Fish Physiol Biochem DOI 10.1007/s10695-013-9881-8
The effect of diet on ontogenic development of the digestive tract in juvenile reared long snout seahorse Hippocampus guttulatus J. Palma • D. P. Bureau • J. P. Andrade
Received: 1 April 2013 / Accepted: 12 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Ontogenetic development of the digestive tract and associated organs in long snout seahorse Hippocampus guttulatus juveniles was morphologically and histologically examined from the time of release from the male’s pouch until 72 h after the first meal. When released from the male’s pouch, juvenile seahorses are small adult replicates. This means that unlike other teleost fish larvae, the first developmental phase has already taken place, and juveniles are morphologically prepared and able to feed on live prey immediately following parturition. At this stage, the buccopharynx, oesophagus, and intestine already appear to be fully developed. The intestine is divided into the midgut and hindgut by an intestinal valve, and intestinal villi are visible in the midgut. When fed with DHA-SelcoÒ enriched Artemia, H. guttulatus juveniles developed a severe condition of overinflation of the gas bladder. The continuous overinflation of the gas bladder forced air into the gut (48 h after the first meal), resulting in overinflation of both the gut and the gas bladder (72 h after the first meal), and death
J. Palma (&) J. P. Andrade CCMAR, FCT, Universidade do Algarve, Edificio 7, Campus de Gambelas, 8005-139 Faro, Portugal e-mail: [email protected] D. P. Bureau UG/OMNR Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1, Canada
occurred within 120 h after the first meal. When fed natural copepods, H. guttulatus juveniles continued a normal feeding activity with no signs of intestinal disorders, and the gas bladder and intestine maintained their normal shape. This is the first study to positively associate gas bladder overinflation of juvenile seahorses with nutritionally unbalanced diets, and not to gas supersaturation alone. It is therefore necessary to develop more adequate feed and/or enrichment products to improve the survival of juvenile seahorses in captivity. Keywords Ontogeny Histology Digestive system Diet Long snout seahorse Hippocampus guttulatus
Introduction The study of fish ontogeny during the larval period has long been the preoccupation of biologists. Understanding the ontogenetic development of the digestive system is crucial for larval fish rearing in any economically important aquaculture species. Knowledge about the structural development of the digestive system is essential in order to understand the digestive physiology and to determine the appropriate time needed to wean fish larvae (Watanabe and Kiron 1994; Baglole et al. 1997; Cahu and Zambonino-Infante 2001). The successful development of the efficient
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digestive system is crucial for the survival and growth of fish larvae because it enables fish to capture, ingest, digest, and absorb food (Kjo¨rsvik et al. 2004). Recent data on physiology have provided a new perspective on nutritional problems, and recently acquired data have facilitated the task of nutritionists in the development of diets adapted for fish during these stages. Understanding the early ontogeny of digestive functionality as well as the nutritional and environmental requisites for properly triggering the coordinate series of hormonal and digestive processes occurring in this sensitive period is of primary importance when designing compound diets or enrichment media for natural diets for first feeding larvae (Yu´fera and Darias 2007). Seahorses are in demand worldwide; dried seahorses are used extensively in traditional Chinese medicine, and, to a lesser extent, as curios, while live seahorses are traded as aquarium fishes (Vincent 1996). The global trade in seahorses and their relatives was estimated to be at least 20 million seahorses a year (Vincent 1996), and nearly 80 countries traded seahorses and their relatives during 1996–2001 (McPherson and Vincent 2004; Baum and Vincent 2005). The number of species of wild-caught seahorses traded internationally remained reasonably constant between 1997 and 2008, while the number of species that were captive-bred increased over the period 2002–2004, indicating increasing participation in captive breeding (Koldewey and Martin-Smith 2010). The long snout seahorse Hippocampus guttulatus Cuvier 1829 is among those wild-caught species traded in large numbers (Koldewey and Martin-Smith 2010); thus, it may represent an opportunity for further diversification within the aquaculture industry. During the last decade, large-scale production of fish species has been hampered by very low larval survival, high incidence of skeletal deformities, nutritional disorders, extreme sensitivity to handling stress, and frequent pathological outbreaks during larval and growing phases (Rueda and Martinez 2001; Koumoundouros et al. 2004; Bermejo-Nogales et al. 2007). Therefore, it is critical to improve fish larviculture in order to synchronize the stage of larval development with the rearing technology. Above all, adequate nutrition is one of the main factors affecting larval survival, and it depends on the effective ingestion, digestion, and assimilation of diets, containing the required essential nutrients (Lazo et al. 2007).
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Persistent problems make seahorse breeding in captivity difficult (Fenner 1998; Hargrove 1998), and an adequate feeding regime is the key to maintaining healthy animals and to achieve enhanced juvenile production. Traditionally, maintaining seahorses has required the culture or collection of live prey, such as rotifers (Otero-Ferrer et al. 2010), Artemia (e.g., Payne and Rippingale 2000; Job et al. 2002; Hilomen-Garcia et al. 2003; Woods 2003; Martinez-Cardenas and Purser 2007; Olivotto et al. 2008; Lin et al. 2009a, b; Palma et al. 2011), copepods (Payne and Rippingale 2000; Gardner 2004; Sheng et al. 2006, 2007), and shrimp larvae (Palma et al. 2011) to feed them in their juvenile stages. Despite some recent studies on husbandry (Faleiro et al. 2008; Palma et al. 2008; Planas et al. 2008, 2009; Faleiro and Narciso 2010), juvenile growth (Palma et al. 2011), and diet effect on growth and reproduction (Palma et al. 2012), little is known regarding H. guttulatus nutrition and ontogenic development. The incorrect formulation of compound diets and enrichment media for natural diets (e.g., Artemia) is still a major bottleneck in fish farming. Compound diets need not only to take in consideration the specificities of fish larvae digestion (Cahu and Zambonino-Infante 1997) but also to provide live prey with the adequate nutritional value for larvae (McEvoy et al. 1998). Thus, incorrect feed formulation along with the natural deficiency of essential fatty acids (EFA) in live prey (e.g., rotifers and Artemia nauplii) commonly used in marine fish hatcheries to feed larvae (Conceic¸a˜o et al. 2010) may result in inadequate early life feeds and consequently poor larval growth and survival. Conversely, high larval growth and survival, normal pigmentation, and low frequencies of skeletal deformities are characteristic of marine fish reared on natural assemblages of marine zooplankton that mainly consist of copepods (Støttrup et al. 1998; Shields et al. 1999; Finn et al. 2002; Hamre et al. 2002). As an example, the use of copepods as feed compared to intensive rearing of cod larvae on rotifers indicated a significant nutritional advantage on juvenile quality and growth (Imsland et al. 2006). The superiority of copepods for larviculture of marine fish has recently increased interest for controlled culture of copepods (Støttrup 2003; Lee et al. 2005). In addition to previously mentioned problems shared with other species, a common issue with dramatic consequences on the juvenile seahorse brood
Fish Physiol Biochem
survival is the occurrence of air bubbles in the abdominal cavity. The hyperinflation of the gas bladder has been attributed to gas supersaturation and may cause extreme buoyancy and movement complications. It is known to affect virtually all seahorse species bred in captivity, and its occurrence was referred to by Woods (2000) when rearing H. abdominalis. In our previous experiments with H. guttulatus, high mortality rates were always associated with the presence of air bubbles in the gas bladder and subsequently in the intestine. Frequently proposed solutions such as decreasing the dissolved gas in water or preventing access to the water surface were ineffective. Air bubbles had a higher prevalence in juveniles that were fed Artemia enriched with commercial products in comparison with fish fed with either Artemia enriched with natural products (microalgae) (Palma et al. 2011) or with copepods (unpublished data). Therefore, the occurrence of internal air bubbles seemed to be due to inadequate or unbalanced juvenile nutrition rather than gas supersaturation in the water. Thus, this study aimed to evaluate the effect of diet on ontogenetic development of the digestive tract of H. guttulatus juveniles reared in captivity.
Materials and methods Feeding experiments F3 generation juveniles used in this experiment were obtained from an F2 generation H. guttulatus broodstock kept at the Ramalhete Aquaculture field station of the Universidade do Algarve, Portugal. Broodstock was maintained in one 500-L plastic square tank assembled in a flow-through system (60 L h-1) and was fed daily with a mix of frozen shrimp (Atlantic ditch shrimp, Palaemonetes varians) and mysids (Mesopodopsis slabberi) at approximately 6 % body weight per day. Holdfasts (see details in Palma et al. 2008) were added to the tanks for the seahorses to attach themselves to. Spawning occurred naturally (July 2011), whereupon juveniles from a single brood were randomly collected for this study. Juvenile-rearing trials were conducted with a completely randomized design, with three replicate tanks assigned to each of the two dietary treatments. Twenty H. guttulatus juveniles were stocked in 10-L glass rectangular tanks at a density of 2 fish per litre. In
each tank, the lateral and back walls were covered with a black adhesive sticker to improve prey detection (Woods 2000). The front wall was left uncovered for observation. Two artificial holdfasts adjusted to juvenile size composed of 5 branches (1.5 mm Ø and 10 cm length) were placed in each tank. Natural seawater flowed into the tanks at a constant flow (10 L h-1) in a closed circuit recirculation system. Seawater was pumped from the reservoir tank through a UV sterilization unit before entering the rearing tanks. The water in the reservoir tank was strongly aerated, and dissolved oxygen was always kept close to saturation. Technical characteristics of the rearing system are described in Palma et al. (2011). Seawater temperature, salinity, and dissolved oxygen were kept at 21.5 ± 0.3 °C, 37.5 ± 0.1 %, and 6.9 ± 0.1 mg L-1, respectively. Tanks were illuminated from above with 2 9 36 W fluorescent tubes, with a light intensity of 900 ± 40 lux at the water surface and a photoperiod controlled by a timer at 16L: 8D (06:00–22:00 h). Seawater quality data (ammonia, nitrates, and nitrites) were recorded at the start of the experiment. Ammonia values were below detectable levels: nitrate, 0.3 mg L-1 and nitrite, 1.25 mg L-1. Two different diets were tested; Artemia and copepods (Oithona nana). Artemia cysts (SandersÒ, Ogden, UT, USA) were hatched according to the procedures described by Sorgeloos et al. (1986). Artemia were enriched for 24 h with DHA-SelcoÒ (0.6 g L-1/24 h/100,000 Artemia) in one 20-L acrylic cylinder-conical tank at a maximum concentration of 50,000 Artemia L-1 held at room temperature (20–22 °C) and under constant moderate aeration. Copepods were naturally produced in the outflow pond of the Ramalhete Aquaculture field station, collected daily, and fed to juvenile seahorses. During the experiment, juvenile seahorses in each dietary treatment were fed 24 h enriched Artemia metanauplii at a density of 5,000 metanauplii L-1 or copepods at the same density. These two diets were chosen based on the fact that H. guttulatus juveniles fed Artemia enriched with DHA-SelcoÒ have a higher incidence of gas disorders [100 % mortality after 10 days post-parturition (DPP)] when compared with those fed Artemia enriched with Chlorella spp. (Palma et al. 2011). When fed natural copepods, the incidence of gas disorders is even lower than the ones fed Artemia enriched with Chlorella spp. (submitted data).
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An initial sample of 10 juveniles was collected just after parturition to be a reference sample. On each sampling day, four juveniles from each replicate tank were randomly sampled at 24, 48, and 72 h after the first feeding. In previous observations, we concluded that the gas bubbles reached their maximum prevalence near 72 h after the first feeding, after which affected juveniles start to die. Sampled juveniles were immediately and rapidly killed with an excess of anaesthetic (2-phenoxyethanol (Sigma-AldrichÒ, Portugal) solution, 0.40 mg L-1). Individuals were placed in the anaesthetic solution for at least 20 min and removed not \10 min after ventilation stopped, and then fixed in 4 % buffered formaldehyde solution. Samples were than dehydrated with gradual alcohol immersions (70–100 %) and embedded in cold-polymerizing resin (TechnovitÒ 7100, Kulzer). Longitudinal sections (3 lm thick) were obtained with a rotatory microtome (Microm HM 340 E, Microm International GmbH, Germany) and stained with a toluidine blue solution (Kuhlmann 2006). Hematoxylin/eosin (Humason 1979) and PAS colouration (Paulete and Bec¸ak 1976) methods were also tested, but failed to provide good staining results. The slide with the sections was mounted permanently using DPX. Observations by light microscopy (Leica Microsystems GmbH, Wetzlar, Germany) were performed at several magnifications in order to examine the development of the digestive tract and the occurrence and development of gas bubbles.
Henderson 1989). Total lipid extracts were subjected to acid-catalysed transmethylation for 16 h at 50 °C, using 1 mL of toluene and 2 mL of 1 % sulphuric acid (v/v) in methanol. The resulting fatty acid methyl esters (FAME) were purified by thin-layer chromatography (TLC) and visualized with iodine in chloroform/ methanol (2:1 v/v) 98 % (v/v) containing 0.01 % BHT (Christie 1982). Prior to transmethylation, heneicosanoic acid (21:0) was added to the TL as an internal standard. FAME were separated and quantified using a SHIMADZU GC 2010 (Kyoto, Japan) gas chromatograph equipped with a flameionization detector (250 °C) and a fused-silica capillary column RTX—WAXTM (10 m 9 0.1 mm I.D.). Helium was used as a carrier gas, and the initial oven temperature was 150 °C, followed by an increase at a rate of 90 °C min-1 to a final temperature of 250 °C for 3 min. Individual FAME were identified by reference to authentic standards and to a well-characterized fish oil. BHT, potassium chloride, potassium bicarbonate, and iodine were supplied by Sigma Chemical Co (St. Louis, USA). TLC (20 9 20 cm 9 0.25 mm) and HPTLC (10 9 10 cm 9 0.15 mm) plates, pre-coated with silica gel (without fluorescent indicator), were purchased from Macheren–Nagel (Du¨ren, Germany). All organic solvents used for GC were of reagent grade and were purchased from PANREAC (Barcelona, Spain). Statistical analysis
Lipid analysis Triplicate samples of Artemia were enriched as described above, and copepods were frozen and stored at -80 °C. The samples were then freeze-dried for 2 days. Total dry weights were recorded, and then, samples were taken for lipid extraction. Total lipid samples were separated into classes by one-dimensional double-development high-performance thinlayer chromatography (HPTLC) using methyl acetate/isopropanol/chloroform/methanol/0.25 % (w/v) KCl (25:25:25:10:9 by vol.), as the polar solvent system, and hexane/diethyl ether/glacial acetic acid (80:20:2 by vol.), as the neutral solvent system. Lipid classes were quantified by charring with a copper acetate reagent followed by calibrated scanning densitometry using a SHIMADZU CS-9001PC (Kyoto, Japan) dual wavelength flying spot scanner (Olsen and
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Differences in fatty acid composition (arc-sine squareroot-transformed data) of the enriched DHA-SelcoÒ enriched Artemia and natural copepods were tested using Kruskal–Wallis ANOVA with post hoc SNK (p = 0.05).
Results As a first observation, Fig. 1a shows a healthy juvenile seahorse with no signs of digestive disorder, whereas Fig. 1b shows a juvenile seahorse with a severe digestive disorder. At this point, the animal is no longer able to maintain normal buoyancy. Observing the samples prior to ingestion of the first meal enabled to characterize the physiology of the digestive tract of H. guttulatus juveniles. Unlike most teleost fish, male
Fish Physiol Biochem
Fig. 1 a Healthy juvenile seahorse with no signs of digestive disorder, and b a juvenile seahorse with a severe digestive disorder
Oesophagus Upon release from the male’s pouch, juveniles already had a buccopharyngeal cavity ending in the oesophagus. The oesophageal lumen, lined with a stratified squamous epithelium, had been formed to connect the buccopharyngeal cavity and intestine (Fig. 2). The oesophagus was surrounded by a layer of striated muscles, and the muscle layer became thicker as the juvenile grew. Stomach
Fig. 2 Morphological observation of a juvenile H. guttulatus prior to the first meal (91.25). B brain, G gills, OE oesophagus, L liver, GB gas bladder, MG midgut, LU lumen, HG hindgut, A anus, N notochord, IV intestinal villus (940 magnification)
seahorses provide the parental care for their brood, and when expelled from the male marsupium, the offspring are fully developed with the capacity to start exogenous feeding within the first 24 h postparturition.
Seahorses do not possess a proper stomach as ingested food enters directly through the oesophagus to the intestine. Intestine After the parturition (Fig. 2) and prior to the first meal, the intestinal maturation of H. guttulatus juveniles was observed to be advanced. The intestine was divided into the midgut and hindgut by an intestinal valve. The midgut was wider, almost sac-shaped, and the hindgut was a straight tube. Intestinal villi in the midgut were already visible (Fig. 2).
Buccopharynx 24 h after first meal The buccopharynx was present and was sided by a squamous epithelium (Fig. 1). It was not possible to observe the pharyngeal tooth, but the tongue was present.
At this stage, it was not possible to identify any major differences between the juveniles fed DHA-SelcoÒ enriched Artemia and those fed natural copepods. In
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tanks, it was observed that some juvenile seahorses had extreme difficulty maintaining neutral buoyancy, and others bumped against the water surface in a constant effort to maintain stable buoyancy. Conversely, juveniles fed natural copepods showed no signs of intestinal disorders, and the gas bladder and intestine maintained their normal shapes. The digestive tract elongated; the gut lumen increased in size, and the intestinal villi were distended (Fig. 5a). The fullness of the whole gut was identified in all sampled fish. In the midgut, it was possible to identify the prey shape (Fig. 5b, c), and the hindgut appeared full with well-digested matter. In the tanks, it was observed that the juvenile seahorses fed natural copepods did not show any signs of swimming disorders and had normal control of their buoyancy. 72 h after first meal
Fig. 3 Morphological observation of a juvenile H. guttulatus 24 h after the first meal (magnification at 91.2). E eye, B brain, G gills, OE oesophagus, L liver, MG midgut, HG hindgut, A anus
the juvenile fish that were fed natural copepods, the hindgut was observed to be completely full, while almost no feed particles were identified in the midgut (Fig. 3). 48 h after first meal At this stage, it was possible to identify morphological differences between H. guttulatus juveniles fed DHASelcoÒ enriched Artemia and those fed natural copepods. In juveniles fed Artemia, the first signs of overinflation of the gas bladder were identified (Fig. 4). Overinflated gas bladders were observed in all sampled fish fed Artemia, but the inflation severity varied between fish. In Fig. 4a), the gas bladder appears overinflated, but the intestine maintains its integrity. In addition, there were still some feed particles in the midgut, and the intestinal villi retained their typical shape. However, other fish displayed a more severe condition as the overinflation of the gas bladder reached a peak, bursting air into the hindgut and starting to fill it up (Fig. 4b). At this stage in the
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At this stage, all sampled fish fed DHA-SelcoÒ enriched Artemia displayed overinflated gas bladders. Overinflation appeared to reach a maximum, and in all observed samples, the air started to burst into the hindgut. Despite the closeness between the hindgut and the anus, fish were unable to excrete the air, which invaded the midgut. In Fig. 6a, it can be seen that the air has burst from the gas bladder to the hindgut causing it to overinflate as well. This condition forces feed to remain in the hindgut, which at this point can only be identified in the upper part of the gut. Finally, the air migrated to the midgut, completing the overinflation of the intestine (Fig. 6b). At this stage in the tanks, juvenile seahorses that were fed Artemia had no control of their swimming ability and were lying horizontally at the water surface. Their movements were erratic with uncoordinated tail attempts to regain normal buoyancy. In juveniles that were fed natural copepods, the digestive process was the same as described above at 48 h after the first meal and no signs of digestive disorder were identified. Again, the fullness of the whole gut was identified in all sampled fish. Preys were identifiable in the midgut, and the hindgut appeared full with well-digested matter. In the tanks, juvenile seahorses fed natural copepods did not show any signs of swimming disorders and maintained perfect control of their buoyancy.
Fish Physiol Biochem
Fig. 4 a Morphological observation of a juvenile H. guttulatus 48 h after being fed with DHA-SelcoÒ enriched Artemia (91.25) and b detail of the intestine and gas bladder (92.5).
B brain, G gills, OE oesophagus, GB gas bladder, MG midgut, HG hindgut, A anus, N notochord, MF muscle fibre
Fatty acid analysis
fish mainly depend on pinocytosis and intracellular digestion and absorption (Watanabe 1982). The third phase begins with the presence of gastric glands and pyloric caeca to metamorphosis onward, indicating the functional maturation of the digestive system (Bisbal and Bengtson 1995). Unlike most marine fish larvae that hatch with a rudimentary digestive system and without mouth (Yu´fera and Darias 2007), seahorses are fully developed when released from the male’s pouch and are small miniatures of the adult stages. In most of the seahorse species, the yolk sac is fully consumed when seahorse larvae are still in the male’s pouch. Therefore, on parturition, seahorse larvae can no longer depend on the yolk sac for energy; their digestive system is fully developed, and they can and need to start feeding just a few hours after being released from male’s pouch. Due to this fact, and as it concerns seahorses, the term ‘‘juvenile’’ is used since the first DPP, and at the moment they are expelled from the male’s pouch, they fit between the second and third stages of normal fish larvae ontogenetic development of the digestive system. Under microscopy, it was possible to identify all the internal organs associated with the digestive system at a fully developed stage. The maturation of the digestive system was also confirmed by the
Both similarities and marked differences in the fatty acid profiles of the enriched DHA-SelcoÒ enriched Artemia and natural copepods were observed (Table 1). Artemia DHA-SelcoÒ enriched had significantly higher (p \ 0.05) amount of both EPA and DHA than natural copepods, but were no different (p [ 0.05) in AA. The percentage of saturated fatty acids was significantly higher in the natural copepods, whereas the percentage of monounsaturated fatty acids was in Artemia DHA-SelcoÒ enriched.
Discussion Ontogenetic development of the digestive system in most teleost fish is generally divided into three major phases (Boulhic and Gabaudan 1992; Bisbal and Bengtson 1995). The first phase begins with hatching and ends at the completion of endogenous feeding, during which time larvae depend on energy reserves in the yolk sac and oil globules. The second phase begins with the onset of exogenous feeding and ends before the formation of gastric glands in the stomach, characterized by the lack of sufficient digestive capabilities (Boulhic and Gabaudan 1992). During this phase, larval
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Fig. 5 a Morphological observation of a juvenile H. guttulatus 48 h after being fed natural copepods (92.). E eye, B brain, G gills, MG midgut, HG hindgut, A anus, N notochord, DF dorsal fin; b prey detail I. Abbreviations: CC copepod cephalothorax,
CR copepod rostrum, CE copepod eye, IL intestinal lumen, IV intestinal villi. c prey detail II. Abbreviations: C copepod, CR copepod rostrum, CE copepod eye, CT copepod telson, IV intestinal villi
simple fact that H. guttulatus juveniles initiated their feeding activity just a few hours after parturition. All of the observed specimens presented full guts within the first 24 h after the first meal, and within that period, no visual differences between the two dietary treatments were found. This changed dramatically in fish fed DHA-SelcoÒ enriched Artemia as 48 h after the first meal, it was observed that all sampled fish had overinflated gas bladders, and in some specimens, air was already present in the hindgut. At
72 h, the condition of the juveniles became even more severe due to the fact that the gas bladder and the gut (both the midgut and hindgut) were overinflated and all other organs were pushed against the upper part of the abdominal cavity. As juveniles are unable to reverse this situation, massive mortality occurred within the next 48 h, and as referred by Palma et al. (2011), when fed DHA-SelcoÒ enriched Artemia, 100 % mortality occurs within the first 120 h after the first meal. Ultimately, this pathology
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Fig. 6 a Morphological observation of a juvenile H. guttulatus 72 h after being fed with DHA-SelcoÒ enriched Artemia (91.25) and b a second specimen with a severe condition of
gut overinflation (91.25). B brain, G gills, MG midgut, HG hindgut, A anus, N notochord, FM feed matter, D diaphragm
prevents the fish from feeding and maintaining controlled buoyancy. Buoyancy is controlled by the gas bladder and in several percoid fishes, including seahorses, a diaphragm separates the posterior, or gas resorbing, part of the gas bladder from the anterior, gas-producing section. Such fish are called ‘‘euphysoclistic,’’ and in these species, the gas resorption is accompanied by contraction of the gas-secreting chamber (Bond 1996). The pathology observed in this study seems to cause an imbalance in the gas exchange between the two parts of the gas bladder and inaptitude to maintain a normal inflation of the gas bladder. Unlike the condition described above, when fed natural copepods, H. guttulatus juveniles maintained a normal feeding activity and no mortality was registered within the same period of time. In this experiment, feed was the only changing factor; thus, it is unlikely that the causes of gas bladder overinflation can be attributed to supersaturation alone or even gas bladder malformations commonly described for cultured marine fish larvae (Chatain 1990, 1994; Battaglene et al. 1994; Trotter et al. 2001). At the present state of knowledge, for most marine species, the inert feed that have been developed is not suitable for the first phases of larval development and larval nutrition should be examined in the light of
ontogenic development of the larvae and juvenile digestive systems (Kolkovski 2001). According to Cahu and Zambonino-Infante (1997), this constrain is due to the fact that the conventional compound diets do not meet the larval nutritional requirements and survival can fall sharply when not all the feeding and environmental requirements are properly met (Yu´fera and Darias 2007). It is therefore necessary to feed these larvae (and juvenile seahorses) with live prey from the point of first feeding up to the post-larval stage/older juvenile seahorses when animals can adapt to inert or frozen food. The nutritional quality of prey has been studied, and attempts have been made to improve it. Progress has been made for several species, but seahorse nutrition poses new challenges to modify and improve the already existing products. The digestion of nutrients occurs in the gastrointestinal tract and is performed by the enzymes of the stomach, exocrine pancreas, and intestine (Zambonino Infante and Cahu 2001), but concerning seahorses, nothing is known about digestive enzymes. As seahorses do not have a proper stomach as other fish species do, it is arguable that stomach enzymes present in other fish species may lack or be diminished in seahorse’s digestive tract, thus additionally reducing their ability to deal with nonnatural diets. Nutritional imbalances are known to play a key role in morphogenesis and skeletogenesis during early stages of development.
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Fish Physiol Biochem Table 1 Fatty acid content (lg of fatty acid per mg of dry weight) of DHA-SelcoÒ enriched Artemia and natural copepods Fatty acid
Natural copepods
DHA-Selco ArtemiaÒ
R 14:0
5.7 ± 0.3a
R 15:0
2.1 ± 0.1
a
1.13 ± 0.1a
18.1 ± 0.3
a
27.31 ± 0.4b
11.1 ± 0.3
a
6.93 ± 0.2a
6.4 ± 0.3
a
6.91 ± 0.2a
18:1n9 18:1n7
a
22.1 ± 0.5 6.5 ± 0.2a
2.22 ± 0.03b 3.24 ± 0.1b
18:2n6
8 ± 0.4a
1.05 ± 0.05b
18:3n3
1.8 ± 0.2
a
1.31 ± 0.004b
4.3 ± 0.1
a
0.97 ± 0.003b
20:4n6
2.1 ± 0.3
a
0.96 ± 0.02a
20:5n3
31.3 ± 0.9a
8.77 ± 0.01b
22:5n6
1 ± 0.1
a
0.49 ± 0.004b
4.3 ± 0.4
a
0.74 ± 0.02b
30 ± 0.3
a
17.18 ± 0.12b
% Saturated
33.4 ± 0.5
a
47.77 ± 0.8b
% Monounsaturated
48.4 ± 0.5a
15.95 ± 0.6b
R 16:0 16:1n7 18:0
18:4n3
22:5n3 22:6n3
% n-3 HUFA DHA/EPA AA/EPA AA/DHA
68.9 ± 0.8
10.02 ± 0.36b
a
27.14 ± 0.14b a
1.96 ± 0.04b
0.96 ± 0.02
a
0.109 ± 0.002a
a
0.056 ± 0.001a
0.067 ± 0.002 0.070 ± 0.001
Data are the mean ± SD, row values with different Superscripts are significantly different (p \ 0.05)
Nutritionally, dietary lipids have been shown to be particularly important for the early development of marine finfish larvae (Sargent et al. 2002). They represent the primary energy source for larvae, and a source of highly unsaturated fatty acids (HUFA) and EFA needed for new cellular structures, as well as required for normal larval growth, morphogenesis, and bone formation (Cahu et al. 2003; Izquierdo et al. 2000; Lall and Lewis-McCrea 2007). Seahorses are no exception, and according to Faleiro and Narciso (2010), the fatty acid consumption during H. guttulatus embryonic development is considerably high, the PUFA constitute the main source of metabolic energy, and the fatty acids 16:0, EPA, and DHA are the main fatty acids that fulfil the energetic demands of H. guttulatus embryos. These patterns of embryonic consumption result in newborn juveniles with exceptionally low lipid content and therefore a need to ingest well-balanced diets to rapidly satisfy their requirements for fatty acids.
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Series n-3 EFA (EFA n-3) content is generally high in copepods, which seem to have the capacity to concentrate them, particularly DHA. In comparison, EFA content as well as the DHA/EPA ratio is low in Artemia. Certain species of copepods have great capacity for elongating and de-saturating n-3 FA’s, but these capacities are low in Artemia (Robin and Gatesoupe 2001). Artemia are not passive carriers of fatty acids, but have specific physiological needs that can metabolically alter the original composition of their diet by retroconverting DHA into EPA, and by redistributing the incorporated fatty acids among lipid classes with high unpredictability (Navarro et al. 1999). The metabolism inherent to Artemia nauplii causes them to accumulate EPA at higher levels than DHA and consequently decreases the DHA/EPA ratio from the enriching products. Sargent et al. (1997) determined an optimal dietary DHA/EPA ratio of 2:1 for newly hatched larvae from the lipid composition of the yolk sac in marine fish eggs. According to these authors, a DHA/EPA ratio inferior or equal to 1 corresponds to a suboptimal diet as it provides larvae with insufficient amounts of DHA or an excess of EPA and could be deleterious in larval fish feeds. These statements were confirmed by the present study, as EPA and DHA values were quite similar in Artemia resulting in a DHA/EPA below 1. However, the higher amount of DHA than EPA on the natural copepod diet resulted in a DHA/EPA ratio of 1:96. Ultimately, the metabolic ability Artemia possess to modify both the FA ratio provided through the enrichment media, and the use of unbalanced enrichments for juvenile seahorse nutritional requirements may cause the digestive disorders observed in this study. Several dietary components have been identified as negative vectors in the correct larval development (Cahu and Zambonino-Infante 1997; Cahu et al. 2003; Lall and Lewis-McCrea 2007) and have been proven in the present study with juvenile seahorse development. Nonetheless, the specific mechanisms that cause the observed pathology remain unknown. Exact biochemical analysis of feeds and different approaches regarding the histological procedures are needed to provide answers to this limitation to seahorse production in captivity. Acknowledgments Jorge Palma was supported by a postdoctoral grant (Foundation for Science and Technology—
Fish Physiol Biochem Portugal, BPD/34816/2007). We thank the reviewers for their valuable comments.
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The effect of diet on ontogenic development of the digestive tract in juvenile reared long snout seahorse Hippocampus guttulatus J. Palma • D. P. Bureau • J. P. Andrade
Received: 1 April 2013 / Accepted: 12 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Ontogenetic development of the digestive tract and associated organs in long snout seahorse Hippocampus guttulatus juveniles was morphologically and histologically examined from the time of release from the male’s pouch until 72 h after the first meal. When released from the male’s pouch, juvenile seahorses are small adult replicates. This means that unlike other teleost fish larvae, the first developmental phase has already taken place, and juveniles are morphologically prepared and able to feed on live prey immediately following parturition. At this stage, the buccopharynx, oesophagus, and intestine already appear to be fully developed. The intestine is divided into the midgut and hindgut by an intestinal valve, and intestinal villi are visible in the midgut. When fed with DHA-SelcoÒ enriched Artemia, H. guttulatus juveniles developed a severe condition of overinflation of the gas bladder. The continuous overinflation of the gas bladder forced air into the gut (48 h after the first meal), resulting in overinflation of both the gut and the gas bladder (72 h after the first meal), and death
J. Palma (&) J. P. Andrade CCMAR, FCT, Universidade do Algarve, Edificio 7, Campus de Gambelas, 8005-139 Faro, Portugal e-mail: [email protected] D. P. Bureau UG/OMNR Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1, Canada
occurred within 120 h after the first meal. When fed natural copepods, H. guttulatus juveniles continued a normal feeding activity with no signs of intestinal disorders, and the gas bladder and intestine maintained their normal shape. This is the first study to positively associate gas bladder overinflation of juvenile seahorses with nutritionally unbalanced diets, and not to gas supersaturation alone. It is therefore necessary to develop more adequate feed and/or enrichment products to improve the survival of juvenile seahorses in captivity. Keywords Ontogeny Histology Digestive system Diet Long snout seahorse Hippocampus guttulatus
Introduction The study of fish ontogeny during the larval period has long been the preoccupation of biologists. Understanding the ontogenetic development of the digestive system is crucial for larval fish rearing in any economically important aquaculture species. Knowledge about the structural development of the digestive system is essential in order to understand the digestive physiology and to determine the appropriate time needed to wean fish larvae (Watanabe and Kiron 1994; Baglole et al. 1997; Cahu and Zambonino-Infante 2001). The successful development of the efficient
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digestive system is crucial for the survival and growth of fish larvae because it enables fish to capture, ingest, digest, and absorb food (Kjo¨rsvik et al. 2004). Recent data on physiology have provided a new perspective on nutritional problems, and recently acquired data have facilitated the task of nutritionists in the development of diets adapted for fish during these stages. Understanding the early ontogeny of digestive functionality as well as the nutritional and environmental requisites for properly triggering the coordinate series of hormonal and digestive processes occurring in this sensitive period is of primary importance when designing compound diets or enrichment media for natural diets for first feeding larvae (Yu´fera and Darias 2007). Seahorses are in demand worldwide; dried seahorses are used extensively in traditional Chinese medicine, and, to a lesser extent, as curios, while live seahorses are traded as aquarium fishes (Vincent 1996). The global trade in seahorses and their relatives was estimated to be at least 20 million seahorses a year (Vincent 1996), and nearly 80 countries traded seahorses and their relatives during 1996–2001 (McPherson and Vincent 2004; Baum and Vincent 2005). The number of species of wild-caught seahorses traded internationally remained reasonably constant between 1997 and 2008, while the number of species that were captive-bred increased over the period 2002–2004, indicating increasing participation in captive breeding (Koldewey and Martin-Smith 2010). The long snout seahorse Hippocampus guttulatus Cuvier 1829 is among those wild-caught species traded in large numbers (Koldewey and Martin-Smith 2010); thus, it may represent an opportunity for further diversification within the aquaculture industry. During the last decade, large-scale production of fish species has been hampered by very low larval survival, high incidence of skeletal deformities, nutritional disorders, extreme sensitivity to handling stress, and frequent pathological outbreaks during larval and growing phases (Rueda and Martinez 2001; Koumoundouros et al. 2004; Bermejo-Nogales et al. 2007). Therefore, it is critical to improve fish larviculture in order to synchronize the stage of larval development with the rearing technology. Above all, adequate nutrition is one of the main factors affecting larval survival, and it depends on the effective ingestion, digestion, and assimilation of diets, containing the required essential nutrients (Lazo et al. 2007).
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Persistent problems make seahorse breeding in captivity difficult (Fenner 1998; Hargrove 1998), and an adequate feeding regime is the key to maintaining healthy animals and to achieve enhanced juvenile production. Traditionally, maintaining seahorses has required the culture or collection of live prey, such as rotifers (Otero-Ferrer et al. 2010), Artemia (e.g., Payne and Rippingale 2000; Job et al. 2002; Hilomen-Garcia et al. 2003; Woods 2003; Martinez-Cardenas and Purser 2007; Olivotto et al. 2008; Lin et al. 2009a, b; Palma et al. 2011), copepods (Payne and Rippingale 2000; Gardner 2004; Sheng et al. 2006, 2007), and shrimp larvae (Palma et al. 2011) to feed them in their juvenile stages. Despite some recent studies on husbandry (Faleiro et al. 2008; Palma et al. 2008; Planas et al. 2008, 2009; Faleiro and Narciso 2010), juvenile growth (Palma et al. 2011), and diet effect on growth and reproduction (Palma et al. 2012), little is known regarding H. guttulatus nutrition and ontogenic development. The incorrect formulation of compound diets and enrichment media for natural diets (e.g., Artemia) is still a major bottleneck in fish farming. Compound diets need not only to take in consideration the specificities of fish larvae digestion (Cahu and Zambonino-Infante 1997) but also to provide live prey with the adequate nutritional value for larvae (McEvoy et al. 1998). Thus, incorrect feed formulation along with the natural deficiency of essential fatty acids (EFA) in live prey (e.g., rotifers and Artemia nauplii) commonly used in marine fish hatcheries to feed larvae (Conceic¸a˜o et al. 2010) may result in inadequate early life feeds and consequently poor larval growth and survival. Conversely, high larval growth and survival, normal pigmentation, and low frequencies of skeletal deformities are characteristic of marine fish reared on natural assemblages of marine zooplankton that mainly consist of copepods (Støttrup et al. 1998; Shields et al. 1999; Finn et al. 2002; Hamre et al. 2002). As an example, the use of copepods as feed compared to intensive rearing of cod larvae on rotifers indicated a significant nutritional advantage on juvenile quality and growth (Imsland et al. 2006). The superiority of copepods for larviculture of marine fish has recently increased interest for controlled culture of copepods (Støttrup 2003; Lee et al. 2005). In addition to previously mentioned problems shared with other species, a common issue with dramatic consequences on the juvenile seahorse brood
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survival is the occurrence of air bubbles in the abdominal cavity. The hyperinflation of the gas bladder has been attributed to gas supersaturation and may cause extreme buoyancy and movement complications. It is known to affect virtually all seahorse species bred in captivity, and its occurrence was referred to by Woods (2000) when rearing H. abdominalis. In our previous experiments with H. guttulatus, high mortality rates were always associated with the presence of air bubbles in the gas bladder and subsequently in the intestine. Frequently proposed solutions such as decreasing the dissolved gas in water or preventing access to the water surface were ineffective. Air bubbles had a higher prevalence in juveniles that were fed Artemia enriched with commercial products in comparison with fish fed with either Artemia enriched with natural products (microalgae) (Palma et al. 2011) or with copepods (unpublished data). Therefore, the occurrence of internal air bubbles seemed to be due to inadequate or unbalanced juvenile nutrition rather than gas supersaturation in the water. Thus, this study aimed to evaluate the effect of diet on ontogenetic development of the digestive tract of H. guttulatus juveniles reared in captivity.
Materials and methods Feeding experiments F3 generation juveniles used in this experiment were obtained from an F2 generation H. guttulatus broodstock kept at the Ramalhete Aquaculture field station of the Universidade do Algarve, Portugal. Broodstock was maintained in one 500-L plastic square tank assembled in a flow-through system (60 L h-1) and was fed daily with a mix of frozen shrimp (Atlantic ditch shrimp, Palaemonetes varians) and mysids (Mesopodopsis slabberi) at approximately 6 % body weight per day. Holdfasts (see details in Palma et al. 2008) were added to the tanks for the seahorses to attach themselves to. Spawning occurred naturally (July 2011), whereupon juveniles from a single brood were randomly collected for this study. Juvenile-rearing trials were conducted with a completely randomized design, with three replicate tanks assigned to each of the two dietary treatments. Twenty H. guttulatus juveniles were stocked in 10-L glass rectangular tanks at a density of 2 fish per litre. In
each tank, the lateral and back walls were covered with a black adhesive sticker to improve prey detection (Woods 2000). The front wall was left uncovered for observation. Two artificial holdfasts adjusted to juvenile size composed of 5 branches (1.5 mm Ø and 10 cm length) were placed in each tank. Natural seawater flowed into the tanks at a constant flow (10 L h-1) in a closed circuit recirculation system. Seawater was pumped from the reservoir tank through a UV sterilization unit before entering the rearing tanks. The water in the reservoir tank was strongly aerated, and dissolved oxygen was always kept close to saturation. Technical characteristics of the rearing system are described in Palma et al. (2011). Seawater temperature, salinity, and dissolved oxygen were kept at 21.5 ± 0.3 °C, 37.5 ± 0.1 %, and 6.9 ± 0.1 mg L-1, respectively. Tanks were illuminated from above with 2 9 36 W fluorescent tubes, with a light intensity of 900 ± 40 lux at the water surface and a photoperiod controlled by a timer at 16L: 8D (06:00–22:00 h). Seawater quality data (ammonia, nitrates, and nitrites) were recorded at the start of the experiment. Ammonia values were below detectable levels: nitrate, 0.3 mg L-1 and nitrite, 1.25 mg L-1. Two different diets were tested; Artemia and copepods (Oithona nana). Artemia cysts (SandersÒ, Ogden, UT, USA) were hatched according to the procedures described by Sorgeloos et al. (1986). Artemia were enriched for 24 h with DHA-SelcoÒ (0.6 g L-1/24 h/100,000 Artemia) in one 20-L acrylic cylinder-conical tank at a maximum concentration of 50,000 Artemia L-1 held at room temperature (20–22 °C) and under constant moderate aeration. Copepods were naturally produced in the outflow pond of the Ramalhete Aquaculture field station, collected daily, and fed to juvenile seahorses. During the experiment, juvenile seahorses in each dietary treatment were fed 24 h enriched Artemia metanauplii at a density of 5,000 metanauplii L-1 or copepods at the same density. These two diets were chosen based on the fact that H. guttulatus juveniles fed Artemia enriched with DHA-SelcoÒ have a higher incidence of gas disorders [100 % mortality after 10 days post-parturition (DPP)] when compared with those fed Artemia enriched with Chlorella spp. (Palma et al. 2011). When fed natural copepods, the incidence of gas disorders is even lower than the ones fed Artemia enriched with Chlorella spp. (submitted data).
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An initial sample of 10 juveniles was collected just after parturition to be a reference sample. On each sampling day, four juveniles from each replicate tank were randomly sampled at 24, 48, and 72 h after the first feeding. In previous observations, we concluded that the gas bubbles reached their maximum prevalence near 72 h after the first feeding, after which affected juveniles start to die. Sampled juveniles were immediately and rapidly killed with an excess of anaesthetic (2-phenoxyethanol (Sigma-AldrichÒ, Portugal) solution, 0.40 mg L-1). Individuals were placed in the anaesthetic solution for at least 20 min and removed not \10 min after ventilation stopped, and then fixed in 4 % buffered formaldehyde solution. Samples were than dehydrated with gradual alcohol immersions (70–100 %) and embedded in cold-polymerizing resin (TechnovitÒ 7100, Kulzer). Longitudinal sections (3 lm thick) were obtained with a rotatory microtome (Microm HM 340 E, Microm International GmbH, Germany) and stained with a toluidine blue solution (Kuhlmann 2006). Hematoxylin/eosin (Humason 1979) and PAS colouration (Paulete and Bec¸ak 1976) methods were also tested, but failed to provide good staining results. The slide with the sections was mounted permanently using DPX. Observations by light microscopy (Leica Microsystems GmbH, Wetzlar, Germany) were performed at several magnifications in order to examine the development of the digestive tract and the occurrence and development of gas bubbles.
Henderson 1989). Total lipid extracts were subjected to acid-catalysed transmethylation for 16 h at 50 °C, using 1 mL of toluene and 2 mL of 1 % sulphuric acid (v/v) in methanol. The resulting fatty acid methyl esters (FAME) were purified by thin-layer chromatography (TLC) and visualized with iodine in chloroform/ methanol (2:1 v/v) 98 % (v/v) containing 0.01 % BHT (Christie 1982). Prior to transmethylation, heneicosanoic acid (21:0) was added to the TL as an internal standard. FAME were separated and quantified using a SHIMADZU GC 2010 (Kyoto, Japan) gas chromatograph equipped with a flameionization detector (250 °C) and a fused-silica capillary column RTX—WAXTM (10 m 9 0.1 mm I.D.). Helium was used as a carrier gas, and the initial oven temperature was 150 °C, followed by an increase at a rate of 90 °C min-1 to a final temperature of 250 °C for 3 min. Individual FAME were identified by reference to authentic standards and to a well-characterized fish oil. BHT, potassium chloride, potassium bicarbonate, and iodine were supplied by Sigma Chemical Co (St. Louis, USA). TLC (20 9 20 cm 9 0.25 mm) and HPTLC (10 9 10 cm 9 0.15 mm) plates, pre-coated with silica gel (without fluorescent indicator), were purchased from Macheren–Nagel (Du¨ren, Germany). All organic solvents used for GC were of reagent grade and were purchased from PANREAC (Barcelona, Spain). Statistical analysis
Lipid analysis Triplicate samples of Artemia were enriched as described above, and copepods were frozen and stored at -80 °C. The samples were then freeze-dried for 2 days. Total dry weights were recorded, and then, samples were taken for lipid extraction. Total lipid samples were separated into classes by one-dimensional double-development high-performance thinlayer chromatography (HPTLC) using methyl acetate/isopropanol/chloroform/methanol/0.25 % (w/v) KCl (25:25:25:10:9 by vol.), as the polar solvent system, and hexane/diethyl ether/glacial acetic acid (80:20:2 by vol.), as the neutral solvent system. Lipid classes were quantified by charring with a copper acetate reagent followed by calibrated scanning densitometry using a SHIMADZU CS-9001PC (Kyoto, Japan) dual wavelength flying spot scanner (Olsen and
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Differences in fatty acid composition (arc-sine squareroot-transformed data) of the enriched DHA-SelcoÒ enriched Artemia and natural copepods were tested using Kruskal–Wallis ANOVA with post hoc SNK (p = 0.05).
Results As a first observation, Fig. 1a shows a healthy juvenile seahorse with no signs of digestive disorder, whereas Fig. 1b shows a juvenile seahorse with a severe digestive disorder. At this point, the animal is no longer able to maintain normal buoyancy. Observing the samples prior to ingestion of the first meal enabled to characterize the physiology of the digestive tract of H. guttulatus juveniles. Unlike most teleost fish, male
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Fig. 1 a Healthy juvenile seahorse with no signs of digestive disorder, and b a juvenile seahorse with a severe digestive disorder
Oesophagus Upon release from the male’s pouch, juveniles already had a buccopharyngeal cavity ending in the oesophagus. The oesophageal lumen, lined with a stratified squamous epithelium, had been formed to connect the buccopharyngeal cavity and intestine (Fig. 2). The oesophagus was surrounded by a layer of striated muscles, and the muscle layer became thicker as the juvenile grew. Stomach
Fig. 2 Morphological observation of a juvenile H. guttulatus prior to the first meal (91.25). B brain, G gills, OE oesophagus, L liver, GB gas bladder, MG midgut, LU lumen, HG hindgut, A anus, N notochord, IV intestinal villus (940 magnification)
seahorses provide the parental care for their brood, and when expelled from the male marsupium, the offspring are fully developed with the capacity to start exogenous feeding within the first 24 h postparturition.
Seahorses do not possess a proper stomach as ingested food enters directly through the oesophagus to the intestine. Intestine After the parturition (Fig. 2) and prior to the first meal, the intestinal maturation of H. guttulatus juveniles was observed to be advanced. The intestine was divided into the midgut and hindgut by an intestinal valve. The midgut was wider, almost sac-shaped, and the hindgut was a straight tube. Intestinal villi in the midgut were already visible (Fig. 2).
Buccopharynx 24 h after first meal The buccopharynx was present and was sided by a squamous epithelium (Fig. 1). It was not possible to observe the pharyngeal tooth, but the tongue was present.
At this stage, it was not possible to identify any major differences between the juveniles fed DHA-SelcoÒ enriched Artemia and those fed natural copepods. In
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tanks, it was observed that some juvenile seahorses had extreme difficulty maintaining neutral buoyancy, and others bumped against the water surface in a constant effort to maintain stable buoyancy. Conversely, juveniles fed natural copepods showed no signs of intestinal disorders, and the gas bladder and intestine maintained their normal shapes. The digestive tract elongated; the gut lumen increased in size, and the intestinal villi were distended (Fig. 5a). The fullness of the whole gut was identified in all sampled fish. In the midgut, it was possible to identify the prey shape (Fig. 5b, c), and the hindgut appeared full with well-digested matter. In the tanks, it was observed that the juvenile seahorses fed natural copepods did not show any signs of swimming disorders and had normal control of their buoyancy. 72 h after first meal
Fig. 3 Morphological observation of a juvenile H. guttulatus 24 h after the first meal (magnification at 91.2). E eye, B brain, G gills, OE oesophagus, L liver, MG midgut, HG hindgut, A anus
the juvenile fish that were fed natural copepods, the hindgut was observed to be completely full, while almost no feed particles were identified in the midgut (Fig. 3). 48 h after first meal At this stage, it was possible to identify morphological differences between H. guttulatus juveniles fed DHASelcoÒ enriched Artemia and those fed natural copepods. In juveniles fed Artemia, the first signs of overinflation of the gas bladder were identified (Fig. 4). Overinflated gas bladders were observed in all sampled fish fed Artemia, but the inflation severity varied between fish. In Fig. 4a), the gas bladder appears overinflated, but the intestine maintains its integrity. In addition, there were still some feed particles in the midgut, and the intestinal villi retained their typical shape. However, other fish displayed a more severe condition as the overinflation of the gas bladder reached a peak, bursting air into the hindgut and starting to fill it up (Fig. 4b). At this stage in the
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At this stage, all sampled fish fed DHA-SelcoÒ enriched Artemia displayed overinflated gas bladders. Overinflation appeared to reach a maximum, and in all observed samples, the air started to burst into the hindgut. Despite the closeness between the hindgut and the anus, fish were unable to excrete the air, which invaded the midgut. In Fig. 6a, it can be seen that the air has burst from the gas bladder to the hindgut causing it to overinflate as well. This condition forces feed to remain in the hindgut, which at this point can only be identified in the upper part of the gut. Finally, the air migrated to the midgut, completing the overinflation of the intestine (Fig. 6b). At this stage in the tanks, juvenile seahorses that were fed Artemia had no control of their swimming ability and were lying horizontally at the water surface. Their movements were erratic with uncoordinated tail attempts to regain normal buoyancy. In juveniles that were fed natural copepods, the digestive process was the same as described above at 48 h after the first meal and no signs of digestive disorder were identified. Again, the fullness of the whole gut was identified in all sampled fish. Preys were identifiable in the midgut, and the hindgut appeared full with well-digested matter. In the tanks, juvenile seahorses fed natural copepods did not show any signs of swimming disorders and maintained perfect control of their buoyancy.
Fish Physiol Biochem
Fig. 4 a Morphological observation of a juvenile H. guttulatus 48 h after being fed with DHA-SelcoÒ enriched Artemia (91.25) and b detail of the intestine and gas bladder (92.5).
B brain, G gills, OE oesophagus, GB gas bladder, MG midgut, HG hindgut, A anus, N notochord, MF muscle fibre
Fatty acid analysis
fish mainly depend on pinocytosis and intracellular digestion and absorption (Watanabe 1982). The third phase begins with the presence of gastric glands and pyloric caeca to metamorphosis onward, indicating the functional maturation of the digestive system (Bisbal and Bengtson 1995). Unlike most marine fish larvae that hatch with a rudimentary digestive system and without mouth (Yu´fera and Darias 2007), seahorses are fully developed when released from the male’s pouch and are small miniatures of the adult stages. In most of the seahorse species, the yolk sac is fully consumed when seahorse larvae are still in the male’s pouch. Therefore, on parturition, seahorse larvae can no longer depend on the yolk sac for energy; their digestive system is fully developed, and they can and need to start feeding just a few hours after being released from male’s pouch. Due to this fact, and as it concerns seahorses, the term ‘‘juvenile’’ is used since the first DPP, and at the moment they are expelled from the male’s pouch, they fit between the second and third stages of normal fish larvae ontogenetic development of the digestive system. Under microscopy, it was possible to identify all the internal organs associated with the digestive system at a fully developed stage. The maturation of the digestive system was also confirmed by the
Both similarities and marked differences in the fatty acid profiles of the enriched DHA-SelcoÒ enriched Artemia and natural copepods were observed (Table 1). Artemia DHA-SelcoÒ enriched had significantly higher (p \ 0.05) amount of both EPA and DHA than natural copepods, but were no different (p [ 0.05) in AA. The percentage of saturated fatty acids was significantly higher in the natural copepods, whereas the percentage of monounsaturated fatty acids was in Artemia DHA-SelcoÒ enriched.
Discussion Ontogenetic development of the digestive system in most teleost fish is generally divided into three major phases (Boulhic and Gabaudan 1992; Bisbal and Bengtson 1995). The first phase begins with hatching and ends at the completion of endogenous feeding, during which time larvae depend on energy reserves in the yolk sac and oil globules. The second phase begins with the onset of exogenous feeding and ends before the formation of gastric glands in the stomach, characterized by the lack of sufficient digestive capabilities (Boulhic and Gabaudan 1992). During this phase, larval
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Fig. 5 a Morphological observation of a juvenile H. guttulatus 48 h after being fed natural copepods (92.). E eye, B brain, G gills, MG midgut, HG hindgut, A anus, N notochord, DF dorsal fin; b prey detail I. Abbreviations: CC copepod cephalothorax,
CR copepod rostrum, CE copepod eye, IL intestinal lumen, IV intestinal villi. c prey detail II. Abbreviations: C copepod, CR copepod rostrum, CE copepod eye, CT copepod telson, IV intestinal villi
simple fact that H. guttulatus juveniles initiated their feeding activity just a few hours after parturition. All of the observed specimens presented full guts within the first 24 h after the first meal, and within that period, no visual differences between the two dietary treatments were found. This changed dramatically in fish fed DHA-SelcoÒ enriched Artemia as 48 h after the first meal, it was observed that all sampled fish had overinflated gas bladders, and in some specimens, air was already present in the hindgut. At
72 h, the condition of the juveniles became even more severe due to the fact that the gas bladder and the gut (both the midgut and hindgut) were overinflated and all other organs were pushed against the upper part of the abdominal cavity. As juveniles are unable to reverse this situation, massive mortality occurred within the next 48 h, and as referred by Palma et al. (2011), when fed DHA-SelcoÒ enriched Artemia, 100 % mortality occurs within the first 120 h after the first meal. Ultimately, this pathology
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Fig. 6 a Morphological observation of a juvenile H. guttulatus 72 h after being fed with DHA-SelcoÒ enriched Artemia (91.25) and b a second specimen with a severe condition of
gut overinflation (91.25). B brain, G gills, MG midgut, HG hindgut, A anus, N notochord, FM feed matter, D diaphragm
prevents the fish from feeding and maintaining controlled buoyancy. Buoyancy is controlled by the gas bladder and in several percoid fishes, including seahorses, a diaphragm separates the posterior, or gas resorbing, part of the gas bladder from the anterior, gas-producing section. Such fish are called ‘‘euphysoclistic,’’ and in these species, the gas resorption is accompanied by contraction of the gas-secreting chamber (Bond 1996). The pathology observed in this study seems to cause an imbalance in the gas exchange between the two parts of the gas bladder and inaptitude to maintain a normal inflation of the gas bladder. Unlike the condition described above, when fed natural copepods, H. guttulatus juveniles maintained a normal feeding activity and no mortality was registered within the same period of time. In this experiment, feed was the only changing factor; thus, it is unlikely that the causes of gas bladder overinflation can be attributed to supersaturation alone or even gas bladder malformations commonly described for cultured marine fish larvae (Chatain 1990, 1994; Battaglene et al. 1994; Trotter et al. 2001). At the present state of knowledge, for most marine species, the inert feed that have been developed is not suitable for the first phases of larval development and larval nutrition should be examined in the light of
ontogenic development of the larvae and juvenile digestive systems (Kolkovski 2001). According to Cahu and Zambonino-Infante (1997), this constrain is due to the fact that the conventional compound diets do not meet the larval nutritional requirements and survival can fall sharply when not all the feeding and environmental requirements are properly met (Yu´fera and Darias 2007). It is therefore necessary to feed these larvae (and juvenile seahorses) with live prey from the point of first feeding up to the post-larval stage/older juvenile seahorses when animals can adapt to inert or frozen food. The nutritional quality of prey has been studied, and attempts have been made to improve it. Progress has been made for several species, but seahorse nutrition poses new challenges to modify and improve the already existing products. The digestion of nutrients occurs in the gastrointestinal tract and is performed by the enzymes of the stomach, exocrine pancreas, and intestine (Zambonino Infante and Cahu 2001), but concerning seahorses, nothing is known about digestive enzymes. As seahorses do not have a proper stomach as other fish species do, it is arguable that stomach enzymes present in other fish species may lack or be diminished in seahorse’s digestive tract, thus additionally reducing their ability to deal with nonnatural diets. Nutritional imbalances are known to play a key role in morphogenesis and skeletogenesis during early stages of development.
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Fish Physiol Biochem Table 1 Fatty acid content (lg of fatty acid per mg of dry weight) of DHA-SelcoÒ enriched Artemia and natural copepods Fatty acid
Natural copepods
DHA-Selco ArtemiaÒ
R 14:0
5.7 ± 0.3a
R 15:0
2.1 ± 0.1
a
1.13 ± 0.1a
18.1 ± 0.3
a
27.31 ± 0.4b
11.1 ± 0.3
a
6.93 ± 0.2a
6.4 ± 0.3
a
6.91 ± 0.2a
18:1n9 18:1n7
a
22.1 ± 0.5 6.5 ± 0.2a
2.22 ± 0.03b 3.24 ± 0.1b
18:2n6
8 ± 0.4a
1.05 ± 0.05b
18:3n3
1.8 ± 0.2
a
1.31 ± 0.004b
4.3 ± 0.1
a
0.97 ± 0.003b
20:4n6
2.1 ± 0.3
a
0.96 ± 0.02a
20:5n3
31.3 ± 0.9a
8.77 ± 0.01b
22:5n6
1 ± 0.1
a
0.49 ± 0.004b
4.3 ± 0.4
a
0.74 ± 0.02b
30 ± 0.3
a
17.18 ± 0.12b
% Saturated
33.4 ± 0.5
a
47.77 ± 0.8b
% Monounsaturated
48.4 ± 0.5a
15.95 ± 0.6b
R 16:0 16:1n7 18:0
18:4n3
22:5n3 22:6n3
% n-3 HUFA DHA/EPA AA/EPA AA/DHA
68.9 ± 0.8
10.02 ± 0.36b
a
27.14 ± 0.14b a
1.96 ± 0.04b
0.96 ± 0.02
a
0.109 ± 0.002a
a
0.056 ± 0.001a
0.067 ± 0.002 0.070 ± 0.001
Data are the mean ± SD, row values with different Superscripts are significantly different (p \ 0.05)
Nutritionally, dietary lipids have been shown to be particularly important for the early development of marine finfish larvae (Sargent et al. 2002). They represent the primary energy source for larvae, and a source of highly unsaturated fatty acids (HUFA) and EFA needed for new cellular structures, as well as required for normal larval growth, morphogenesis, and bone formation (Cahu et al. 2003; Izquierdo et al. 2000; Lall and Lewis-McCrea 2007). Seahorses are no exception, and according to Faleiro and Narciso (2010), the fatty acid consumption during H. guttulatus embryonic development is considerably high, the PUFA constitute the main source of metabolic energy, and the fatty acids 16:0, EPA, and DHA are the main fatty acids that fulfil the energetic demands of H. guttulatus embryos. These patterns of embryonic consumption result in newborn juveniles with exceptionally low lipid content and therefore a need to ingest well-balanced diets to rapidly satisfy their requirements for fatty acids.
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Series n-3 EFA (EFA n-3) content is generally high in copepods, which seem to have the capacity to concentrate them, particularly DHA. In comparison, EFA content as well as the DHA/EPA ratio is low in Artemia. Certain species of copepods have great capacity for elongating and de-saturating n-3 FA’s, but these capacities are low in Artemia (Robin and Gatesoupe 2001). Artemia are not passive carriers of fatty acids, but have specific physiological needs that can metabolically alter the original composition of their diet by retroconverting DHA into EPA, and by redistributing the incorporated fatty acids among lipid classes with high unpredictability (Navarro et al. 1999). The metabolism inherent to Artemia nauplii causes them to accumulate EPA at higher levels than DHA and consequently decreases the DHA/EPA ratio from the enriching products. Sargent et al. (1997) determined an optimal dietary DHA/EPA ratio of 2:1 for newly hatched larvae from the lipid composition of the yolk sac in marine fish eggs. According to these authors, a DHA/EPA ratio inferior or equal to 1 corresponds to a suboptimal diet as it provides larvae with insufficient amounts of DHA or an excess of EPA and could be deleterious in larval fish feeds. These statements were confirmed by the present study, as EPA and DHA values were quite similar in Artemia resulting in a DHA/EPA below 1. However, the higher amount of DHA than EPA on the natural copepod diet resulted in a DHA/EPA ratio of 1:96. Ultimately, the metabolic ability Artemia possess to modify both the FA ratio provided through the enrichment media, and the use of unbalanced enrichments for juvenile seahorse nutritional requirements may cause the digestive disorders observed in this study. Several dietary components have been identified as negative vectors in the correct larval development (Cahu and Zambonino-Infante 1997; Cahu et al. 2003; Lall and Lewis-McCrea 2007) and have been proven in the present study with juvenile seahorse development. Nonetheless, the specific mechanisms that cause the observed pathology remain unknown. Exact biochemical analysis of feeds and different approaches regarding the histological procedures are needed to provide answers to this limitation to seahorse production in captivity. Acknowledgments Jorge Palma was supported by a postdoctoral grant (Foundation for Science and Technology—
Fish Physiol Biochem Portugal, BPD/34816/2007). We thank the reviewers for their valuable comments.
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