Developmental changes of digestive enzymes in Persian sturgeon (Acipenser persicus) during larval ontogeny

Aquaculture 318 (2011) 138–144

Contents lists available at ScienceDirect

Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Developmental changes of digestive enzymes in Persian sturgeon (Acipenser persicus) during larval ontogeny Seyedeh Sedigheh Babaei a, Abdolmohammad Abedian Kenari a,⁎, Rajabmohammad Nazari b, Enric Gisbert c a b c

Fisheries Department, Tarbiat Modares University, Noor, P.O. Box 64414-356, Noor, Mazandaran, Iran Shahid Rajaee Sturgeon Hatchery Center, Sari, P.O. Box 833, Sari, Mazandaran, Iran IRTA, Centre de Sant Carles de la Ràpita, Unitat de Cultius Experimentals, Crta. de Poblenou km 5.5, 43450 Sant Carles de la Ràpita Tarragona, Spain

a r t i c l e

i n f o

Article history: Received 11 December 2010 Received in revised form 6 April 2011 Accepted 15 April 2011 Available online 30 April 2011 Keywords: Acipenser persicus Larval growth Digestive enzyme Ontogeny Weaning

a b s t r a c t The development of digestive enzymes from the stomach (pepsin), pancreas (trypsin, chymotrypsin, αamylase and lipase) and intestine (alkaline phosphatase) was studied in Persian sturgeon (Acipenser persicus) from hatching to the juvenile stage at 40 days post hatching (dph). Larvae were obtained from artificial propagation of one male and one female and transferred to larval culture tanks where, after yolk sac absorption (9 dph at 17–18 °C), they were fed with Artemia urmiana and Daphnia sp. The assessment of the activity of digestive enzymes showed that at the onset of exogenous feeding, gastric glands were already functional as indicated by the increase in pepsin specific activity. In contrast, alkaline proteases like trypsin and chymotrypsin decreased their specific activity after the onset of exogenous feeding, indicating the importance of these types of enzymes in the cleavage of yolk proteins during the endogenous feeding phase and the replacement of the larval alkaline-type digestion by a juvenile-type acid digestion. After the first feeding, amylase and lipase specific activities increased. Such increments in the activity of amylase might be genetically programmed to better digest carbohydrates in diets with the goal of sparing proteins during the larval stage, whereas the increase in lipase was related to changes in the lipid content of live prey and the progressive maturation of the pancreatic function during larval development. Changes in enzyme activities from the stomach and pancreas were coupled with that in the intestine (brush border membrane), where the specific activity of alkaline phosphatase progressively increased until 19–24 dph and remained constant thereafter, indicated the maturation of the intestine and the achievement of a juvenile-like mode of digestion. Considering these data on the digestive enzymes from the pancreas, stomach and intestine, Persian sturgeon larvae might be weaned around 19–24 dph, as larvae have achieved the complete maturation of their digestive functions by this date. This developmental process, and particularly for the digestive functions, can be considered as a reference to evaluate the effect of a formulated micro diets feeding on larvae. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Persian sturgeon (Acipenser persicus) inhabits in the southern part of the Caspian Sea, being one of the most important components of the Caspian Sea ichthyofauna. In the mid-1980s, this sturgeon species constituted 85% of the standing stock of the world sturgeon population (Abhari and Tavakkoli, 1999), although the population collapsed in the 1970s (Moghim et al., 2006). Persian sturgeon is among the most vulnerable fish species because of overfishing for meat and caviar production, destruction of their spawning grounds and water pollution; this species is currently included in the IUCN Red Data List. For these reasons, most research in recent years has been focused on the culture of Persian sturgeon for restocking and commercial ⁎ Corresponding author. Tel.: + 98 1226253101 3; fax: + 98 1226253499. E-mail addresses: [email protected] (S.S. Babaei), [email protected], [email protected] (A. Abedian Kenari), [email protected] (R. Nazari), [email protected] (E. Gisbert). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.04.032

aquaculture programs. In this sense, there is a sturgeon stock replenishment program in Iran since 1972. The development of hatchery technology for sturgeon larvae rearing has become necessary in order to grow these fishes up to fingerling size for restocking and aquaculture purposes. Traditionally, hatchery-produced sturgeon larvae and fingerlings were raised on live food organisms, e.g., oligochaetes (Enchytraeus sp. and Tubifex sp.), daphnia (Daphnia sp. and Moina sp.) and Artemia sp. (see review in Gisbert and Williot, 2002). However, the production of live food is a labor-intensive and expensive process, since their production and enrichment require of considerable space, manpower and labor. Moreover, the nutritional supplies of live food are often inadequate to complete the growth-out phase. Although moist diets based on natural products (blood and bone meals, silk worms, mineral and vitamin supplements) were formulated in the former U.S.S.R. during the early development of sturgeon hatcheries, their use had limited success and did not replace cultured live feeds. During the last twenty years, several studies have demonstrated that artificial larval diets can be used successfully for

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

intensive commercial culture of several sturgeon species (e.g., Acipenser baerii, A. transmontanus, A. oxyrinchus and A. medirostris) from the onset of exogenous feeding (Bardi et al., 1998; Buddington and Doroshov, 1984; Dabrowski et al., 1985; Gisbert and Doroshov, 2006). However, limited success has been observed when feeding Persian sturgeon larvae with compound diets. The efficiency of food depends on physiological capacities in fish to digest and transform ingested nutrients (Furne et al., 2008). Digestion mechanisms in fish larvae have been particularly studied during the last 2 decades. Similar to most fish species, sturgeon larvae are not fully developed at hatch and must undergo further development and differentiation before external foods can be properly ingested and digested (Gisbert and Sarasquete, 2000). The ontogenetic development of digestive enzymes reflects the development of the digestive tract and digestive capability of the organism under study and can thus be used as an indicator of nutritional status at an early life stage (Yúfera and Darias, 2007a) and can provide information for determining the appropriate time for weaning in fish culture (Zambonino-Infante and Cahu, 2007). A comprehensive analysis of the ontogenic changes during the early life stages of fish is essential for the design of feeding strategies and formulation of dry diets (Verreth and Segner, 1995). The analysis of digestive enzyme activities is an easy and reliable biochemical method that can provide insight into the digestive physiology in fish larvae, their nutritional condition (Bolasina et al., 2006) and assist in defining the nutritional requirements for several nutrients like proteins, lipids or carbohydrates (Twining et al., 1983). As Buddington and Doroshov (1986) stated in their review, the composition of digestive enzymes in sturgeons depends on the fish age, diet composition, genetic and other factors. The early development of sturgeons includes three phases: the yolk sac stage, the actively feeding larval period and metamorphosis, during which enzymatic activity reaches a level typical for juveniles and adults (Buddington, 1985; Zółtowska et al., 1999). The ontogenetic development of the digestive enzymes has been described in many marine and freshwater fish species (see review in Zambonino-Infante et al., 2009); however, there is very limited information on acipenserid species with partial information on A. transmontanus (Buddington and Doroshov, 1986), A. baerii (Gisbert et al., 1999; Zółtowska et al., 1999), Huso huso and Acipencer ruthenus (Timeiko and Bondarenko, 1988). No data are available on the ontogenetic development of digestive enzymes in Persian sturgeon. The aim of this study was to describe the onset and development of the main digestive enzymes (stomach, pancreas and intestine) in Persian sturgeon fed live prey to provide data on the digestive features of larvae and juveniles (from hatching to 40 days after hatching), which will be useful for the development of a formulated compound diet specific for this sturgeon species.

139

larval culture were similar to those recorded during egg incubation. Tanks were indoors and the photoperiod was 12L:12D. Every day, all tanks were cleaned and siphoned to remove debris and dead animals. After yolk sac absorption on the 9th day post hatching (dph), fish larvae were fed with Artemia urmiana (Artemia Research Center Jahad Ministry, Oromieh, Iran) between 9 and 14 dph and then until 40 dph with Daphnia sp. (Fig. 1). In all cases, different live preys were administered ad libitum. 2.2. Sample preparation Samples for analysis of digestive enzyme activity were taken at 1 (hatching), 5, 9 (onset of exogenous feeding), 14, 19, 24, 29, 34 and 40 dph. No food was added to the rearing tank at night on the day prior to sampling, and larvae were sampled in the morning to minimize the effects of exogenous enzymes from live food in fish guts (Kolkovski, 2001). For sampling purposes, two hundred larvae were washed with distilled water and after removing freshwater with a filter paper, frozen in liquid nitrogen and stored at −80 °C until further analysis. Growth measurements were obtained from a pool of 20 larvae at the sampling days. 2.3. Treatment of the samples Whole larvae were homogenized at 0–4 °C in an electric homogenizer (WIGGEN, D500, Germany). For the homogenization, a 100 mM Tris–HCl buffer with 0.1 mM EDTA and 0.1% Triton X-100, pH 7.8, was used at a proportion of 1 g tissue in 9 ml of buffer. The homogenates were centrifuged at 30,000 g for 30 min at 4 °C (Hermle Z36HK, Germany). After centrifugation, the supernatant was collected and frozen at −80 °C (Furne et al., 2008). Determinations of the intestinal brush border membrane enzyme were in accordance with Cahu et al. (1999) methods. The samples were homogenized in 30 v/w fractions of Tris (2 mM)–mannitol (50 mM), pH 7.0 for 30 s at 19,000 g. Brush border extracts were

2. Materials and methods 2.1. Rearing procedures Persian sturgeon larval culture was performed at Shahid Rajaee Sturgeon Hatchery Center, (Sari, Mazandaran, Iran; Lat 36°37′ N, Long 53°05′ E). Broodstock was selected from wild breeders originating from the Caspian Sea and stocked in a 75.4 m 3 tank with a freshwater supply. Persian sturgeon larvae were obtained from artificial propagation of one male (19 kg) and one female (26 kg) in April–May 2009. The type and dose of hormone administration for artificial propagation were LH-RH-A2 and 5 μg kg − 1, respectively. After stripping, eggs were de-adhesived with bole and then transferred to incubators (Russian incubator, 39 × 29 × 18.5 cm 3). Water temperature, oxygen content and pH were maintained at 17–18 °C, N5.1 mg l − 1 and 7.9, respectively during incubation. Eggs hatched after 96 h of incubation and were transferred into 3 culture tanks (4 m 3). Fish were reared with a constant flow-through system. The water quality parameters during

Fig. 1. Growth in total length (■) and wet body weight (▲) and morphological development of Persian sturgeon (Acipenser persicus) from hatching to the juvenile stage (40 dph) reared at 18 °C. Main feed types and associated feeding schedule are marked by a solid gray line, whereas the arrow indicates the onset of exogenous feeding.

140

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

prepared as described by Crane et al. (1979). Briefly, tissue homogenates were centrifuged at 9000 g for 10 min after the addition of 0.1 M CaCl2. The supernatants were transferred to new vials and stored frozen (−80 °C) until analysis of enzyme activity or protein content. Pepsin (E.C.3.4.23.1) activity quantification followed according to the method published by Worthington (1991) and based on Anson (1938). In brief, the enzymatic extract was mixed with the substrate (2% hemoglobin solution in 0.3 N HCl at pH = 2.0) and incubated for 10 min at 37 °C. The reaction was stopped with 5% Trichloroacetic acid (TCA), and the assay tubes were centrifuged at 4000 g for 6 min at 4 °C. The absorbance of the supernatant was recorded at 280 nm. One unit of pepsin activity was defined as the μg of tyrosine released at 37 °C min −1 mL − 1, considering the extinction coefficient (∈280 = 1250 M −1 cm − 1). Trypsin (E.C.3.4.21.4) activity was measured with N-α-benzoyldlarginine-p-nitroanilide (BAPNA) as substrate. BAPNA (1 mM in 50 mM Tris–HCl, pH 7.5, 20 mM CaCl2) was incubated with the enzyme extract at 37 °C. Absorbance was recorded at 410 nm (Erlanger et al., 1961). Chymotrypsin (EC. 3.4.21.1) activity was measured by using 0.1 mM Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAPNA) in 50 mM Tris–HCl, pH 7.5, 20 mM CaCl2. The enzyme preparation was incubated with substrate at 37 °C and monitored at 410 nm for 3 min. The molar extinction coefficient of p-nitroanilide is 8800 cm 2 mg − 1. Chymotrypsin activity units were expressed as change in absorbance per minute per mg protein (Erlanger et al., 1961). Trypsin and chymotrypsin activity units were calculated by the following equation:

2.4. Statistical analysis Data were checked for normality (Kolmogorov–Smirnov test) and homogeneity of variances (Bartlett's test) prior to their comparison. All data are expressed as the mean ± SD (n = 3). Digestive enzyme activities were compared by means of a one-way ANOVA, and the mean comparison was performed with a Duncan's test at a reliability level of 5%. Data were analyzed using SPSS statistical software (release 15). 3. Results The mean wet body weight and total length (TL) evolution of Persian sturgeon from hatching to the juvenile stage (40 dph) are given in Fig. 1. Newly hatched larvae weighed 16.7 mg and measured 11.2 mm in TL; at the end of the study they measured 921.3 mg and 56.1 mm, respectively. Larval growth in weight showed rapid exponential growth from hatching to 40 dph. 3.1. Gastric enzyme Pepsin activity was not detected until 5 dph (15.8 mm TL). Concomitantly with first feeding at 9 dph (18.9 mm TL), the specific activity of pepsin increased until 29–34 dph, reaching maximum values of activity (5.77 ± 0.27 U mg protein − 1). Subsequently, pepsin activity decreased to the end of the study (4.68 ± 0.44 U mg protein − 1; P b 0.05) (Fig. 2a). 3.2. Pancreatic enzymes

ðAbs410 = minÞ × 1000 × ml of reaction mixture : unit = mg protein = 8800 × mg protein in reactin mixture Amylase (E.C.3.2.1.1) activity was determined by the 3, 5dinitrosalicylic acid (DNS) method (Bernfeld, 1951; Worthington, 1991). Starch substrate (1% w/v) was diluted in a buffer at pH 6.9, 0.02 M Na2HPO4 and 0.006 M NaCl. The substrate (250 μl) was incubated with crude extract (50 μl) and buffer solution (250 μl) for 3–4 min at 25 °C. Then 0.5 ml of 1% dinitrosalicylic acid (DNS) solution was added and boiled for 5 min. After boiling, 5 ml of distilled water was added to the mixture and the absorbance of the cooled solution was recorded at 540 nm. Blanks were similarly prepared, but without the crude enzyme extracts. Maltose (0.3–5 μM ml − 1) was used for the preparation of the standard curve. The α-amylase specific activity was defined by the μmol of maltose produced per min per mg protein at the specified condition. Lipase (E.C.3.1.1) activity was determined by hydrolysis of nnitrophenyl myristate. Each assay (0.5 ml) contained 0.53 mM nnitrophenyl myristate, 0.25 mM 2-methoxyethanol, 5 mM sodium cholate and 0.25 M Tris–HCl (pH 9.0). Incubation was carried out for 15 min at 30 °C, and the reaction was terminated by adding 0.7 ml of acetone/n-heptane (5:2, v/v). The reaction mixture was vigorously mixed and centrifuged at 6080 g for 2 min. The absorbance at 405 nm in the resulting lower aqueous layer was measured. The extinction coefficient of n-nitrophenol was 16,500 M −1 cm − 1 l − 1. One unit of enzyme activity was defined as 1 μmol of n-nitrophenol released per min (Iijima et al., 1998). Alkaline phosphatase (AP) (E.C.3.1.3.1) was quantified at 37 °C using 4-nitrophenyl phosphate (PNPP) as substrate in 30 mM Na2CO3 buffer (pH 9.8). One unit (U) was defined as 1 μg BTEE released per min per ml of brush border homogenate at 407 nm (Bessey et al., 1946). Total soluble protein was measured by the Bradford (1976) method using bovine serum albumin as a standard. Enzyme activities were expressed as specific activity (U mg protein − 1). All the enzymatic assays were run in triplicate.

Trypsin was detected in Persian sturgeon larvae at hatching and before mouth opening. The specific activity of trypsin reached the highest values at the onset of first feeding (9 dph) (0.05 ± 0.00 U mg protein − 1) (P b 0.05). Trypsin activity decreased gradually from 9 to 29 dph (41.5 mm TL) (P b 0.05) and remained constant until 40 dph (Fig. 2b). At hatching, chymotrypsin specific activity was very high (0.25 ± 0.11 U mg protein − 1) (P b 0.05) and tended to decrease along larval development (P b 0.05). At the onset of first feeding, chymotrypsin specific activity slightly increased (0.13 ± 0.05 U mg protein − 1) (P b 0.05) and then progressively decreased after 29 dph (0.26 ± 0.00 U mg protein − 1; P b 0.05), remaining constant without a significant difference until 40 dph (P N 0.05) (Fig. 2c). Amylolytic activity fluctuated considerably over the study period (Fig. 2d). Alpha-amylase activity was lower in newly hatched larvae (17.72 ± 0.41 U mg protein − 1) and then increased, remaining constant between 5 and 9 dph (P N 0.05). At first feeding, α-amylase activity showed a sharp increase at 14 dph (21.9 mm TL), reaching a maximum value of 94.58 ± 12.77 U mg protein − 1 at 29 dph (P b 0.05). After 29 dph, amylase decreased until the end of the study (P b 0.05) (Fig. 2d). The specific activity of lipase in Persian sturgeon larvae steadily increased with larval age (P b 0.05). At hatching, lipolytic activity had a low value (0.3 ± 0.53 mU mg protein − 1) and then increased gradually (P b 0.05). The specific activity increased in end of experiment (4.48 ± 1.02 mU mg protein − 1). In 4 weeks, lipolytic activity reached 4.5 folds relative to the activity levels recorded during the first week of exogenous feeding (Fig. 2e). 3.3. Intestinal enzyme Alkaline phosphatase specific activity was not detected at hatching. The specific activity of AP in Persian sturgeon larvae abruptly increased from 5 to 9 dph (exogenous feeding). A rapid increase in AP specific activity from 0.50± 0.01 U mg protein− 1 to 0.89 ± 0.06 U mg protein− 1 occurred between 14 and 19 dph (27.5 mm TL), coinciding with the

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

141

Fig. 2. Specific activity (U mg protein− 1) of pepsin (gastric enzyme), trypsin, chymotrypsin, α-amylase, lipase (pancreatic enzymes) and alkaline phosphatase (intestinal brush border enzyme) in Persian sturgeon from hatching to 40 dph. Different values of enzyme activity (mean ± SD, n = 3) with different superscript letters are statistically significant (P b 0.05). The arrow indicates the onset of exogenous feeding.

feeding of larvae with Daphnia sp. After 19 dph, AP activity decreased and reached a plateau until 40 dph (P N 0.05) (Fig. 2f). 4. Discussion Physiological studies during the early stages of development of fish, as well as the evolution of the digestive enzyme activities, are valuable tools for better understanding the nutritional capabilities of young larvae and establishing adequate feeding protocols for optimizing larval mass rearing production (Diaz et al., 1997). In this sense, it is very important to synchronize the physiological digestive status of the larva, measured by its digestive capabilities, with the feeding protocol and weaning process, because the success of larval

rearing is highly dependent upon it. In the present study, pancreatic enzymes like trypsin, chymotrypsin, α-amylase and lipase were detected in Persian sturgeon larvae at hatching. This early detection of enzymes responsible for protein, lipid and carbohydrate digestion has been reported in many marine and freshwater fish species (see reviews in Rønnestad and Morais, 2007; Zambonino-Infante et al., 2009), showing that enzyme synthesis at early stages of development is not triggered by food ingestion but rather is genetically programmed (Zambonino-Infante and Cahu, 2001). Protein digestion occurs mainly by the action of alkaline proteases such as trypsin and chymotrypsin in combination with intestinal cytosolic peptidases (Zambonino-Infante and Cahu, 2001). During larval stages, these enzymes have limited capacity for digesting

142

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

macromolecules that are absorbed by the pinocytotic activity of the enterocytes in the posterior intestine for their intracellular digestion. The activity of trypsin and chymotrypsin in the newly hatched A. persicus larvae reveals the importance of these alkaline pancreatic enzymes during organogenesis and early larval development because both types of proteases might be involved in the cleavage of proteins contained in the yolk and in live feeds (Gisbert et al., 2009), as well as during the hatching process in which the hatching gland participates in the digestion and breakage of the egg chorion (Dettlaff et al., 1993). In this sense, the high activity of these two alkaline pancreatic enzymes during early larval development confirmed their important digestive role in chondrosteans, as well as previously described in modern teleosts (Zambonino-Infante and Cahu, 2001). In addition, the decline in specific activity of both enzymes along larval development might be mainly explained by the normal increase of tissue proteins in growing larvae, reflecting anatomical and physiological changes in fish larvae, but not corresponding to a lowering in the amount of digestive enzymes (Zambonino-Infante et al., 2009), as well as the progressive transformation of the digestion mode from an alkaline larval digestion, mainly characterized by pancreatic proteases like trypsin and chymotrypsin, to a juvenile acid digestion mode. A progressive slow shift in the relative activity from alkaline (trypsin and chymotrypsin) to acid (pepsin) was observed in Persian sturgeon during larval development coinciding with the onset of exogenous feeding at 9 dph. In this sense, the development of the stomach generally involves an acid digestion and consequently a more efficient extracellular digestion of proteins (Segner et al., 1994). The first gastric glands can be detected a few days or weeks after hatching and their number increases progressively partially or completely covering the stomach epithelium depending on the species (see review in Zambonino-Infante et al., 2009). Accordingly, the secretions of these glands, pepsinogen and hydrochloric acid, induce a progressively lower pH environment in the lumen of the stomach and the conversion of pepsinogen into pepsin (Darias et al., 2007; Yúfera and Darías, 2007b). The time sequence of gastric gland apparition, their final number and regions of stomach covered vary among families and species (Zambonino-Infante et al., 2009). Their characteristics are obviously related to feeding preference and habits. In addition to the apparition of acid digestion, the increase of intestinal brush border enzymes and the progressive decrease of cytosolic peptidases characterize the progress of the enterocytes maturation (Zambonino-Infante and Cahu, 2001). These results are in agreement with previous studies on the histological organization of the digestive tract in Persian sturgeon larvae because first gastric glands were detected at similar stages of development, between 8 and 10 dph (Pahlevanyaly et al., 2004). In lake sturgeon (Acipenser fulvescens), the initiation of gastric secretion was simultaneous with the establishment of active feeding at 14–18 dph (Buddington, 1985). Carboxylic proteinases (e.g., pepsin) were reported to be one of the main contributors to the proteolytic activity in Acipenser gueldenstaedti, A. stellatus and H. huso eggs (Kopylenko et al., 1984). In Caspian brown trout (Salmo caspius), pepsin activity was detected on the first day after hatching (Zamani et al., 2009), whereas Buddington and Doroshov (1986) reported pepsin activity in the gut of endogenously feeding A. transmontanus and A. fulvescens yolk-sac larvae. However, it seems very plausible that in the former studies, lysosomal cathepsins related to the mobilization of protein from the yolk-sac and body reserves were mistaken by pepsin (Carnevali et al., 2001; Lazo et al., 2007). Although pepsin activity was detected coinciding with the transition to exogenous feeding in Persian sturgeon, activity values were not stabilized until 24 dph, which seems to indicate that the complete change to a juvenile/adult mode of digestion was not achieved until then. These observations are supported by the lowest values of alkaline proteases recorded from 24 to 40 dph, reflecting the temporal changes and stabilization of the different alkaline and acid proteases (Lazo et al., 2007).

Previous studies revealed trace levels of α-amylase activity in embryos and 3–4 day-old sturgeon larvae, whereas amylase activity sharply increased after the transition to exogenous feeding (Kuzmina and Gelman, 1998). These results are somehow different from those reported in the present study with Persian sturgeon, wherein α-amylase activity values were quite elevated during the endogenous feeding phase. High α-amylase activity values during the consumption of yolk reserves might be due to the presence of large glycogen deposits accumulated in the yolk sac in this group of chondrostean species (Gisbert et al., 1999; Gisbert and Doroshov, 2006). The high activity of α-amylase during the larval stage in Persian sturgeon might indicate that dietary carbohydrates may fill the energy gap between endogenous and exogenous protein requirements in larvae as suggested by Tanaka (1973). In addition, α-amylase activity also reflected the relatively high carbohydrate content (6–10%) in live preys (Ma et al., 2005) used for feeding larvae, which might have differentially stimulated the synthesis and secretion of α-amylase during larval development. These changes in activity patterns of α-amylase along larval ontogeny are similar to those reported in A. baerii (Zółtowska et al., 1999) and in some teleost marine species like Sciaenops ocellatus (Lazo et al., 2007) and Pseudosciaena crocea (Ma et al., 2005). As a rule of thumb, α-amylase specific activity has been shown to be high during larval stages and generally decreases during development when the juvenile stage is reached, which is considered as an indicator of pancreas maturation in carnivorous fish species (Cahu et al., 2004). In this study, the decrease in α-amylase observed in A. persicus from 29 to 40 dph might be attributed to the acquisition of a juvenile-like digestion mode, rather than a dietary induced change in α-amylase production and secretion, since the type of live prey remained stable during this period. Despite major advances in the understanding of lipid digestion in juvenile and adult fish, the knowledge of lipolytic enzymes in larvae is scarce, particularly in terms of luminal digestion of dietary lipids (Zambonino-Infante and Cahu, 1999). In this study, the specific activity of lipase was detected at hatching and progressively increased with larval age. A similar pattern in lipase activity has been reported in Theraga chalcogramma (Davis and Olla, 1992), S. ocellatus (Lazo et al., 2007) and among other species (see review in Rønnestad and Morais, 2007). However, these results are different from those reported in Lates calcarifer by Walford and Lam (1993). The former authors found high lipase activities at early stages that decreased during larval development. However, such changes in lipase activity might be attributed to changes in food quality and quantity rather than a decrease in the lipolytic digestive capabilities of fish (Morais et al., 2004). Previous studies in European sea bass larvae demonstrated that lipase activity was not influenced by an increase in dietary lipid content but by different lipid classes (Morais et al., 2004). In this sense, the large amount of wax esters, phospholipids and triacylglycerols of zooplanktonic preys (Shields et al., 1999) might also have stimulated the production of lipase in the pancreas of fish larvae (Rønnestad and Morais, 2007). However, under present experimental conditions and considering the fact that the change in the type of live prey (brine shrimp and cladocerans) during the feeding schedule of Persian sturgeon did not match changes in lipase activity, we considered that the observed changes in lipase activity were more related to the acquisition of the typical digestive capacities of the species and rather than dietary induced by type of live prey. Two groups of digestive enzymes are found in enterocytes: cytosolic enzymes (mainly peptidases) found in the cytoplasm, and brush border membrane enzymes, which are linked to the cell membrane. Under this context, different types of membranous enzymes can be detected: peptidases, alkaline phosphatase and aminopeptidase-N. These different enzymes found in the gastrointestinal tract lead to the final digestion of dietary components, allowing their absorption or transport by enterocytes (Zambonino-Infante and Cahu, 2001). The development of a functional intestine implies different maturational

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

and morphological events that are very well preserved among vertebrates (Henning, 1987). From an enzymatic point of view, the appearance of a functional microvillus membrane in enterocytes constitutes a crucial step during larval development of fish for the acquisition of an adult mode of digestion (see review in ZamboninoInfante et al., 2009). According to the former authors, this change has been reported to occur around the 3rd and 4th week after hatching in temperate marine teleost fish species, whereas in the present study, it was detected after 24 dph. Although in the present study the activity of intestinal cytosolic enzyme was not analyzed, it seems plausible that the observed increase in alkaline phosphatase activity was coupled with a progressive decline of cytosolic enzyme activities, as this process characterizes the normal development of the intestine and maturation of the enterocytes in vertebrates (Zambonino-Infante et al., 2009). 5. Conclusion This study showed that the live prey feeding schedule commonly used in hatcheries for many freshwater species also allowed the proper development and growth in Persian sturgeon larvae. During larval development, trypsin and chymotrypsin showed decreasing trends in specific activity that were coupled with increasing levels of pepsin activity. These results indicated a progressive transformation of the digestion mode from an alkaline larval digestion, mainly characterized by pancreatic proteases like trypsin and chymotrypsin, to a juvenile acid digestion mode. These results were also supported by the ontogenetic changes in α-amylase, lipase and alkaline phosphatase activities that were also attributed to the maturation of the digestive system at the end of the larval period as previously reported in modern teleosts. Considering data of digestive enzymes from the pancreas, stomach, intestine and morphological development, Persian sturgeon larvae could be progressively weaned around 19–24 dph. This developmental process, and particularly for the digestive functions, can be considered a reference to evaluate the effect of a formulated micro-diet feeding on larvae. Acknowledgments The authors wish to thank to the Tarbiat Modares University (Noor, Iran) for their financial support. Thanks are also extended to the technical staff of Shahid Rajaee Sturgeon Hatchery Center (Sari, Iran) for their valuable practical assistance. References Abhari, S., Tavakkoli, M., 1999. Study of Diets in Sturgeon in Kheirud Kenar Fishery Catch Station. University of Tehran, Press, Tehran. Anson, M.L., 1938. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Gen. Physiol. 22, 79–89. Bardi, R.W., Chapman, F.A., Barrows, F.T., 1998. Feeding trials with hatchery-produced Gulf of Mexico sturgeon larvae. Prog. Fish Cult. 60, 25–31. Bernfeld, P., 1951. Amylases α and β. In: Colowick, P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic Press, New York, pp. 149–157. Bessey, O.A., Lowry, O.H., Brock, M.J., 1946. Rapid coloric method for determination of alkaline phosphatase in five cubic millimeters of serum. J. Biol. Chem. 164, 321–329. Bolasina, S., Perez, A., Yamashita, Y., 2006. Digestive enzymes activity during ontogenetic development and effect of starvation in Japanese flounder, Paralichthys olivaceus. Aquaculture 252, 503–515. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Buddington, R.K., 1985. Digestive secretions of lake sturgeon, Acipenser fulvescens, during early development. J. Fish Biol. 26, 715–723. Buddington, R.K., Doroshov, S.I., 1984. Feeding trials with hatchery produced white sturgeon juveniles (Acipenser transmontanus). Aquaculture 36, 237–243. Buddington, R.K., Doroshov, S.I., 1986. Digestive enzyme complement of white sturgeon (Acipenser transmontanus). Comp. Biochem. Physiol. 83A, 561–567. Cahu, C., Zambonino-Infante, J.L., Quazuguel, P., Le Gall, M.M., 1999. Protein hydrolysate vs. Fish meal in compound diets for 10-day old sea bass Dicentrarchus labrax larvae. Aquaculture 171, 109–119.

143

Cahu, C., Rønnestad, I., Grangier, V., Zambonino-Infante, J.L., 2004. Expression and activities of pancreatic enzymes in developing sea bass larvae (Dicentrarchus labrax) in relation to intact and hydrolyzed dietary protein; involvement of cholecystokinin. Aquaculture 238, 295–308. Carnevali, O., Mosconi, G., Cambi, A., Ridolfi, S., Zanuy, S., Polzonetti-Magni, A.M., 2001. Changes of lysosomal enzyme activities in sea bass (Dicentrarchus labrax) eggs and developing embryos. Aquaculture 202, 249–256. Crane, R.K., Boge, G., Rigal, A., 1979. Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small dogfish Scyliorhinus canicula. Biochim. Biophys. Acta 554, 264–267. Dabrowski, K., Kaushik, S.J., Fauconneau, B., 1985. Rearing of sturgeon (Acipenser baeri Brandt) I. Feeding trial. Aquaculture 47, 185–192. Darias, M.J., Murray, H.M., Gallant, J.W., Douglas, S.E., Yúfera, M., Martínez-Rodríguez, G., 2007. Ontogeny of pepsinogen and gastric proton pump expression in red porgy (Pagrus pagrus): determination of stomach functionality. Aquaculture 270, 369–378. Davis, M.W., Olla, B.L., 1992. Comparison of growth, behavior and lipid concentrations of walleye Pollock Theraga chalcogramma larvae fed lipid-enriched, lipid-deficient field-collected prey. Mar. Ecol. Prog. Ser. 90, 23–30. Dettlaff, T.A., Ginsburg, A.S., Schmalhausen, O.I., 1993. Development of prelarvae. Sturgeon Fishes. Developmental Biology and Aquaculture. Springer-Verlag Ed, Berlin, Germany, pp. 155–221. Diaz, M., Moyano, F.J., Garcia-Carreño, L.F., Alarcon, F.J., Sarasquete, M.C., 1997. Substrate-SDS-PAGE determination of protease activity through larval development in sea bream. Aquac. Int. 5, 461–471. Erlanger, B., Kokowsky, N., Cohen, W., 1961. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271–278. Furne, M., García-Gallego, M., Hidalgo, M.C., Morales, A.E., Domezain, A., Domezain, J., Sanz, A., 2008. Effect of starvation and refeeding on digestive enzyme activities in sturgeon (Acipenser naccarii) and trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 149, 420–425. Gisbert, E., Sarasquete, M.C., Williot, P., Castello-Orvay, F., 1999. Histochemistry of the development of the digestive system of Siberian sturgeon (Acipenser baeri, Brandt) during early ontogeny. J. Fish Biol. 55, 596–616. Gisbert, E., Sarasquete, C., 2000. Histochemical identification of the blackbrown pigment granules found in the alimentary canal of Siberian sturgeon (Acipenser baeri) during the lecitotrophic stage. Fish Physiol. Biochem. 22, 349–354. Gisbert, E., Williot, P., 2002. Advances in the larval rearing of Siberian sturgeon. J. Fish Biol. 60, 1071–1092. Gisbert, E., Doroshov, S.I., 2006. Allometric growth in green sturgeon larvae. J. Appl. Ichthyol. 22, 202–207. Gisbert, E., Gimenez, G., Fernandez, I., Kotzamanis, Y., Estevez, A., 2009. Development of digestive enzymes in common dentex Dentex dentex during early ontogeny. Aquaculture 287, 381–387. Henning, S.J., 1987. Functional development of the gastrointestinal tract, In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract, 2nd edition. Raven Press, New York, pp. 285–300. Iijima, N., Tanaka, S., Ota, Y., 1998. Purification and characterization of bile saltactivated lipase from the hepatopancreas of red sea bream, Pagrus major. Fish Physiol. Biochem. 18, 59–69. Kolkovski, S., 2001. Digestive enzymes in fish larvae and juveniles—implications and applications to formulated diets. Aquaculture 200, 181–201. Kopylenko, L.R., Mitskevich, L.G., Vaitman, G.A., Mosolov, V.V., 1984. Proteinases in the spawn of sturgeon. Prikl. Biohim. Mikrobiol. 20, 373–377. Kuzmina, V.V., Gelman, A.G., 1998. Traits in the development of the digestive function in fish. J. Ichthyol. 39, 106–115. Lazo, J.P., Mendoza, R., Holt, G.J., Aguilera, C., Arnold, C.R., 2007. Characterization of digestive enzymes during larval development of red drum (Sciaenops ocellatus). Aquaculture 265, 194–205. Ma, H., Cahu, C., Zambonino-Infante, J.L., Yu, H., Duan, Q., Le Gall, M.M., Mai, K., 2005. Activities of selected digestive enzymes during larval development of large yellow croaker (Pseudosciaena crocea). Aquaculture 245, 239–248. Moghim, M., Kor, D., Tavakolieshkalak, M., Khoshghalb, M.B., 2006. Stock status of Persian sturgeon (Acipenser persicus Borodin, 1897) along the Iranian coast of the Caspian Sea. J. Appl. Ichthyol. 22, 99–107. Morais, S., Cahu, C., Zambonino-Infante, J.L., Robin, J., Rnnestad, I., Dinis, M.T., Conceicao, L.E.C., 2004. Dietary TAG source and level affect performance and lipase expression in larval sea bass (Dicentrarchus labrax). Lipids 39, 449–458. Pahlevanyaly, M., Mojazi amiri, B., Posty, A., Bahmani, M., 2004. Study of histological development in Persian sturgeon (Acipenser persicus) during early ontogeny. Iran Fisheries J. 2, 33–50 (in Iran with English abstract). Rønnestad, I., Morais, S., 2007. Digestion. In: Fin, R.N., Kapoor, B.G. (Eds.), Fish Larval Physiology. Enfield, Science Publishers, pp. 201–262. Segner, H., Storch, V., Reinecke, M., Kloas, W., Hanke, W., 1994. The development of functional digestive and metabolic organs in turbot, Scophthalmus maximus. Mar. Biol. 119, 471–486. Shields, R.J., Bell, J.G., Luizi, F.S., Gara, J., Bromage, N., Sargent, J.R., 1999. Natural copepods are superior to enriched Artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr. 129, 1186–1194. Tanaka, M., 1973. Studies on the structure and function of the digestive system of teleost larvae. Ph.D. Dissertation, Kyoto University, Japan, 136 pp. Timeiko, V.N., Bondarenko, L.G., 1988. A study of digestive enzymes in bester (giant sturgeon sterlet hybrid) during postembryonic period. Vopr. Ikhtiol. 1, 117–123. Twining, S.S., Alexander, P.A., Huibregste, K., Glick, D.M., 1983. A pepsinogen from rainbow trout. Comp. Biochem. Physiol. 75B, 109–112.

144

S.S. Babaei et al. / Aquaculture 318 (2011) 138–144

Walford, J., Lam, T.J., 1993. Development of digestive tract and proteolytic enzyme activity in seabass (Lates calcarifer) larvae and juveniles. Aquaculture 109, 187–205. Worthington, C.C., 1991. Worthigton Enzyme Manual Related Biochemical, 3th Edition. Freehold, New Jersey. Verreth, J., Segner, H., 1995. The impact of development on larval nutrition. In: Lavens, P., Jasper, E., Roelants, I. (Eds.), Larvi' 95, Fish and Shellfish Larviculture Symposium, Europe. Aquacult. Society, Special publication, 24, Ghent, Belgium. Yúfera, M., Darias, M.J., 2007. The onset of exogenous feeding in marine fish larvae. Aquaculture 268, 53–63. Yúfera, M., Darías, M.J., 2007b. Changes in the gastrointestinal pH from larvae to adult in Senegal sole (Solea senegalensis). Aquaculture 267, 94–99. Zamani, A., Hajimoradloo, A., Madani, R., Farhangi, M., 2009. Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius. J. Fish Biol. 75, 932–937.

Zambonino-Infante, J.L., Cahu, C.L., 1999. High dietary lipid levels enhance digestive tract maturation and improve Dicentrarchus labrax larval development. J. Nutr. 129, 1195–1200. Zambonino-Infante, J.L., Cahu, C., 2001. Ontogeny of the gastrointestinal tract of marine fish larvae. Comp. Biochem. Physiol. 130, 477–487. Zambonino-Infante, J.L., Cahu, C.L., 2007. Dietary modulation of some digestive enzymes and metabolic processes in developing marine fish: applications to diet formulation. Aquaculture 268, 98–105. Zambonino-Infante, J., Gisbert, E., Sarasquete, C., Navarro, I., Gutiérrez, J., Cahu, C.L., 2009. Ontogeny and physiology of the digestive system of marine fish larvae. In: Cyrino, J.E.O., Bureau, D., Kapoor, B.G. (Eds.), Feeding and Digestive Functions of Fish. Science Publishers, Inc, Enfield, USA, pp. 277–344. Zółtowska, K., Kolman, R., Lopienska, E., Kolman, H., 1999. Activity of digestive enzymes in Siberian sturgeon juveniles (Acipenser baeri Brandt), a preliminary study. Arch. Polish Fisheries 7, 201–211.