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The Proteolytic Digestive Activity and Growth During Ontogeny of Parachromis Dovii Larvae (Pisces: Cichlidae) Using Two Feeding Protocols

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The Proteolytic Digestive Activity and Growth During Ontogeny of Parachromis Dovii Larvae (Pisces: Cichlidae) Using Two Feeding Protocols Fish Physiol Biochem DOI 10.1007/s10695-014-9920-0 The proteolytic digestive activity and growth during ontogeny of Parachromis dovii larvae (Pisces: Cichlidae) using two feeding protocols Jose´ R. Quiro´s Orlich • Silvia Valverde Chavarrı´a • Juan B. Ulloa Rojas Received: 22 October 2013 / Accepted: 10 February 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract The proteolytic digestive activity and proteases was higher in larvae reared with Artemia growth of Parachromis dovii larvae during the ontog- after 12 DAH, whereas the specific enzymatic activity eny were evaluated in a recirculation system using two was similar for both enzyme types in the two feeding strategies during a 28-day period. Larvae were treatments. The results suggest that P. dovii larvae reared using two feeding protocols (three replicates were capable to digest formulated diets from the each): (A) Artemia nauplii (at satiation), fed from beginning of exogenous feeding and that they could be exogenous feeding [8 days after hatching (DAH)] until reared with formulated feeds. However, the formu- 15 DAH followed by nauplii substitution by formu- lated feed used should be nutritionally improved lated feed (20 % day-1) until 20 DAH and then because of the poor growth obtained in this research. formulated feed until 28 DAH; (B) formulated feed (100 % BW daily) from exogenous feeding until 28 Keywords Parachromis dovii Á Fish larvae Á DAH. Levels of acid (pepsin type) and alkaline Fish digestive enzymes Á Enzymes ontogeny Á digestive proteases as well as growth and survival of Larvae feeding larvae were measured along the feeding period. Survival was high and similar between treatments: 98.9 ± 0.0 for Artemia, 97.3 ± 0.0 % for formulated Introduction feed. The specific growth rate for length and weight was higher in larvae fed with Artemia nauplii than in The cichlid Parachromis dovii is a freshwater fish that larvae reared with formulated feed: 3.4 ± 0.1 versus has been exploited for a long time as a resource for 1.8 ± 0.1 % day-1 for body length (P = 0.009) and sport fishing and food by the Central America 12.2 ± 0.1 versus 6.5 ± 0.3 % day-1 for body population. This autochthonous fish species showed weight (P = 0.002). The acid and alkaline proteolytic good traits for culture: excellent flesh flavor, easy activity was detected, in both treatments, from the reproduction in captivity, high resistance to diseases, beginning of the experiment, at 8 DAH. The total manipulation and acceptance to formulated feeds enzymatic activity (U larva-1) for acid and alkaline (Gu¨nther 1996; Bussing 2002). As a carnivorous species, it requires diets with high protein content along its life cycle, but especially J. R. Quiro´s Orlich Á S. Valverde Chavarrı´a Á during the first stages of development. The larvae & J. B. Ulloa Rojas ( ) stage is one of the most critical in fish culture, and Escuela de Ciencias Biolo´gicas, Universidad Nacional, 86-3000 Heredia, Costa Rica generally, they need live food for adequate growth e-mail: [email protected] during the first days. The first feeding is one of the 123 Fish Physiol Biochem biggest problems in larval rearing of many fish species The objective of this research was to evaluate the because they do not accept formulated feeds easily or effect of two feeding protocols on the digestive the feed do not meet all the larvae requirements. proteolytic activity, growth and survival during the Further, these feeds must meet several criteria such as ontogeny of P. dovii larvae. easy ingestion, digestion and assimilation of nutrients (Lazo et al. 2007). In the present time, live food still plays an important Materials and methods role as feed for fish larvae; however, it can represent about 50 % of production costs and possess a variable Larvae nutritional quality (Kanazawa 2000; Suzer et al. 2007). For these reasons, formulated diets are now Parachromis dovii larvae were obtained from sponta- more widely used reducing significantly the feeding neous spawns at the hatchery unit of the Escuela de costs but resulting in lower growth and, in some cases, Ciencias Biolo´gicas of Universidad Nacional, Costa in higher mortalities than live foods, especially with Rica. After hatching, all larvae from one spawn (about marine larvae (Civera-Cerecedo et al. 2004). Further, 680) were transferred from the spawners’ tank to partial substitution of live feed (co-feeding) is a widely aquaria (11 l) in a recirculation system until beginning used practice (Hamza et al. 2007). of experiment. The water temperature was kept at The use of artificial feeds in larval rearing provides 25.2–29.4 °C, dissolved oxygen at 6.0–6.8 ppm and some advantages such as greater availability, easy NH at 0.05 ppm maximum. storage and lower costs. However, their success 3 depends on the digestive capacity of the fish larvae Feeding to be fed (Uscanga-Martı´nez et al. 2011). In this context, studies on the digestive physiology allow a Larvae were randomly distributed into six aquaria and better diet formulation according to fish larvae reared with two feeding protocols (by triplicates each). development, which in turn produce better growth Each aquarium contained 113 larvae. In both protocols, and health, a reduction on feed wastes and an increase larvae were fed in excess at the same feeding frequency. in profitability for the producers (Tengjaroenkul et al. The feeding schedules used were as follows: 2002; Drossou et al. 2006; Gonza´lez-Fe´lix et al. 2010). (A) Artemia franciscana nauplii (Great Salt Lake, Several works have been focused on the study of Artemia InternationalÒ) from 8 to 15 days after digestive enzyme variability during larvae ontogeny hatching (DAH), followed by a 20 % daily and on the digestive system development and its substitution with formulated feed until 20 DAH functionality. These studies also demonstrated that the and continued with formulated feed until 28 DAH digestive system is not completely developed at first (end of larval development). Larvae were fed four feeding (Yu´fera and Darias 2007). times daily (0900, 1130, 1430 and 1700 hours). Digestive enzymes ontogeny of larvae has been Artemia nauplii were harvested from 1 l contain- studied in many freshwater fish groups, such as ers (17 ppm) with 3 g cysts after 24 h. The mean salmonids (Golchinfar et al. 2011), cyprinids (Chak- nauplii hatching rate was 254 per ml. rabarti et al. 2006; Chakrabarti and Rathore 2010), (B) Formulated feed (45 % protein and 10 % lipid, catfishes (Pradhan et al. 2013), sturgeons (Babaei et al. Table 1), fed from 8 DAH (at the beginning of 2011; Sanz et al. 2011), gars (Comabella et al. 2006), exogenous feeding) to the end of experiment (28 tilapias (Tengjaroenkul et al. 2002; Lo and Weng DAH). Larvae were fed 100 % of body weight 2006) and Neotropical cichlids (Lo´pez-Ramı´rez et al. as in protocol A. The feed was prepared by using 2011; Uscanga-Martı´nez et al. 2011). These studies a pelletizer provided with a 2 mm die. It was have detected digestive enzyme activity in larvae since oven-dried at 60 °C for 24 h, and then, the first feeding. However, the level of enzyme activity, spaghetti-like feed was crushed with a labora- the larvae growth and survival depend on feed quality tory mill. The different feed particles used (Drossou et al. 2006; Aguilera et al. 2012; Kamarudin (powder, 300 lm, 600 lm and 1 mm diameter) et al. 2011). were separated using appropriate sieves. 123 Fish Physiol Biochem Table 1 Ingredient composition of dieta fed P. dovii larvae every period) before feeding and kept 1 h in clear Ingredientsb (g kg-1 as feed) water to allow the emptiness of digestive tract (Gisbert et al. 2009). Viscera samples of about 20 mg larvae-1 Tuna meal 480 were removed and homogenized with 1 ml distilled Soymeal 210 water in an ice bath using a tissue homogenizer Wheat feed flour 150 (Contes). Samples from the smallest larvae were taken Tankage meal 90 by removing their head and tail. Next, the homoge- Soy oil 20 nized sample was centrifuged at 14,000g,4°C for Cod liver oil 20 30 min (Hettich Mikro 200). The supernatant was kept Vitamin premix 20 at -20 °C for further enzymatic analysis. Salt 10 Soluble protein quantification a Nutrient content: 45 % protein and 10 % lipid b Tuna meal (IFN: 5-02-023, processing meal, min. 49 % protein), Soybean meal (IFN: 5-20-637, min. 44 % protein), wheat Total soluble protein was measured by the Bio-Rad flour (IFN: 4-05-199, 11 % protein), tankage (IFN: 5-00-388, min. kit, based on Bradford method using bovine serum 50 % protein) albumin as the standard (Bradford 1976). Mixtures of 5 ll enzymatic extract, 795 ll distilled water and Before feeding, all feed and feces wastes remaining 200 ll Bradford reagent were incubated for five min at were removed from the bottom of the aquaria, and room temperature. The absorbance was read at during feeding, the water flow was stopped for 30 min 595 nm, using a blank without extract, with a Jasco to avoid feed losses. V-630 spectrophotometer. Growth parameters and survival Proteolytic activity determination Total length (mm) and weight (mg) were determined The measurement of alkaline and acid proteolytic from 10 larvae at 8, 12, 16, 20, 24 and 28 DAH for activities was done according to Sarath et al. (1989). each aquaria. Previously, larvae were anesthetized To determine total alkaline proteolytic activity, enzy- with tricainemetansulfonate (MS-222), measured and matic extract was incubated with azocasein (2 %) pH weighted with a stereoscopy (Olympus 329345 with 9.0 for 30 min at 37 °C in a flux water bath.
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