Abstract: Lordosis, which is one of the skeletal deformations, was frequently encountered in the culture of sea bass (Dicentrarchus labrax L. 1758) and it affects the biological performance and marketing image of the fish negatively. Thus it causes big economic losses. In larger natural environments, this abnormality in wild-caught individual is rare, whereas the case is the other way round in hatchery-reared sea bass. This problem can be caused by swim bladder malformations, environmental conditions, bacterial factors, water flow rate and swimming activity. However, many researchers reported that all of these factors cause these malformations in different regions of the skeletal system, commonly known as V-shaped curvatures. But in this study, it is not aimed to investigate the possible causes of the observed abnormalities. We focused on whether the abnormal individuals can be transformed into individuals with normal skeletal systems. In this study, a total number of 5636 juvenile individuals (0.09-1.93 g of live weight) were used. Lordosis rates were identified between the days 54 and 110 by randomly taking at least 30 samples from every group on a weekly basis, at triplicate. In this study, 100% of the individuals with this type of deformation were successfully transformed into individuals with the normal skeletal system in a large volume and rectangular ponds with semi-intensive rearing method.
INTRODUCTION
Skeletal malformations and their incidence are one of the most important factors affecting marine larviculture, also of species consolidated for aquaculture, with effects on production costs, taking into account that as many as 50-60% of hatchery juveniles present at least one severe, externally detectable skeletal malformation. In the aquaculture industry, losses due to malformations also impact on-growing farms, where malformed market size fish have to be discarded or sold at lower values than market prices. Thus, the reduction of the incidence of larval deformities would reduce the economic cost of production, both in the hatcheries and in the outgrowing production sectors and improve the image of aquaculture products in consumers (Boglione et al., 2009).
Skeleton abnormalities include neurocranial, spinal and apendicular aberrations. Environmental factors, such as light, temperature and salinity have been reported to be involved in their origin (Cobcroft et al., 2001; Sfakianakis et al., 2004, 2006). Furthermore, mechanical traumas related with the management of eggs and larvae (Chatain and Dewavrin,1989), hydrodynamic variables (Kihara et al., 2002) and composition of diet (Cahu et al., 2003; Lall and Levis-McCrea, 2007) have been related with these abnormalities. The fact that hatchery-reared fish show a higher proportion of these abnormalities than wild-caught fish, strongly suggests an environmental component related with rearing conditions at farm facilities (Boglione et al., 2001; Mana and Kawamura, 2002). Genetic data on this ground are scarce (Gjerde et al., 2005) and sometimes these abnormalities have been related with inbreeding (Ando et al., 1995).
Lordosis is an abnormal ventral curvature of the vertebral column and is one of the most severe deformities developing in reared fish (Divanach et al., 1997; Beraldo et al., 2003; Sfakianakis et al., 2006; Verhaegen et al., 2007).
According to the anatomical centre of development, lordosis can affect the pre-haemal or the haemal part of the vertebral column. Haemal lordosis was first differentiated from pre-haemal by Divanach et al. (1997) in Dicentrarchus labrax Linnaeus (1758) juveniles and by Kihara et al. (2002) in Pagrus major andwas attributed to the intense swimming effort of fish with inflated swimbladder. Haemal lordosis can reach high prevalence levels (to even 70%), especially in D. labrax (Divanach et al., 1997), but also in fish with less elongated body shape like P. major (21-25%, Kihara et al., 2002). Although water-current decreasing devices have proved a valuable tool for the lowering of swimming intensity and thus lordosis in D. labrax (Divanach et al., 1997). Given the high temperature-induced plasticity of the systems implicated in the haemal lordosis (muscles and bones) (Stickland et al., 1988; Koumoundouros, 2001; Johnston and Temple, 2002; Campinho et al., 2004; Sfakianakis et al., 2004), as well as the well documented temperature-induced ontogenetic plasticity of D. labrax (Pavlidis et al., 2000; Koumoundouros et al., 2001; Sfakianakis et al., 2006) (a) tested the hypothesis of the effect of developmental temperature on the sensitivity of D. labrax to the lordosis inducing factor, acting at the juvenile stage and (b) provided the anatomical criteria for the early quality control of the fish (Fig. 1a, b).
Andrades et al. (1996) suggested an underlying genetic component based on the observation that lordotic larvae could proceed from surviving lordotics of the previous generation. But Castro et al. (2008) reported that the idea of a weak additive genetic control for the lack of operculum, if any, but no such evidence for lordosis.
Up to now, many researchers have reported that all of these factors cause these malformations in different regions of the skeletal system, commonly known as V-shaped curvatures. However, some researchers investigated the morphometric characteristics of it (Coban et al., 2009; Lewis et al., 2004; Loy et al., 2000). But in this study, it is not aimed to investigate the possible causes of the observed abnormalities or morfometrics. We have focused on whether the abnormal individuals can be transformed into the individuals with normal skeletal systems.
| Fig. 1: | (a) Lordotic and (b) normal juveniles of sea bass |
This is the first study, which is investigated whether any abnormalities in fish can be turn to normal individuals. Finding the reason of these problems and trying to prevent before they take place is so important. In addition, it is also important to treat those abnormalities when they take place.
In nature the environmental factors are more suitable than intensive aquaculture conditions. Sea bass juveniles can swim normally and bacterial load is low in wild. There is less stressor (for example, cannibalism, competing for food etc.). On aquaculture production, in cylindrical small tanks, juveniles always turn around the tanks whereas they don’t in large rectangular ponds. Swimming activity and muscle activity of juveniles in larger ponds are similar to they are in wild. Therefore, we tested the lordotic larvae with semi-intensive rearing technique in the large rectangular ponds because swimming activity, muscle activity and psychology are more relaxed than they are in cylindrical small tanks with intensive rearing method. So our thesis is lordotic individuals can be cured in juvenile stages in large rectangular ponds with semi-intensive rearing method.
MATERIALS AND METHODS
Rearing
Experiments were conducted at a special hatchery facility in Hunkar Sea
Product Inc. in Seferihisar (Izmir-Turkey) in 2007. The eggs were obtained from
a brood stock held in captivity for 5 years. Frozen cuttlefish (Sepia officinalis),
leander squilla (Palaemon elegans), deepwater rose shrimp (Parapaneaus
longirostris) and common octopus (Octopus vulgaris) were provided
daily as the primary food source for breeders. No hormonal treatment applied
to broodstock, maturation and spawning occurred spontaneously under local photoperiod
and natural thermic conditions.
The hatchery phase was conducted in indoor 3 m3 cylinder-conical tanks, 1.9 m diameter, with the intensive technique (100 eggs-1) in clear water. Eggs diameter of sea bass were established as 1.185±0.76 mm in treatment. During the egg incubation and yolk-sac larval stage, individuals were kept in the dark, while at the onset of feeding a photoperiod of 14 h light (10-50 Lux). Water temperature was 15-19.5°C (maintained by the use of chillers and heaters), salinity 25-35 ppt, oxygen saturation over 90%. The other rearing conditions for all phases were summarized in Table 1.
The larvae were fed newly hatched instar I Artemia until they reached 6 mm and enriched (Selco INVE) nauplii Instar II thereafter (3 ind mL-1). The surface was kept free from any lipid film, a requisite for good swimbladder inflation, using a skimmer cleaned (Chatain and Ounais-Gushumann, 1990) and normal swim bladder inflation was achieved at 98% incidence. The nursery phases were conducted in 12 m3 cylindrical outdoor tanks with open sea water circulation, under intensive conditions and progressive transition from live to inert food (PROTON series INVE) manually distributed ad libitum. At the weaning phases fish were organized to triplicate two different groups. The group A is cylindrical outdoor tanks of 12 m3 with intensive rearing method and the group B is rectangular outdoor ponds of 400 m3 with semi intensive rearing method (Table 1). Fish age was shown as dph (day post hatch). Feeding has done manually ad libitum with NRD series (INVE) at this stage.
All the amounts (numbers) of fish used in this study were calculated by weighing at the beginning and counted by TPS Micro Fish Counter at the end of the experiment. Meanwhile 1000 samples for each of the groups were measured for mean TL±SD and weighed for mean WW±SD at the beginning and at the end. In this way, the growth and survival rate were calculated. Besides the length-weight relation of the populations was calculated during the Table 1: Technical conditions during the rearing phases juvenile phase by measuring weekly. Survival rate was assessed with the deformation rate by sorting and observing the mortality during the study. Also, daily mortality was estimated by counting all dead fish removed from the tanks during surface and bottom cleaning (Hatziathanasiou et al., 2002).
| Table 1: | Technical conditions during the rearing phases |
Behavioral observations (events of aggressiveness between siblings, the way of population dispersion, attacks due to cannibalism) were made early in the morning before the first food supply and during photophase before and after feeding. In case of morphological abnormalities or lesions indicating cannibalism, photos were taken of live and dead fish under an Olympus CX21 stereoscope with an Olympus C-500 camera (Hatziathanasiou et al., 2002).
Bone Observation
In this study, a total number of 5636 individuals (0.09-1.93 g of live weight)
were used. In order to assess the degree of lordosis on sea bass juveniles and
establish a potential relationship with the rearing volume during the juvenile
stages the deformation rates were observed between the days 54 and 110 taking
at least 30 samples randomly by dip net from every group on a weekly basis,
at triplicate. Fish were anesthetized with 0.2-0.5 mL-1 of phenoxyethanol
(ethylenglycol-monophenyl ether, Merck) and measured for total length and weight.
Then, fish were fixed with 10% (v/v) formalin in neutral phosphate buffer solution.
Juveniles were radiographed with X-ray (Koumoundouros et
al., 2001) and the negatives were observed by transparency on an illuminated
table.
Statistical Analysis
Results are given as means and standard deviations. Data expressed as percentage
(survival, incidence of skeletal deformities) were previously arcsin (x1/2)-transformed.
Results were compared by means of One Way ANOVA and when significant differences
were detected the Tukey multiple-comparison test was used to detect differences
amongst experimental groups (Zar, 1974). Statistical comparisons
of the mean TL between the different fish groups were made by means of Mann-Whitney
U-test. In all statistical analyses, the level of significant difference was
set at p<0.05.
RESULTS
Growth and Survival Rate
No difference was observed between treatment and control groups in terms
of parameters related to water environment used in the experiment (p<0.05,
except temperature). The desired criteria were maintained for sea bass rearing
during larval, post-larval and juvenile stages.
| Table 2: | Larval size in Total Length (TL) and Wet Weight (WW) and survival rate of sea bass larvae at different tank volumes (Group A, 12 m3; Group B, 400 m3) |
| Values are Mean±SD. Different superscripted letters within the same column show statistical significant differences (p<0.05) | |
| Fig. 2: | Mean individual weight and total length of the experimental populations of sea bass during juvenile phase under different rearing methods (Week 1 indicates dph 54 and week 6 indicates dph 110) |
| Fig. 3: | The length-weight relationship of the group A during the juvenile phase |
| Fig. 4: | The length-weight relationship of the group B during the juvenile phase |
At the end of the experiment (110 dph), in group A the mean weight was 1.66±0.02 g while in group B this value was found as 1.93±0.09 g. As a result of these findings it was found that there are differences between the group (p<0.05) and this was showed in Table 2. Measured values of total length during the study were found to be different between the groups (p<0.05), as well (Table 2).
Mean individual weight and total length of the experimental populations of sea bass during juvenile phase under different rearing methods shown in Fig. 2. The relationship of body length and weight of the fish larvae are shown in Fig. 3 and 4. The mean body length and weight of the larvae increased progressively from the day 54 to the day 110. The increase over the mean value of the previous age group was always statistically significant (p<0.05).
| Table 3: | Mortality and lordosis rate during the study between the groups |
| N: Normal; +: Light; ++: Severe lordosis | |
| Fig. 5: | Transformation of hatchery-reared abnormal juveniles of sea bass to the normal individuals under semi-intensive rearing conditions |
Lordotic Fish Frequency with Mortality and Age (dph) Distribution
The higher increase of mortality in the group A was due to the higher mortality
of the animals with abnormal vertebra and cannibalism in these tanks. But, there
is no relation between cannibalism and lordosis. Mortality and lordosis rate
during the study (from day 54 to 110) between the groups were shown in Table
3 at different categories.
Final Balance Sheet of Production
At the end of the 110 day, the population reared with intensive (group A) produced
115416 fish with 1.66 g mean weight, of which 38% had a lordotic vertebra and
the population reared with semi intensive (group B) produced 553740 juveniles
with 1.93 g mean weight of which 0% had a lordotic vertebra (Fig.
5).
DISCUSSION
At the end of the experiment, mean total length, growth rate and body weight were found different in all treatments during the rearing period. Nejedli et al. (2006) reported that Mean±SEM values for body length were 11.15±0.17 mm (40 days), 18.99±0.16 mm (50 days), 18.53±0.44 mm (60 days), 26.35±0.21 mm (70 days), 32.54±0.30 mm (80 days) and 34.03±0.30 mm (90 days) in sea bass. Present findings shows similarity with theirs, but our study started the experiment on the day of 54 and initial body length was 20.90±0.96 mm for the group A and 21.91 for the group B differently from theirs. Roncarati et al. (2001) reported that, at the same age, wild sea bass have a slimmer body shape and a smaller abdominal circumference than reared sea bass. Similarly, we observed that the group B had a slimmer body shape than the group A at the end of our experiment. We can say that our treatment group with semi intensive method had similar environmental parameters with in wild in comparison the intensive one. But the growth in semi intensive reared group was higher than the group A.
In sea bass, survival was strongly affected by stocking density during the weaning (Hatziathanasiou et al., 2002). Similarly, our results revealed that survival significantly improved at the group B (semi intensive rearing method). For the group A (intensive rearing method), cannibalism was identified as the most important factor inducing mortality during this phase. It was dependent on the population density, increasing drastically during the transition period from live to artificial feed (Hatziathanasiou et al., 2002; Can, 2006). Also bacterial load may be secondary factor because higher stocking densities cause the higher bacterial bloom (Vadstein, 1997; Queiroz and Boyd, 1998; Austin, 1998; Can, 2006). Timing of transition in rearing and its effects on mortality and cannibalism have already been described for other species (Paller and Lewis, 1987; Folkvord, 1991; Hatziathanasiou et al., 2002). Daily observations showed that all dead fish removed from the rearing tanks over the experimental period was cannibalized. The increased mortality recorded in the first 10 days of this experiment was probably due to complete weaning when live food was totally replaced by artificial dry food. Also the higher increase of mortality in the group A was due to the higher mortality of the animals with lordotic vertebra and cannibalism in this tank during 54-64 dph. However, mortality continued to the end of the experiment in this group. It may be cause of the increased stocking density and/or absence of high ratio of abnormalities in this group. So, juveniles siphoned from the tank bottom had an abnormal vertebra.
In present study survival rate was rather high in the group A (with semi intensive rearing) and it was similar to the findings of Boglione et al. (2009), they reported that survival rates were found to be modulated by rearing conditions, reaching the highest value in semi-intensive (large volumes) methodology.
Boglione et al. (2009) reported that their analysis actually show that severe anomalies were the main discriminating factor between large volumes and green waters reared groups, while the most frequent anomaly typologies (that is, excluding rare anomalies and considering only those anomalies that were observed in more than 10 individuals) allowed a degree of discrimination among all the lots. Similarly, in present study severe abnormality (investigated for only lordosis) in the group B was found less than group A. However, the differences in the morphological quality in the different reared lots are very low compared with that observed in juveniles of sea bass (D. labrax) and gilthead sea bream (Sparus aurata) siblings reared using intensive vs semi-intensive methodologies (Boglione et al., 2003a, b; Cataudella et al., 2006; Koumoundouros et al., 1997).
Some researchers reported that lordosis induction has been attributed to increased muscle activity (Chatain, 1994; Kihara et al., 2002), yet a mechanistic coupling between muscle activity and the abnormal vertebral curvature is largely lacking (Divanach et al., 1997). Both an increased tailbeat frequency and amplitude require increased muscle power output. This causes an increase in the magnitude of the bending moment and compressive load on the axis of animals that become lordotic. (Kranenbarg et al., 2005). This study was conducted on the small scale tanks during the nursery stage and during the weaning for group A, with intensive rearing method. And we think that the individuals especially in nursery stage can be increased muscle activity because of competition for food. Tank shape (the fish tend to swim around in the cylindrical small tank) with high stocking density may be lead to this case. However Akiyama et al. (1986a, b) have reported that occurrence of scoliosis (an another vertebral abnormality) caused by tryptophan (Trp) deficiency may be related to the depletion of serotonin (5-HT) in the body, probably in the central nervous system. Also in this study high stocking density, competition for food and cannibalism with intra-specific may cause the depletion of serotonin or not enough releasing of it in the body, as well. Akiyama et al. (1989) said that catecholamine may be also involved in an occurrence of scoliosis. Same case may consist of the release of the high quantities dopamine from the fish brain when there are cannibalistic behaviors or another stressor because of high stocking density in their environment. We think that fish psychology may be one of the major factors for the abnormal vertebral formation. But, Kranenbarg et al. (2005) were reported that the abnormal calcification of lordotic vertebrae can be an adaptive response to the increased swimming activity. The abnormal calcification can also be a response to the local change in curvature due to buckling. We think that both of these can be true. Even they may be related to each others. Also swimbladder non-inflation may cause of the stress absence of a functional swimbladder. And it is another reason of the lordosis (Paperna, 1978; Daoulas et al., 1991; Chatain, 1994). Similarly, a reason of the hypertrophy that swim bladder problem is presences of any stressor in their environment, too.
Several aquaculture-related factors have been reported to increase skeletal deformities, unnatural environmental conditions (such as hard hydrodynamic conditions temperature, pH, stocking density, water flow, deficient alimentation, heavy metals, bacteria, parasites,…) (Chatain, 1994; Boglione et al., 2001, 2003a, b; Koumoundouros et al., 2001, 2002a,b; Lewis et al., 2004), nutritional imbalances including vitamins deficiency or excess (Sato et al., 1978; John et al., 1979; Mahajan and Agrawal, 1979; Chavez de Martínez, 1990; Kanazawa et al., 1992; Dabrowski et al., 1996), tryptophan deficiency and tyrosine excess (Nacario, 1983; Walton et al., 1984; Akiyama et al., 1986a, b). Afonso et al. (2000) reported that, homogeneity in breeding condition for all studied families indicate differences among families that were not due to environmental or nutritional factors. However, present study was not aimed to find any reason for any abnormalities. Our aim was firstly to investigate, whether lordotic fish can be transformed. Thus we investigated whether any abnormalities in fish can be turn to normal individuals. It is important that finding the reason of these problems and trying to prevent before and to treat them when they take place. However, the idea of present study, semi intensive method is close to natural environmental parameters. And fish in this group were more relaxed than the ones in they intensive one psychologically. This is an important difference between in intensive and semi intensive rearing.
CONCLUSIONS
In this study, 100% of the individuals with this type of deformation were successfully transformed into individuals with the normal skeletal system in the large volume and rectangular ponds with semi-intensive rearing method. It is suggested that the transformation of the lordotic species of the sea bass to the normal individual during juvenile stages may be achieved. Future studies require by investigating other deformation types in the sea bass and or in the other species to support our result. Also the other deformation types of this species and of the other species may be corrected with similar studies at the future.
ACKNOWLEDGMENTS
The authors wish to thank the staff of the Hunkar Sea Products Inc., Seferhisar, Izmir, Turkey where the experiments were conducted with their most efficient technical assistance.