Mat
a
a
Spaw
b
b
Post
a
b
Male
Figure 11 : Broodstock SGRW (A) A.lupus, B) A.minor-QC, C) A.minor-NW), SGRl (D) A.lupus, E) A.minor-QC, F) A.minor-NW) and condition factor (k) (G) A.lupus, H) A.minor-QC, I) A.minor-NW) reared under two photoperiod regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod according to female prematuration (Imm), maturation (Mat), spawning (Spaw), post-spawning (Post) and male (Male). Results are given in mean ± S.E. Different lettes show significant difference beetween each period or group.
a
a
a
a
0.6
0.8
1.0
1.2
CP SNP
b
0.4
b
0.2 0.0
Food conversion efficiency (FCE)
1.4
65
A.minor QC
A.Lupus
A.minor NW
Figure 12 : Food conversion efficiency (FCE) for wolffishes reared under two photoperiods regimes: CP = 8 months compressed photoperiod and SNP = simulated natural photoperiod Results are given in mean ± S.E. Different letters show significant difference beetween each photoperiod treatment and group.
Discussion générale Les résultats de cette étude proposent que la photopériode à un effet significatif sur le contrôle du cycle reproducteur des deux espèces de loup de mer (A. minor et A. lupus). La photopériode serait un signal majeur dans le déclenchement de la maturation du loup de mer en captivité et elle permettrait l’installation d’une production annuelle constante en instaurant une fraie hors saison. Les résultats suggèrent aussi la présence chez les deux espèces d’un dimorphisme de croissance lié au sexe : les mâles ont un taux de croissance et une masse finale supérieurs aux femelles. Ces travaux ont permis une meilleure compréhension du cycle biologique du loup de mer (A. minor et A. lupus) et l’intégration des résultats de zootechnie en aquaculture commerciale semble prometteur.
Compression du cycle reproducteur En regard des résultats obtenus, la photopériode serait un facteur de premier plan dans le déclenchement de la maturation sexuelle chez le loup de mer (A. minor et A. lupus) en captivité. L'augmentation homogène des concentrations plasmiques en stéroïdes sexuels (E2 et 11-KT) et la différence significative dans le calendrier de ponte démontrent que le traitement photopériodique est un outil efficace afin d’instaurer une fraie hors saison (Prat et al., 1999; Howell et al., 2003). La réponse du loup de mer (A. lupus et A. minor) au traitement
photopériodique
est
similaire
a
celle
observée
chez d’autres
espèces
commerciales (Duston and Bromage, 1986; Hansen et al., 1992; Björnsson et al., 1994; Davis et al., 1999; Prat et al., 1999; Bromage et al., 2001; Howell et al., 2003; Norberg et al., 2004). Les résultats sont en accord avec Foss et al., (2004) qui rapportent qu’aux installations de Trom Steinbit AS (Troms county, northern Norway) le cycle reproducteur de loup de mer aurait été compressé en exposant les géniteurs à une photopériode saisonnière sur 9 mois. La compression du cycle naturel de la photopériode sur une période de huit mois a permis d’avancer de 3 — 6 mois la période de fraie, comparativement au groupe témoin SNP en 2007. Selon la littérature, la période de reproduction de A. lupus semble varier en fonction de l’origine du cheptel. Par exemple, une souche de A. lupus russe aurait frayé de juin — juillet jusqu’en septembre (Scott et Scott 1988; Pavlov et Radzikhovskaya 1991;
67 Dzerzhinskiy et Pavlov 1992), Johannessen et al., (1993) rapportent qu’un cheptel norvégien aurait frayé de la mi-octobre à la mi-février tandis qu’un autre le cheptel norvégien aurait frayé de novembre à janvier (Tveiten et al., 2001). En complément à ces observations, l’absence de différence significative dans la période d’ovulation entre les souches de A. minor QC et NW suggère que le cycle reproducteur du loup de mer aurait une oscillation circannuelle endogène qui pourrait être entraînée par une modification des variations saisonnières photopériodiques.
Les résultats de cette recherche corroborent cette
hypothèse et proposent que la photopériode serait un outil efficace dans la manipulation du cycle reproducteur du loup de mer (A. lupus, A. minor QC et NW). La photopériode agirait sur les variations nycthémérales de l’hormone mélatonine qui aurait un impact majeur sur l’axe pituitaire-hypothalamus gonadique (Porter et al., 2001a, b ; Bromage et al., 2001). Par conséquent, un profil photopériodique modulant vers de longues journées (solstice d’été) serait perçu comme un stimulant pour amorcer le développement de la reproduction, et à l’inverse un profil photopériodique modulant vers de courtes journées (solstice d’hiver), serait perçu comme un signal pour le déclenchement des stades ultérieurs de la gamétogénèse et de l’ovulation (Bromage et al., 1984 ; Takashima et Yamada, 1984; Bromage et al., 2001). Selon le régime photopériodique de cette étude (e.i. 48° 24′ 00″ Nord, 64° 30′ 00″ Ouest), la maturation sexuelle du loup de mer (A. minor et A. lupus) aurait été stimulée par l’augmentation de la durée des journées associée au solstice d’été et la ponte aurait lieu 1 à 2 avant le solstice d’hiver. Ceci est en accord avec le cycle naturel de ces espèces pour une latitude similaire (Brander, 1995) Nos résultats proposent que la reproduction du loup de mer (A. lupus, A. minor QC et NW) ne soit pas annuelle et que certains individus puissent sauter une ou deux années de ponte. Johannessen et al., (1993) ont observé ce phénomène et proposent que la maturation sexuelle du loup de mer en captivité serait limitée par la complexité du cycle de reproduction et à l’environnement spécifique nécessaire à l’expression du comportement reproducteur. Chez plusieurs espèces commerciales, il semble que la compression du cycle reproducteur pourrait affecter le pourcentage de maturation durant les premières années d’exposition au traitement (Duston et Bromage, 1988; Blythe et al., 1994 ; Björnsson et al., 1998). Chez les poissons marins, la période d’acclimatation à la compression du cycle
68 reproducteur par la manipulation de la photopériode peut s'échelonner sur une période de deux ans avant d’observer un effet sur le cycle biologique (Björnsson et al., 1998 ; MartinRobichaud et Berlinsky, 2004). Selon Foss et al., (2004), la période d'acclimatation à la compression de la photopériode du loup de mer serait de 18 mois. Les résultats obtenus dans cette recherche sont en accord avec cette observation. Dans cette étude, l’effet de la photopériode a été davantage marqué lors de la deuxième année d‘expérimentation (18 — 20 mois d’exposition). Il est documenté que la compression du cycle reproducteur par la photopériode peut entraîner une diminution du diamètre des œufs, du taux de maturation et du taux d’ovulation chez les femelles (Blythe et al., 1994; Bon et al., 1999; Morehead et al., 2000; Foss et al., 2004). Selon la littérature, les effets de la photopériode sur la qualité des produits sexuels ne sont pas faciles à clarifier. Ils impliquent généralement la modification de plusieurs paramètres tout au long de l’ovogenèse (Frantzen et al., 2004 ; Bobe et Labé, 2010). La compression du cycle reproducteur ne modifie pas seulement la durée entre les périodes de fraye, mais affecte également l’ensemble des paramètres endogénique de l’animal (comportement alimentaire, endocrinologie de la reproduction, métabolisme, etc.). Les résultats suggèrent que la réponse du loup (A. lupus et A. minor) au traitement photopériodique serait positive et aucun effet du traitement photopériodique n’a entrainé de diminution dans le diamètre des œufs, dans le taux de maturation et/ou dans le taux d’ovulation des femelles. Les loups de mer (A. lupus et A. minor) ont été en grande proportion spermiant toute l’année. Cette observation préliminaire supporterait l'hypothèse que le loup mâle serait spermiant toute l’année pour attirer les femelles en éjaculation de faible quantité de sperme contenant des phéromones (Johannessen et al., 1993 ; Pavlov et al., 1997 ; Foss et al., 2004). Ceci a été observé chez d’autres espèces telles que le bar (Dicentrarchus labrax) (Prat et al., 1999) et la sole (Solea senegalensis) (García-López et al., 2007). À la lumière des travaux de Tveiten et al., (2001), afin de prioriser la réponse au traitement photopériodique, la température a été maintenue à une température constante de 8 °C. La température n’est pas considérée comme le facteur principal dans la synchronisation de la maturation, mais le loup de mer est sensible à la température, comme l’ont démontré les
69 travaux de Tveiten et Johnsen (1999, 2001). Le protocole expérimental a été conçu afin de contrer les effets possibles de la température sur la maturation sexuelle. Toutefois, il convient de souligner que malgré les effets de la température sur la qualité des produits démontrés par plusieurs travaux antérieurs, la température de l'eau a été maintenue à une température légèrement supérieure celle optimale (6 °C ; Pavlov et Moksness, 1995; Hansen et Falk-Petersen, 2001; Tveiten et al., 2001 ; Tveiten et Johnsen, 2001; Imsland et al., 2006 ; Sund et Falk-Petersen, 2005) en raison des contraintes liées aux installations de refroidissement du CAMGR. La température de 8 °C a été choisie, car elle correspondait à la température minimale que le système été en mesure de fournir sur l’ensemble de l’expérience. Le profil décalé de la 11-KT démontre clairement un effet de la photopériode sur le cycle reproducteur des mâles chez les deux espèces (A. lupus et A. minor). Les profils plasmiques des stéroïdes sexuels du loup de mer (A. lupus, A. minor QC et NW) du groupe SNP sont semblables à ceux observés chez les téléostéens à maturation annuelle synchrone (Pankhurst, 1998). Le profil de la 11-KT propose une augmentation des teneurs en stéroïdes plasmiques lors de la période de fraye ce qui est en accord avec la littérature (Billard et al., 1982; Fostier et al., 1983; Borg 1994; Bromage et al., 2001). En 2006, les A. minor QC et NW de notre étude étaient à leur première ponte. Les mâles A. minor QC et NW sous photopériode CP n'ont pas eu de cycle de maturation clairement défini. Les faibles teneurs observées seraient liées à l’initiation du système endocrinien de la reproduction associé à la puberté (Holland et al., 2000). Pour les poissons en culture, il est souvent observé que plus d'un cycle est nécessaire avant d’obtenir des teneurs en stéroïdes maximales (Holland et al., 2000; Martin-Robichaud et al., 2004; Norberg et al., 2004). Les faibles teneurs observées pourraient être attribuées aux statuts de jeune géniteur et/ou à une longue période d’acclimatation. Depuis cette étude les produits sexuels sont de très bonne qualité et les taux de fertilisation sont élevés (Com. pers. Le François, 2008). En 2007, les teneurs maximales de E2 ont été observées aux mois d’août — octobre (A. lupus, A. minor QC et NW) tandis que celles soumises à la photopériode comprimée ont été devancées au mois de juin. Les teneurs sont demeurées faibles pour chacune des souches A. minor CP comparativement au groupe SNP. D’ordre général les données d’E2 ont été plus
70 faibles que les teneurs observées chez d’autres espèces comme le saumon de l’Atlantique dont les teneurs avoisinent les 30 ng ml-1 (Taranger et al., 1998). L’E2 induit la synthèse par le foie de la protéine précurseur du vitellus, la VTG (Idler et Ng Bun, 1983; Specker et Sullivan, 1994). Nagahama (1994) rapporte que l’E2 et la VTG sont positivement corrélées. Le profil décalé de l’E2 du groupe CP est un indice puissant indiquant que le profil de la VTG serait aussi affecté par la manipulation de la photopériode. Le signal ultime de la modification du cycle se traduit par un décalage dans la date d’ovulation du groupe CP comparativement au groupe témoin SNP. Les faibles teneurs d’E2 observée pourraient avoir influencer la gamétogénèse et cela suggère la possibilité d’un développement incomplet des gamètes et un taux de fécondation ultérieure plus bas. En conclusion, cette section a permis de démontrer que la manipulation de la photopériode serait un outil puissant dans le contrôle de la reproduction du loup de mer. Par conséquent, l’application de cette méthode en aquaculture commerciale permettrait d’instaurer une production annuelle constante et d’augmenter l’accessibilité en œufs et en larve. Suite à cette étude, le traitement a été poursuivi afin d’obtenir une deux pontes annuelles avec un groupe décaler en mai et un groupe naturel en octobre.
Croissance Nos résultats proposent la présence d’un dimorphisme de croissance lié au sexe chez les loups de mer des deux espèces (A. lupus et A. minor): les mâles auraient un taux de croissance et une masse finale supérieure à celui des femelles. À notre connaissance, c’est la première fois qu’un dimorphisme lié au sexe est clairement démontré pour cette espèce en captivité. Ces résultats sont renforcés par les observations indirectes en milieu naturel et les conclusions proposées par Templeman en 1986 (a, b). Les résultats suggèrent que le dimorphisme sexuel de croissance serait directement lié à la maturation. Lors de cette étude, deux groupes bien distincts ont été suivis : un groupe de géniteur bien établi (6+, A. lupus) et un autre composé de jeunes géniteurs qui complétaient leur première maturation (3+, A. minor). Chez A. lupus, les mâles étaient significativement plus gros que les femelles sur l’ensemble du suivi, tandis que chez les A. minor aucune différence dans la masse n’a été observée avant la maturation. Cette différence entre les
71 deux groupes propose que la maturation sexuelle soit le facteur principal expliquant la présence d’un dimorphisme de croissance lié au sexe. Selon cette hypothèse, deux principaux phénomènes seraient impliqués dans l’émergence d’un dimorphisme chez le loup: les modifications physiologiques et la différence comportementale entre les sexes. Il est
largement documenté chez les espèces gonochoriques que l’investissement
physiologique lié à l’ovogenèse et à la spermatogenèse peut être très différent (Malison et al., 1988). Chez A. lupus, les ovaires représentés de 0.7 — 28% de la masse de la femelle comparativement aux testicules qui représentent 1.3% de la masse maximale des ovaires (Templeman, 1986a). Suite à cette observation, Templeman (1986a) propose la possibilité d’un dimorphisme de croissance avantageant les mâles en raison de la grande disparité dans l’affectation énergétique entre les sexes. Nos résultats abondent dans ce sens. L’absence de différence significative entre les sexes observés chez les A. minor avant la maturation suggère que les différences physiologiques entre les sexes lors de la gamétogénèse seraient un facteur déterminant dans l’émergence du dimorphisme de croissance. La deuxième hypothèse est basée sur les différences comportementales entre les loups de mer sauvages et captifs. En captivité, le mâle ne participe pas à la reproduction. Cette différence majeure serait un élément déterminant dans la présence d’un dimorphisme de croissance lié au sexe observé en captivité. A. lupus est connue pour perdre sa dentition quelques semaines avant l’ovulation (Barsukov, 1959; Jönsson, 1982). Après la ponte, la femelle quitte le nid et le mâle fournira des soins parentaux à la masse d’oeufs jusqu’à l’éclosion des alevins. Durant cette période, le mâle jeûne jusqu’à l’éclosion des alevins (5 à 10 mois). Jönsson (1982) a observé que durant la période de jeûne le mâle passe d’un état replet à un état amaigri. Cette perde de masse corporelle pourrait justifier l’absence d’observation d’un dimorphisme de croissance lié au sexe en milieu naturel. En captivité, la femelle garde un comportement sensiblement similaire à celui des loups de mer sauvages. La femelle captive investit dans la reproduction tant sur le plan physiologique que comportemental. Cependant, l’absence de jeûne chez le mâle captif et son comportement passif lors de la reproduction semble lui conférer un cycle de croissance plus régulier que celui des femelles. Cette différence comportementale serait l’un des facteurs expliquant la présence d’un dimorphisme de croissance lié au sexe chez les loups (A. minor et A. lupus).
72 Selon nos observations, le dimorphisme de croissance observé entre les mâles et les femelles serait donc lié aux différences physiologiques entre les mâles et les femelles lors de la gamétogénèse ainsi qu’aux particularités comportementales du loup de mer en captivité. En juillet — août, les loups de mer ont entrepris leur maturation. Aucun comportement naturel de reproduction n’a été observé chez les mâles tandis que les femelles ont complété l’ensemble de leur cycle. Suite à la fraie par collecte des gamètes, les femelles ont réduit leur alimentation durant quelque semaine (1 — 3 semaines) avant de reprendre leur alimentation normale. Même après la collecte du sperme, aucune modification comportementale et aucun changement dans leur patron d’alimentation n’ont été observés chez les mâles. Nos résultats proposent clairement que la présence du dimorphisme de croissance lié au sexe avantageant les mâles serait attribuable à la dénaturation du comportement reproducteur des mâles ainsi qu’à leur faible investissement physiologique caractéristique au loup de mer lors de la spermatogenèse en comparaison à l'ovogenèse Les
observations
proposent
aussi
l’absence
d’effet
significatif
du
traitement
photopériodique sur la croissance et la prise alimentaires des loups tacheté et atlantique. Ceci suggère donc que durant la période d’avril 2006 à juillet 2007, aucune modification du métabolisme de croissance des loups n’a été observée entre les groupes CP et SNP. L’effet de la photopériode sur la croissance a été largement étudié chez plusieurs espèces (Hansen et al., 1992; Barlow et al., 1995). L’objectif de ces travaux visait à déterminer les conditions optimales d’élevage afin de maximiser la croissance et de réduire la période du stade larvaire ou de modifier le patron de maturation sexuelle (Rowe et Thorpe, 1990; Thorpe et al., 1990; Duston and Saunders, 1997). En gestion du cheptel de géniteur, l’objectif est bien différent. Pour cette étude, la photopériode a été modifiée afin de compresser le cycle reproducteur et non afin de maximiser la croissance. Dans ce contexte, toute modification dans la croissance était à éviter. Aucun dimorphisme de croissance n’avait été clairement observé chez A. minor et A. lupus. Les résultats proposent que la croissance des femelles mature A. minor et A. lupus soit cyclique en raison de la gamétogénèse. La dénaturation comportementale ainsi que le faible investissement lors de la spermatogenèse caractéristique à ces espèces avantageraient la
73 croissance des mâles. Aucun schéma de croissance n’avait encore été proposé pour les géniteurs de ces espèces. En captivité, la maturation du loup de mer débuterait vers l’âge de 3 ans lorsque le loup atteindrait 900 — 1000 g. Afin de maximiser la rentabilité en aquaculture commerciale, une gestion du sexe pour les produits de plus de 1 kg (post maturation) pourrait augmenter la productivité de 20 — 25%.
Condition d’élevage Entre février et avril 2006, un taux de mortalité élevé lors des prélèvements mensuels et un comportement agressif ont été observés chez exclusivement chez le loup atlantique. Seuls les loups atlantiques de l’étude photopériode semblaient démontrer un comportement agressif en comparaison avec les autres stocks du CAMGR et aucune observation similaire n’a été rapportée dans la littérature et/ou par d’autres groupes de recherche sur le loup (Com. pers. Tveiten, 2006). Deux hypothèses ont été explorées afin d’expliquer les mortalités observées chez le loup atlantique durant la période de février à avril 2006; le choix de l’anesthésiant et l’intensité lumineuse. En premier lieu, nous avons reconsidéré le choix de l'anesthésiant (benzocaïne) ainsi que la méthodologie utilisée. Les diverses manipulations sont reconnues pour avoir des effets importants sur la physiologie et le comportement des poissons (Ross et Ross, 1999). L’anesthésie est une méthode utilisée afin de minimiser le stress lors des manipulations (Palic et al., 2006), rendre l’animal inconscient et/ou d’enlever toute sensation sans affecter les mécanismes physiologiques (Brown, 1988; Ross et Ross, 1999; Bowser, 2001). Plusieurs
anesthésiants
bloquent
l’activation
de
l’axe
hypothalamus pituitaire-interne
associé au stress (Iwama et al., 1989; Thomas et Robertson, 1991). Dans le cas d’un anesthésiant sans inhibiteur, la réponse se traduira par l’activation des inducteurs de stress dont un des principaux signaux est l’augmentation du cortisol (Barton, 2002; Palic et al., 2006). Plusieurs anesthésiants comme le MS-222, l'eugénol, le métomidate et la benzocaïne ont été testés. Malgré les changements d’anesthésiants, aucune observation n’a permis de conclure à un effet négatif de la benzocaïne et/ou de la méthodologie. L’utilisation de la benzocaïne n’a jamais occasionné de problème dans les travaux antérieurs de l’équipe poisson marin (Le François et al., 2010).
74 En second lieu, il a été suggéré que l’intensité lumineuse utilisée de février à avril était inappropriée pour la biologie de l’espèce. En février 2006 lors du début de l’expérience, l’intensité lumineuse a été calibrée à 100 lux au niveau de la surface de l’eau, pour ensuite être réduite en avril 2006 à 50 lux et finalement à 25 lux en juin 2006. En réponse à la première diminution de l’intensité en avril 2006, les mortalités lors des prélèvements et le comportement
agressif
des
loups
atlantiques
ont
complètement
arrêté.
À
notre
connaissance, l’intensité et la nature de la lumière n’ont fait l’objet d'aucune recherche chez les géniteurs de loup. Il est par contre largement proposé que le loup préfère des conditions d’élevage équivalentes à celles de son habitat naturel (Foss et al., 2004). L’intensité lumineuse est un paramètre important affectant la croissance de plusieurs téléostéens d’élevage (Dowding et Litvak, 1999, 2002). Hoel et Pittman (1995) ont observé que les meilleurs taux de croissance chez le flétan (Hippoglossus) avaient été obtenus avec une intensité lumineuse de
1-10 lux comparativement à 500 lux. Les différents indices
permettent de supposer que l’intensité lumineuse aurait été une source de stress chronique. Les comportements agressifs des loups ont complètement disparu suite à la première diminution
de
l’intensité
lumineuse.
Les
mortalités
lors
des
prélèvements
étaient
probablement reliées à un état de stress très élevé et les animaux n’étaient pas en état de supporter un stress supplémentaire. Cette hypothèse serait en lien direct avec la littérature qui rapporte qu’un stress sévère et chronique est généralement associé à de mauvaises performances,
à
des
troubles
d’immunosuppressions,
à
des
modifications
comportementales et à une augmentation des mortalités (Beitinger, 1990; Pickering, 1998). En milieu naturel, le mâle choisirait une tanière et attirerait la femelle. La qualité du nid pourrait être un facteur déterminant dans l’attraction de la femelle et dans le succès reproducteur du mâle (Johannessen et al., 1993). En bassin, il est probable que l’abondance d’individus dans un environnement restreint lié à l’état de stress causé par l’intensité lumineuse aurait augmenté la compétition intraspécifique pour l’espace. Johannessen et al., (1993) proposent que la compétition intraspécifique augmenterait à l’approche de la période de reproduction et que le loup atlantique serait territorial lors de la formation des couples. Dans cette étude, ceci se serait traduit par des agressions répétées sur les individus de petite taille qui, après autopsie, s’avéraient être principalement des femelles. C’est la première fois que l’intensité lumineuse est suggérée comme un facteur pouvant affecté le
75 comportement (cannibalisme et agressivité) du loup de mer atlantique (A. lupus). L’intensité
lumineuse
devrait
être
investigué d’avantage afin validé les conditions
optimales d’élevage d’un cheptel de géniteur de loup atlantique.
Conclusion générale Le projet comportait deux hypothèses de recherche : 1. La compression du cycle saisonnier par manipulation de la photopériode sur une période de huit mois permettra l’obtention de deux pontes par cycle de deux ans chez les populations captives de A. lupus et A. minor. Le profil des hormones sexuelles impliquées dans la gamétogénèse sera modifié. 2. La compression du cycle reproducteur par manipulation de la photopériode n’affectera pas la croissance, le taux de conversion alimentaire et l’indice corporel des géniteurs comparativement à la population sous photopériode normale. En premier lieu, l’hypothèse la première hypothèse de recherche a été confirmée et en regard aux résultats obtenus, la photopériode est un outil efficace dans la gestion du cycle reproducteur du le loup de mer (A. lupus et A. minor). La plasticité du loup au traitement photopériodique permet de moduler la période de ponte et d’augmenter leur fréquence. L’utilisation de la photopériode permet de pallier à deux problèmes fondamentaux liés à l’élevage du loup: 1) accessibilité limitée à des juvéniles de qualité et 2) la grande variabilité dans qualité des produits sexuels. La diminution du cycle sur une période de 8 mois permet d’obtenir 3 pontes sur une période de 24 mois. Ce qui permet d’augmenter la productivité des géniteurs en place et l’accessibilité à des juvéniles. La compression du cycle et l’instauration de ponte hors saison permettent aussi de réduire les coûts liés au refroidissement de l’eau en instaurant une ponte au printemps. Comme la température est un facteur déterminant dans lors de l'ovogenèse, une ponte printanière permet de profiter des basses températures hivernales. Ce qui permet un approvisionnement en eau froide propice au bon développement des gamètes. En complément à la première hypothèse de recherche, la caractérisation du cycle hormonale du loup permet une meilleure compréhension du cycle reproducteur et s’est avéré un outil efficace dans le suivie de la maturation. La présence d’un pic unique de E2
76 permet d’affirmer que le suivie du cycle hormonal permettrait de déterminé précisément la période du cycle et l’avancement de la maturation. Cet outil pourrait permettre d’évaluer la période d’ovulation. Comme la reproduction du loup en captivité est entièrement faite artificiellement, un tel outil pourrait permettre d’identifier la période optimale pour la collecte des gamètes et ainsi réduire les risques de surmaturation. En second lieu, la deuxième hypothèse de recherche a été validée et les résultats proposent l’absence d’effet du traitement photopériodique sur la croissance, le taux de conversion alimentaire et l’indice corporel des géniteurs comparativement à la population sous photopériode normale. En lien avec cette hypothèse de recherche, les résultats ont permis d’observer éloquemment un dimorphisme de croissance lié au sexe. Cette caractéristique du loup en captivité est d’une grande pertinence pour le développement commercial du loup de mer. Le gain potentiel en croissance lié à une gestion du sexe pourrait permettre une augmentation de 10 — 15% sur la croissance pour des produits élevés postmaturation. Une monoculture de mâle devrait donc être envisageable pour la production d’individus de plus de 1.5 kg. Suite à cette étude, l’exposition des loups tachetés à la photopériode comprimée a été continuée au CAMGR. Le cycle de reproduction de ces poissons a été réduit, avec une production accrue d’œufs et de sperme de très bonne qualité. Présentement, un groupe fraie au mois de mai tandis que les animaux sous photopériode naturelle fraient en septembre. Ce compromis permet de produire deux fraies annuelles sans affecter la qualité des produits sexuels en conservant une période de 12 mois entre chaque reproduction.
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