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Latin american journal of aquatic research

On-line version ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.42 no.1 Valparaíso Mar. 2014

http://dx.doi.org/103856/vol42-issue1-fulltext-12 

Research Article

 

Growth and survival of Hippocampus erectus (Perry, 1810) juveniles fed on Artemia with different HUFA levels

Crecimiento y sobrevivencia de juveniles de Hippocampus erectus (Perry, 1810) alimentados con Artemia con diferentes niveles de HUFA

 

Nicolás Vite-Garcia1,2,4, Nuno Simoes2, Olivia Arjona3, Maite Mascaro2 & Elena Palacios3

1 Programa de Posgrado en Ciencias del Mar y Limnología Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, Ciudad de México
2
Programa de Investigación en Ecología y Cultivo de Especies Marinas de Ornato (PIECEMO) Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán, México
3
Programa de Acuicultura, Centro de Investigaciones Biológicas del Noroeste Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, BCS, C.P. 23096 México
4
Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Agropecuarias Villahermosa, Tabasco, C.P. 86280, México
Corresponding author: Nicolás Vite (nic.vite@gmail.com)


ABSTRACT. Survival during first months after birth is one of the bottlenecks for consolidating the seahorse farming industry. In this work, Artemia metanauplii enriched with two highly unsaturated fatty acids (HUFA) rich commercial emulsions with different docosahexaenoic acid (DHA) levels (63% and 14% of total lipids), a vegetable oil with no DHA, and non-enriched Artemia as control, were used to feed 5-day-old juvenile Hippocampus erectus for 60 days. Enriched Artemia had similar levels of DHA (13% and 9%), despite great differences of DHA in the emulsions, with traces of DHA in non-enriched and vegetable oil enriched Artemia. More than 20% of DHA was found in 24 h starved juveniles fed both DHA-enriched treatments, similar to values in newly born juveniles, but those fed vegetable oil enriched Artemia or non-enriched Artemia had 5% of DHA. Total lipid and protein levels were similar in juveniles from the four treatments. The n-3/n-6 ratio was almost four-fold higher in seahorses fed DHA-enriched treatments compared to juveniles fed the non-enriched treatments. Survival of seahorses only partially reflected the DHA levels: it was lower in the vegetable oil treatment, similar in the seahorses fed Artemia with higher DHA and in the control treatment, and higher in seahorses fed the HUFA-enriched Artemia with lower DHA levels, although growth was similar in the two DHA-enriched Artemia treatments. Juvenile H. erectus seahorses perform better when they have at least 20% of DHA in their tissues, and these levels can be attained with no more than 14% of DHA in emulsions, eliminating the need for more expensive emulsions with higher DHA levels.

Keywords: Hippocampus erectus, seahorse, DHA, growth, fatty acids, survival, aquaculture.


RESUMEN. La sobrevivencia durante los primeros meses de vida es uno de los cuellos de botella para la consolidación de la industria del cultivo del caballito de mar (Hippocampus erectus). En este trabajo se utilizaron metanauplios enriquecidos con dos emulsiones comerciales, con diferentes niveles de ácido docosahexaenóico (DHA) (63% y 14%), con aceite vegetal sin DHA, y metanauplios sin enriquecimiento como control, para alimentar caballitos de mar de 5 días de vida durante 60 días. La Artemia enriquecida tuvo niveles similares de DHA (13% y 9%), a pesar de las grandes diferencias en las emulsiones, con trazas de DHA en la Artemia no enriquecida y la enriquecida con aceite vegetal. Se obtuvo más de 20% de DHA en juveniles, muestreados con 24 h de ayuno, de los tratamientos con enriquecimiento de DHA, con valores similares a los de caballitos recién nacidos, pero los caballitos que se alimentaron con Artemia enriquecida con aceite vegetal y con Artemia no enriquecida tuvieron 5% de DHA. Los lípidos y proteínas totales fueron similares en los caballitos de los cuatros tratamientos. La razón n-3/n-6 fue casi cuatro veces mayor en caballitos alimentados con los tratamientos ricos en DHA comparados con los otros dos tratamientos. La sobrevivencia de caballitos de mar estuvo reflejada parcialmente por los niveles de DHA: fue mínima en caballitos del tratamiento de aceite vegetal, intermedia y similar en caballitos del tratamiento control y en los del tratamiento con enriquecimiento de HUFA (ácidos grasos altamente insaturados) con mayores niveles de DHA, y resultó máxima en caballitos del tratamiento enriquecido con HUFA con menores niveles de DHA. Juveniles de H. erectus tienen un mejor desempeño cuando tienen en sus tejidos 20% de DHA, y estos niveles pueden ser alcanzados con no más de 14% de DHA en las emulsiones, eliminado la necesidad de utilizar emulsiones con mayores niveles de este ácido graso, que tienen un precio más elevado.

Palabras clave: Hippocampus erectus, caballito de mar, DHA, ácidos grasos, crecimiento, sobrevivencia, acuicultura.


 

INTRODUCTION

Seahorses have low fertility, defined social structures (strict monogamy in many species), low mobility, small home ranges, limited distribution, and close association with habitat, traits that cause their populations to be highly vulnerable to human impact on the coastal zone they inhabit (Foster & Vincent, 2004; Vincent et al., 2005). Hippocampus erectus populations in the Gulf of Mexico are affected mainly by-catch (Baum et al., 2003), but also by habitat degradation, and capture for trade. Among the protective measures proposed for seahorse protection are trade restriction of wild specimens (included in Convention on International Trade in Endangered Species of Wild Flora and Fauna CITES appendix II), establishment of protection zones, environmental education for raising awareness of populations in coastal areas, and more recently, implementation of aquaculture programs. Seahorse culture can be a partial solution to replace wild animal capture and provide alternative economic opportunities to fishermen in developing countries (Payne & Rippingale, 2000; Job et al., 2006; Koldewey & Martin-Smith, 2010). Substantial efforts have been made to develop a suitable technology to culture different species of seahorses worldwide (Wilson & Vincent, 1998; Woods, 2000; Job et al., 2006; Lin et al., 2008; Planas et al., 2008), but one of the biggest bottlenecks for the development of the seahorse culture industry is still low survival of young seahorses, frequently attributed to inadequate nutrition of broodstock and brood.

Seahorses are gestated by the male. When they are born, they are referred to as juveniles and are an exact replica of the adult, which is a considerable advantage, because, contrary to most other cultured fish, newborn seahorses have a relatively large mouth; in the case of H. erectus, juveniles can be fed on Artemia metanauplii, avoiding the need of other live prey (Vite-García et al., 2009). An additional advantage of Artemia metanauplii is that they can be easily enriched using oils and emulsions (Leger et al., 1986). Enrichment of Artemia to feed juvenile seahorses is important, because it is not known if juvenile seahorses can synthesize long-chained highly unsaturated fatty acids (HUFA) from shorter precursors (PUFA: polyunsaturated fatty acids). Most marine fish studied so far cannot synthetize HUFA, and freshwater fish that can, are not able to in early stages of larval development (Sargent et al., 2002). H. hippocampus broodstock fed a docosahexaenoic acid (DHA, 22:6n-3) depleted diet produce newborns that have 7% of DHA, compared to 16% in broodstock fed mysids with more DHA (Otero-Ferrer et al., 2012). Newly born seahorse have high levels of HUFA, particularly DHA with levels above 20% relative to total fatty acids in H. kuda (Saavedra et al., 2013) and levels of 16-17% for H. guttulatus (Faleiro & Narciso, 2011, 2013). These high DHA levels decrease as juveniles grow if given a DHA-depleted diet, as is the case with non-enriched Artemia nauplii, and this decrease has been associated to a decrease in survival (Chang & Southgate, 2001; Shapawi & Purser, 2003; Wong & Benzie, 2003), with the exception of juvenile H. guttulatus, which did worse when fed with enriched Artemia nauplii (Palma et al., 2011). Consequently, most studies use enriched Artemia metanauplii as a standard for seahorse juvenile culture (Chang & Southgate, 2001; Lin et al., 2008; Olivotto et al., 2008; Hora & Joyeux, 2009; Otero-Ferrer et al., 2010). Woods (2003) found a significant better growth and survival in H. abdominalis fed diets containing 13 and 8% of DHA (relative concentration) compared with a diet having 4% of DHA. Chang & Southgate (2001) found juveniles grew more when fed Artemia metanauplii enriched with 8% of DHA, compared to 4 or 6% of DHA. No nutritional studies have been done for H. erectus, but since apparently the higher the DHA content in the emulsions used to enrich the Artemia metanauplii, the better the survival of juvenile seahorses of other species, we here tested an emulsion that has an excess DHA content and compared it to a standard commercial emulsion for fish. We also included an isolipidic treatment with no DHA to test if the positive effect of the emulsion was not a result of an increased caloric intake, and the traditions non-enriched Artemia metanauplii for comparison purposes to other studies.

MATERIALS AND METHODS

Origin of juveniles

Three wild pregnant male seahorses (Hippocampus erectus) were captured in Laguna de Chelem, Yucatán, México, (21°15׳21°17-׳N, 89°39´-89°48W) (under scientific license granted in official letter "SGPA/DGVS/03153/10" from SEMARNAT, Mexican Ministry of the Environment and Natural Resources). The seahorses were transferred (no more than 2 h after capture) to Unidad Multidisciplinaria de Docencia e Investigación (UNAM), Sisal, Yucatán, México. Until the time of parturition (1-5 days), pregnant seahorses were kept in 100-L glass aquaria (70H×50L×30W cm) with a recirculation system. Temperature was maintained at 26 ± 2°C, salinity at 33-36, pH at 8.1-8.3, NO2- < 0.3 mg L-1, NO3- < 5 mg L-1, NH3/NH4+ < 0.1 mg L-1, and 12:12 photoperiod. Aquaria were cleaned daily. Inside the tanks, a polypropylene rope structure was provided for seahorse attachment. Adults were fed ad libitum twice a day on live adult Artemia with a typical fatty acid composition, high in eicosapentaenoic acid (EPA) (18.4%), 18:1n-7 (15.7%), 16:1n-7 (13.4%), 18:0 (11.0%), 16:0 (10.7%), 18:2n-6 (5.4%), 18:3n-3 (3.4%), and arachidonic acid (ARA) (2.5%), and no DHA.

Three broods (average 370 juveniles per brood) from three males (average height = 141.3 ± 5.75 mm) were used, born with a 3-day difference between the first and the last brood. At the time of birth juveniles were 11.3 ± 0.2 mm (height), and the relative fatty acid composition at birth was (only values above 1% are shown): 16:0 = 11.9 ± 0.82, 18:0 = 9.0 ± 0.6, 16:1n-7 = 2.4 ± 0.3, 18:1n-9 = 6.0 ± 2.9, 18:1n-7 = 4.3 ± 0.3, 18:2n-6 = 1.6 ± 0.3, ARA = 8.8 ± 0.6, EPA = 7.1 ± 1.1, and DHA = 22.9 ± 2.7. Total saturated fatty acids (SFA) = 28.9 ± 1.2, monounsaturated fatty acids (MUFA) = 21.1 ± 2.1, and polyunsaturated fatty acids (PUFA) = 48.7 ± 1.4. The n-3/n-6 ratio was = 2.6 and DHA/EPA ratio was = 3.3. No differences between newly-born juveniles sampled from the three broods were obtained for individual fatty acid composition.

Each brood was placed individually in 100-L tanks with the same conditions as described for the broodstock. From birth until the beginning of the experiment (4-7 days), juveniles were given enriched Artemia metanauplii with Selco emulsion (see below) three times a day; survival of the three broods was over 90% from birth until the beginning of the experiment.

Experimental design

For the experiment three dietary treatments and a control were used to test survival and growth of juvenile seahorses. It is well known that although DHA levels were high in the emulsions, they tended to decrease in the enriched metanauplii, probably a due to fatty acid retroconversion (Navarro et al., 1999; Han et al., 2001; Palacios et al., 2004). Since we wanted to further increase DHA levels supplied to juvenile seahorses, we used commercial oil designed for human consumption containing more than 60% of DHA, compared to 10-15% of DHA generally present in the emulsions designed for fish. The control treatment consisted on Artemia metanauplii without enrichment. Two high-HUFA commercial emulsions with different DHA levels were used for metanauplii enrichment on treatments, and one more emulsion without HUFA was used: (1) DHA Protein Selco® INVE, Belgium (developed for marine fish), hereinafter referred to as Selco, which had: ARA = 0.7%, EPA = 7.2%, DHA = 14.4%; (2) EPAX1050TG EPAX® Norway, AS (designed to increase omega 3 in humans), hereinafter referred to as EPAX which had: ARA = 9.2%, EPA = 18.2%, DHA = 63.3%; (3) A treatment consisting in canola oil, rich in omega 3 and 6 PUFA, specifically 18:2n-6 = 20% and 18:3n-3 = 9% but no omega 3-HUFA, hereinafter referred to as Canola. Canola oil and EPAX were emulsified in seawater using a blender. Artemia metanauplii were enriched twice (24 h after hatching and 24 h later) and harvested at 26 h. This double enrichment was found to be the best in order to retain HUFA in metanauplii in preliminary tests where several enrichment times were compared. Artemia samples from the four diets were taken for biochemical and fatty acid analyses every week during the experiment.

For the experiment, two interconnected systems with six-100 L tanks each were used (total 12 tanks). Aquarium conditions were the same as described for the broodstock. From each brood (n = 3), 240 juvenile seahorses were distributed in four tanks (60 seahorses each) with a different dietary treatment; thus, the three broods were taken as replicates of each dietary treatment. Seahorses were fed twice a day at 10:00 and 17:00 h, with 10 metanauplii mL-1. Leftover food and feces were collected daily with a siphon. Dead juveniles were registered daily. A sample of 20 juveniles from each brood was measured (height and weight) prior to the experiment, and then a sample of 20 seahorses were measured after 15 and 30 days; all surviving seahorses were measured at 60 days. Considering measurements provided by Foster & Vincent (2004), height (HT) was chosen to use instead of the standard length to minimize stress on the fishes. The Fulton s condition index K was calculated as follows: K = 100xWW (g)/HT (cm3) (Zhang et al., 2011).

For fatty acid and biochemical analyses, 30 juveniles from each brood were sampled prior the experiment, and five juveniles from each treatment and brood were sampled at the end of the experiment (4 diets × 3 broods = 12 individual samples of 5 seahorses), and then kept at -80°C until the biochemical and fatty acid analyses were performed. Before being sampled, seahorses were starved for 24 h to make sure they had their guts empty. The experiment was concluded after 60 days. The initial fatty acid composition (day 0) is shown in Table 3. No differences between juveniles sampled from the three broods were obtained for individual fatty acid composition.

Biochemical and fatty acid analyses

The biochemical and fatty acid analyses of Artemia metanauplii and juvenile seahorses were done at CIBNOR-La Paz, México. Both biochemical and fatty acid analyses, juveniles sampled at the start of the experiment were analyzed pooling five juveniles in a sample, in order to attain an adequate weight for the tests; juveniles after 60 days were analyzed individually. Total proteins, carbohydrates, and lipid analyses were performed in lyophilized samples, and data are referred to as mg g-1 dw. These analyses were done according with the methodology described in Palacios et al. (2000) and Vite-García & Saucedo (2008).

For fatty acid analyses frozen seahorses were opened longitudinally and cut into four transversal segments with a scalpel, placed in a glass vial, 6 mL Chloroform:methanol (2:1 v/v), 10 μL antioxidant (BHT), and 10 μL of 23:0 as internal standard were added to each sample. The segments were macerated directly in the solvent using a glass mortar. All the lipids were transesterified using boron-trifloride methanol (BF3 14% methanol, Supelco, Bellefonte, PA, USA), and analyzed in a Hewllet-Packard CG 6890-N gas chromatography equipped with a DB-23 (30 min length×0.25 mm inner diameter, 0.25 μm-thick-film) fused silica capillary column (J. & W Scientific, Folsom, CA, USA), flame ionization detector (FID) at 280°C, helium as a carrier gas and a temperature gradient 110-220°C at 3°C min-1. The identification of FAME was made by comparison to retention time of standards (Supelco, Bellefonte, PA, USA) (Palacios et al., 2007).

Statistical analyses

For final survival (%), growth (mm), weight (mg), biochemical (lipids, proteins, carbohydrates, mg g-1 dw) and fatty acid (% of total fatty acids) composition of juveniles and Artemia metanauplii, one-way ANOVA was used to test significant differences among the four dietary treatments and a Tukey test for mean comparisons was performed when significant differences were found. All statistical analyses were made using StatisticaTM v. 6.0.

RESULTS

Survival and growth of juvenile seahorses

Survival of juvenile seahorses fed the four treatments for 60 days is depicted in Figure 1a. A higher mortality was observed in the Canola treatment from the second week. A more gradual decrease was found for juveniles fed non-enriched metanauplii and EPAX treated metanauplii. The final survival after 60 days was significantly higher (F = 5.22; P < 0.05) for juveniles fed Selco (82%), followed by EPAX-enriched metanauplii (58%) and juveniles fed non-enriched metanauplii (49%), and the lowest for the Canola-enriched metanauplii (16%).

 

Figure 1. a) Survival (%), b) K condition index of H. erectus juveniles during treatment. Black squares: Artemia without enrichment; white rectangles: Artemia + Canola oil; black triangles: Artemia + Selco; white circles: Artemia + EPAX. Different letters are significantly different at 60 days (P < 0.05).

Condition index was higher in seahorses at the start of the experiment (0.38), ending after 60 days with 0.32 at the EPAX treatment and a minimum of 0.30 in seahorses fed Canola treatment (Fig. 1b). Seahorses' weight (Fig. 2a) at the start of the experiment was 17 ± 5 mg for all treatments, but weight was significantly different (F = 106.3; P < 0.001) by the end of the experiment with bigger juveniles fed Selco (249 ± 53 mg) and EPAX (231 ± 55 mg), followed by juveniles fed non-enriched metanauplii (141 ± 50 mg), and the smallest juveniles were obtained when fed Canola (109 ± 39 mg).

 

Figure 2. a) Weight (mg), b) height (mm) gain during treatment. Black squares: Artemia without enrichment, white rectangles: Artemia + Canola oil, black triangles: Artemia + Selco, white circles: Artemia + EPAX. Different letters are significantly different at 60 days (P < 0.05).

Height (Fig. 2b) followed a similar behavior with initial values of 16.4 ± 1.8 mm for all treatments, then significantly bigger juveniles were obtained by the end of the experiment when fed Selco (43.4 ± 0.2 mm) or EPAX (41.6 ± 0.6 mm) compared to significantly (F = 76.45; P < 0.001) smaller juveniles fed non-enriched metanauplii (35.4 ± 0.4 mm) and still smaller juveniles fed Canola (32.9 ± 0.1 mm).

Biochemical and fatty acid composition of metanauplii in the four treatments

Total protein in Artemia metanauplii was higher in non-enriched metanauplii and lower in Selco, with intermediate values for EPAX and Canola. Total carbohydrates were also higher in non-enriched metanauplii, with lower levels in EPAX, and intermediate levels in Selco and Canola. There were no significant differences between the total lipid content in Selco, Epax and Canola, but in the non-enriched metanauplii were significantly lower (Table 1).

Table 1. Biochemical (mg g-1 dw) levels (mean ± SE) in non-enriched and enriched Artemia metanauplii with canola oil, Selco, and EPAX (n = 9 samples per treatment), in juveniles at the start of the experiment, and in juveniles at the end of the experiment. Different letters in the same row indicate significant differences (P < 0.05) in metanauplii.

 

One of the work objectives to enrich Artemia metanauplii with DHA to levels above those reached with Selco enrichment was attained, as the EPAX had significantly higher levels of DHA (13.2%) compared to Selco (9%). Nevertheless, we expected the enrichment to be more pronounced, considering the very high levels of DHA in the EPAX oil (63% vs 14% in Selco). Canola and non-enriched metanauplii had traces of DHA (Table 2).

Table 2. Fatty acid (% of total fatty acids) levels (mean ± SE) in non-enriched and enriched Artemia metanauplii with canola oil, Selco, and EPAX (n = 9 samples per treatment). Different letters in the same row indicate significant differences (P < 0.05) in metanauplii.

 

The EPA level was also higher in the EPAX (10.9%) compared to Selco (7.7%), and both were higher than in Canola (1.7%) or non-enriched metanauplii (1.4%). The level of ARA was significantly higher in Selco (1.6%) than in any other treatment (0.8-1.1%).

We expected the levels of 18:1n-9, 18:2n-6 and 18:3n-3 to be higher in the Canola, which was true for the 18:1n-9 (36.7%), almost double than that of the other treatments. The level of 18:2n-6 was also higher in the Canola and doubled the levels in the EPAX. However, the level of 18:3n-3 was higher in the non-enriched metanauplii (29.8% compared to 19.3% in Canola).

Biochemical and fatty acids composition of juveniles after 60-days treatment

After the 60 day-treatment, total protein and lipid levels in juveniles were not significantly different among treatments regardless of differences among enriched and non-enriched metanauplii (Table 1). However, total carbohydrates were significantly higher in the juveniles fed the Selco followed by the EPAX. The lowest carbohydrate levels were found in juveniles fed the non-enriched and the Canola.

All fatty acids analyzed in juvenile seahorses were affected by treatments at the end of the experimental period (Table 3). The level of DHA was four-fivefold higher in juveniles fed Selco and EPAX, compared to juveniles fed Canola or non-enriched metanauplii. In juveniles fed EPAX, EPA was significantly higher (7%) in comparison to the juveniles fed the Selco (5%), and these levels were significantly higher compared to the juveniles from Canola (2%) or non-enriched metanauplii (3%). Hence, the DHA/EPA ratio was significantly higher in juveniles fed the Selco (4.8), followed by juveniles fed EPAX (3.4), with a much lower ratio for the other two treatments (2.1). The high DHA and EPA levels affected the total PUFA levels, which reached more than half of the total fatty acids, and the n-3/n-6 ratio, which was fourfold higher in juveniles fed the Selco and EPAX compared to the other two treatments.

Table 3. Fatty acid (% of total fatty acids) levels (mean ± SE) in juvenile seahorses (n = 15 organisms per treatment) fed non-enriched and enriched Artemia metanauplii for 60 days. Different letters in the same row indicate significant differences (P < 0.05) among treatments.

 

In all treatments ARA relative concentration decreased at 60 days. Starting with 4.6% the lowest concentration occurred in EPAX (2%) and the highest in juveniles from non-enriched treatment (3.2%). Juveniles fed the Canola treatment had significantly higher levels of 18:1n-9 and 18:2n-6, compared to the other treatments. The highest values of 18:1n-9 was also reflected in the total MUFA content in this treatment. Juveniles fed the non-enriched metanauplii had significantly higher levels of 16:0 and 18:0 and, therefore, higher levels of total saturated fatty acids (Table 3).

DISCUSSION

The objective of this work was to test the rearing performance (survival and growth) of juvenile seahorses by giving them different HUFA levels, particularly DHA. We usually use Selco emulsion with 14% of DHA (according to our analysis) for Artemia metanauplii enrichment, so to increase DHA levels we used EPAX, a commercial oil produced from raw Peruvian fish for human consumption with more than 60% of DHA, which was emulsified and given to Artemia metanauplii. Canola oil was used as an isolipidic treatment to enrich metanauplii, but devoid of HUFA. Contrary to our expectations, the DHA level in the metanauplii was not much higher when using EPAX (13%) compared to Selco (9%), although there was a significant difference between both. DHA is particularly difficult to increase in Artemia metanauplii as it has been previously reported; for example, Faleiro & Narciso (2011) managed to increase DHA levels in Artemia metanauplii to 4% when using Algamac 2000 emulsion containing 27% of DHA. This decrease of DHA in enriched Artemia metanauplii can be a result of retroconversion from DHA to EPA, which has been proposed before in Artemia (Navarro et al., 1999; Han et al., 2001). However, it could also be a result of the Artemia strain used, since different strains might present different retroconversion capacities, and of different enrichment methodologies, such as enriching twice, washing after the enrichment, light and temperature, etc. Using double enrichment and ICES emulsions with 9% DHA, we had previously obtained 13% of DHA in Artemia metanauplii (Palacios et al., 2004). Part of the DHA in EPAX could have been lost by oxidation during emulsification and enrichment, since it is not designed as commercial emulsion to be used dissolved in water. In any case, we started with a 4-fold DHA in EPAX compared to Selco, and ended with 50% increase in DHA levels in metanauplii. As expected, Canola-enriched and non-enriched meta-nauplii had traces of DHA and less than 2% of EPA, but the three treatments with enriched metanauplii had similar lipid levels.

When comparing performance to biochemical and fatty acid composition of juveniles, we found that even if there were differences in the DHA proportion in metanauplii between Selco and EPAX, juveniles had similar DHA levels in their tissues in both treatments (23-26%). Interestingly, 23% of DHA was also what we found the newborn juveniles had before first feeding (see methodology). It is possible that DHA upper levels are regulated in juvenile H. erectus seahorse, as DHA is not further accumulated when given extra DHA in the diet. By contrast, in seahorses fed non-enriched metanauplii and Canola, DHA content had a strong decrease (more than 80%), till reaching 4-5% of total fatty acids. Faleiro & Narciso (2011) found similar levels of DHA in H. guttulatus juveniles at the beginning of the experiment (17%) compared to our levels in H. erectus (20%) in this study; DHA levels in H. guttulatus fell to 3-7% feeding Algamac-enriched Artemia with 4% of DHA after 30 days of experiment.

Lower levels of n-3 HUFA might be affecting growth, as juveniles fed non-enriched metanauplii or Canola were smaller; this is further supported by a significant correlation between DHA tissue levels of juveniles and weight (r = 0.89) and a lower correlation of EPA in tissues and weight (r = 0.78). Other studies have also concluded that higher DHA content has a positive effect on fish performance during early stages (Mourente et al., 1993; Olivotto et al., 2011; Trushenski et al., 2012). On these stages there is a great energy demand because the rapid growth of fishes. DHA promotes a better development and function of motocondria, and a higher production of enzymes and coenzimes with a central position in the metabolism (Olivotto et al., 2011; Yin & Tang, 2012).

In relation to survival, we found juveniles fed Canola that had 4% DHA started dying by the second week, and end of the experiment had the highest mortality, consistent with the lower condition index. The lowest survival for juveniles fed the Canola treatment could be observed from the second week of culture, which is consistent with the results for Morone saxatilis, where a mass mortality after two or three weeks of culture was observed but only in organisms fed low levels of HUFA in diets (Tuncer & Harrell, 1992). However, the juveniles fed non-enriched metanauplii also lacked HUFA, and juveniles were left with 5% of DHA after 60 days of treatment; nonetheless, survival was similar to the juveniles from the EPAX, that had 23% DHA. Therefore it is possible that Canola oil might have anti-nutritional factors that not only hamper growth (Manajan et al., 1997; Ali et al., 2009) but also survival, in spite of the loss of most anti-nutritional factors during canola oil commercial processing.

In contrast to growth, survival was not so strongly associated to DHA levels in juveniles (r2 = 0.44; F = 7.9; P < 0.05) and no correlation was found to any other fatty acid or to the general biochemical composition of seahorse tissues. Juveniles fed Selco had excellent survival rates in spite of similar DHA levels (25%) with EPAX treated juveniles (23%). It should be pointed out that Selco is not solely composed of fatty acids, it is also enriched in vitamin C, amino acids, and phospholipids (according to the manufacturer), which is not the case of EPAX.

Selco-treated juveniles had more ARA (both in the diet and in the tissues), more carbohydrates and a higher DHA/EPA ratio compared to EPAX-treated juveniles that could account for some differences in survival. In Sparus aurata larvae, increasing levels of ARA from 0.1 to 1% in diets significantly improves survival (Bessonart et al., 1999). Has been discussed the importance of ARA with DHA and EPA by Sargent et al. (2002), who proposed that, in diets, the ratio DHA/EPA should also consider ARA levels, and be on the order of 8:1 EPA/ARA, which is very similar to that occurring in the Selco treatment. ARA can be a precursor for the synthesis of eicosanoids through the COX and LOX enzymes. These enzymes also used EPA as substrate to produce other eicosanoids, therefore, if ARA levels are too low in comparison with those of EPA, the synthesis of certain prostaglandins may be inhibited and biased in favor of eicosanoids derived from EPA, with reduced biological activity (Bransden et al., 2004). In this experiment the highest EPA/ARA ratio occurred in EPAX and this may have been detrimental to their survival.

Thus, juvenile H. erectus seahorses, which have above 20% of DHA in tissues, are expected to perform better during culture, particularly in reference to growth. This relative concentration in tissues could be attained with approximately 10% of DHA in Artemia metanauplii used as diet. Survival is less dependent on DHA levels and less dependent on EPA levels in tissues. In this research, the work was focused on the DHA role, because seems to be the most important HUFA for fish in early stages, further research is recommended regarding the role of EPA/ARA ratio in performance of seahorses rearing.

ACKNOWLEDGMENTS

The authors are grateful to M.Sc. Gemma Martínez M., technician in charge of Area Experimental de Ecología y Conducta, UMDI Sisal, where seahorses were maintained and cultured and to Diana Dorantes for English editing. This research was funded by projects PAPIIT No. IN208009 and SEP-CONACYT No. 101610 granted to M. Mascaró and SEP-CONACYT No. 4081 granted to Elena Palacios. Nicolas Vite-García is a doctoral CONACYT fellowship recipient, No. 42594.

 

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Received: 1 August 2013; Accepted: 18 December 2013

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