Manuel Yúfera

Histology and histochemistry of the development of the digestive system of larval gilthead seabream, Sparus aurata L.

Resorption of the yolksac and development of the digestive tract and associated organs, including the swim bladder, were studied in Sparus aurata larvae from hatching until day 30 using histological and histochemical procedures. At the onset of exogenous feeding three regions could be easily distinguished in the gut: the foregut including the oesophagus and a primordial stomach, the midgut and lastly the hindgut. At this time, the digestive tract was functional even though the stomach was not yet completely developed; gastric glands, for instance, were not present in the period studied. Glycogen and zymogen grains were stored in the liver and pancreas, respectively between days 4 and 6 after hatching. Proteins were observed in the pancreas, hepatic vascular system and to a lesser extent the hepatocytes. Once feeding had commenced, the anterior intestinal epithelium developed the capacity for the absorption of lipids, most of which were included in large lipid droplets. Simultaneously, acidophilic supranuclear inclusions containing proteins were observed in the posterior intestinal epithelium. All oesophageal mucous cells, intestinal goblet cells, epithelial columnar cells in the stomach, and enterocytes of the digestive epithelium were rich in carboxylated, sulphated and/or neutral mucosubstances. Proteins were not present in digestive goblet cells.

Digestive enzyme activity during larval development of the Senegal sole (Solea senegalensis)

The activities of the main digestive enzymes (proteases, amylase, lipase) as well as those of acid and alkaline phosphatases were assessed during the larval development of the Senegal sole, Solea senegalensis. Important variations in specific activities of all the enzymes were observed during the period of study and were mostly related to the beginning or the end of metamorphosis. Both acid and alkaline protease activities were identified at early stages of development. Acid proteases accounted for only 10% of total protease activity in a 9 days-old larva, but exceeded 75% in a 33 days-old individual. Maximum lipase activity was related to the development of exocrine pancreas (days 6 to 10) and to metamorphosis. It is suggested this can be related to the metabolism of lipid reserves taking place during this morphological change. Activity of alkaline phosphatase decreased from day 5 to day 20 but therafter revealed a sharp increase, possibly linked to the development of enterocytes. The pattern of development of the main digestive enzymes found in S. senegalensis is similar to that described in other flatfish species.

A highly efficient microencapsulated food for rearing early larvae of marine fish

Abstract This paper describes the process of elaboration and the characteristics of an inert microencapsulated diet which provides much improved growth and development in the rearing of gilthead seabream larvae. The microencapsulated diet was composed of casein, fish protein hydrolysate, octopus meal, dextrin, emulsified lipids and a vitamin mixture. Following 4 days feeding on rotifers, three rearing treatments were used from day 8 to day 15 after hatching: Regime A (control): larvae fed on rotifers in semi-open system; Regime B: larvae fed on microcapsules and reared in a semi-open system; Regime C: larvae fed on microcapsules and reared in a closed recirculating water system. Growth rate and survival were similar in the three treatments (growth rates: 0.13, 0.09 and 0.12 day −1 , respectively), and notably higher than those obtained with the previous prototype. The results clearly show that the present microencapsulated diet was able to substitute for live food during the early stages of larval rearing and hence can be used as an excellent tool for investigation of the nutritional requirements of marine larval fish.

Feeding behaviour and prey size selection of gilthead seabream, Sparus aurata, larvae fed on inert and live food

Prey selection shortly after the onset of feeding by laboratory-reared gilthead seabream, Sparus aurata L., larvae was studied using larvae fed on two types of microcapsule (hard- and soft-walled) having diameters ranging from 25 to 300 μm. Preferences between inert food and live prey (rotifers and Artemia sp. nauplii) were also studied. Seabream larvae were able to ingest inert food from first feeding. Larvae of all size classes ingested hard microcapsules with diameters in the range 25 to 250 μm. However, larvae with a total length (TL) below 4 mm preferentially selected particles 25 to 50 μm in diameter, larvae of TL 4 and 5 mm preferred particles 51 to 100 μm in diameter, while larvae above 5 mm TL preferred particles 101 to 150 μm in diameter. With soft microcapsules, larvae always preferred particles larger than in the previous case, and above 4.5 mm TL they preferentially selected particles 201 to 250 μm in diameter. In addition, the gradual increase of preferred diameters with increasing TL was more pronounced when larvae were increasing TL was more pronounced when larvae were fed on soft particles. Mean values for prey width/mouth width ratios were approximately 0.24 and 0.30 when larvae were fed on hard-walled and soft-walled microcapsules, respectively, irrespective of the absolute value of larval length. When a mixed diet of live and inert food items was offered, live prey were always preferentially selected, even if the prey width/mouth width ratio was apparently not favourable. Only a physical constraint such as excessive prey width could counter this preference for living prey vs inert microcapsules. These results contribute to our knowledge in larval feeding behaviour, especially in the presence of inert food, and represent a fundamental step in developing prepared food for marine fish larvae.

Effects of temperature on egg and larval development of Sparus aurata L.

Abstract The optimal temperature range for embryonic development of Sparus aurata is estimated. Hatching rate was maximal (91–95%) from 14 to 26°C. Eggs did not hatch at 12 and 30°C. Abnormalities at hatching are usual outside the 16–22°C range. Higher survival at the stage of mouth opening occurred between 16 and 22°C; outside this range mortality and abnormalities increased considerably. Growth rate and yolk absorption rate increased with increasing temperature. The conversion efficiency index (yolk volume to larval length) was higher at 16°C. However, when the complete period from fertilization to mouth opening is taken into account, 19°C may be considered as the optimal temperature for development, since larvae reached the greatest length at 19°C.

Effect of starvation on the feeding ability of gilthead seabream (Spams aurata L.) larvae at first feeding

The effect of delayed initial feeding on the ingestion ability of gilthead seabream Spams aurata larvae was studied. The percentage of larvae with visible gut contents was maximal when food was first provided on the 5th day after hatching and decreased to half this value when provision of food was delayed until Day 8, indicating the point at which starvation became irreversible. Morphological and histological changes were studied in growing and starving larvae. Sizes of different organs increased in actively feeding larvae but decreased in unfed larvae, particularly in the case of the liver. In unfed larvae, development due to yolk reserves attained a maximum on Day 5. The digestive tract was functional in 4-day-old larvae, which had food in the gut. The absorption process was obvious from Day 6, although it could be detected in 5-day-old larvae. Starving larvae exhibited widespread histological degeneration, especially in the digestive tract and associated glands.

Free amino acid leaching from a protein-walled microencapsulated diet for fish larvae

The leaching loss of free amino acids (FAA) from an experimental protein-walled microencapsulated diet (MC) for larval fish after immersion in water was measured and compared with leaching loss from a gelatin microbound diet (MB) containing the same dietary ingredients. The loss of FAA (as a percentage of total dietary FAA) was significantly higher in the MB diet compared to the MC diet. After 5 min of re-hydration, 22% of FAA were leached from the MB diet compared with 8% from the MC diet. After 60 min, 85% of FAA from the MB diet were leached into the water compared to 17% from the MC diet. The addition of free lysine to protein-walled microcapsules (MC-L) was also investigated to determine the suitability of MC for delivering specific FAA. Lysine was incorporated into the particles with an efficiency of 7.5% and accounted for approximately 60% of the total FAA in the diet. The loss of this amino acid from MC-L after 60 min of immersion was 1.4%. The loss of specific FAA from MB and MC diets was found to be negatively and positively correlated to the hydrophobic character (hydropathy index) of each FAA, respectively. These results support the use of protein-walled microcapsules as a vector for specific dietary amino acids using a macronutrient-balanced diet.

Detecting growth in gilthead seabream, Sparus aurata L., larvae fed microcapsules

Abstract The ability to grow Sparus aurata fed a prototype microencapsulated food in relation to larval age was examined. The contribution of live prey in co-feeding experiments (live + inert food) was also determined. To attain these objectives several feeding regimes differing in the time that inert food was introduced were tested during the first 2 weeks of larval life. Larvae fed microcapsules alone from first feeding did not increase their dry weight during the experimental period, but after the point of irreversible starvation (day 8 after hatching), the larvae were able to grow (specific growth rate 0.034) and recover their initial dry weight. The larvae fed microcapsules from day 8 or day 12, after previously being fed rotifers (from day 4 to day 8 or 12), showed similar growth rates (specific growth rate 0.053 and 0.034, respectively), although in these cases the final dry weight was higher. Survival of larvae fed only microcapsules ranged from 11%, when the capsules were added from first feeding, to over 50% when pre-fed rotifers. The addition of live prey (5% of the total food supplied on dry weight basis) improved the survival (42%) when the microcapsules were supplied from the start of feeding. Feeding incidence on microcapsules was similar to that obtained with rotifers and the physiological status of the surviving larvae at end of the experiments was acceptable. Water quality was similar between the controls fed rotifers and the treatments fed microcapsules, indicating that no negative effect on larval growth could be attributed to water quality. This study indicates that S. aurata larvae are able to grow when fed only microcapsules, although with a low growth rate probably due to a lower assimilation of the diet. Further experiments testing other sources of protein and additives are needed in order to understand the factors that are limiting larval growth.

Feeding, physiology and growth responses in first-feeding gilthead seabream (Sparus aurata L.) larvae in relation to prey density

Abstract The effects of prey density (0.1, 1 and 10 rotifer ml −1 ) on feeding, oxygen uptake, growth, survival and energetic efficiencies in Sparus aurata larvae were investigated. Dry weights and total lengths at day 15 after hatching were similar in larvae fed with 10 and 1 rotifer ml −1 but were significantly higher than in larvae fed with 0.1 rotifer ml −1 . These differences were a consequence of a delay in starting feeding and growth in larvae fed with the lowest rotifer concentrations tested. Growth rates were similar in larvae fed on 0.1 and 1 rotifer ml −1 , while larvae fed with 10 rotifers ml −1 showed the smallest growth rate. Survival decreased markedly (from 91 to 7%) when prey density decreased. Ingestion rates of the actively feeding larvae increased with age at the three prey densities but larvae fed with 0.1 rotifer ml −1 showed lower ingestion rates. The specific oxygen uptake increased from hatching up to 35–40 μg dry weight; above this size the specific oxygen consumption remained almost constant. Survival at the end of the experimental period was closely related to the percentage of larvae able to start feeding at day 6. In larvae fed with 0.1 rotifer ml −1 , the energy ingested during the first days of feeding roughly met maintenance requirements. Therefore, the survival of larvae able to start feeding is not guaranteed at such low prey density.

Feeding rates of gilthead seabream (Sparus aurata), larvae on microcapsules

Abstract Food consumption rates of gilthead seabream larvae from first feeding to 20 days of age were studied by feeding larvae on two types of microcapsule (hard- and soft-walled) having diameters ranging from 25 to 400 μm. Experiments were carried out in 15-litre beakers using larvae cultured with standard techniques and fed on rotifers and Artemia sp. nauplii. Ingestion rate ranged from 3 to 35 particles · larva−1 · h−1. but, except for some increase during the first days of feeding, no consistent trend was observed in relation to larval weight. However, when values were converted to volume or dry weight units, food consumption rate increased progressively with increasing larval weight. On a volume basis, the rate of this increase was different for the two microcapsule types, being generally higher with soft microcapsules, probably due to its better accessibility and to their easier progress through the digestive tract. However, on a dry weight basis the rates of food consumption were similar with both types of microcapsule because of the higher density of hard-walled microcapsules. Ingestion rates increased from 0.5–3 μg · larva−1 · h−1 at first feeding up to 18–25 μg · larva−1 μg · h−1 in older larvae. The daily specific ration increased from 0.5 to 2.5 μg food · μg larva−1 · d−1 at first feeding to a maximum of 3–5 μg food · μg larva−1 · d−1 at a larval dry weight of 70–110 μg. Above this larval weight, the specific ration tended to remain constant. The results show that gilthead seabream larvae can be transferred directly from live to microencapsulated food without appreciable variation in the incidence of feeding or in the rate of food consumption from first feeding onwards.