Zootechnical Parameters and Enzyme Activity in the Species Brycon moorei (Steindachner 1878)

: Brycon moorei is an opportunistic omnivorous species. It is not known what its nutritional requirements are at any stage of its development; this study determined the inﬂuence of diet on the zootechnical parameters and enzyme activity of these ﬁsh. In a completely randomized quintuplicate design, an ad libitum dietary protocol was applied, which included Prochilodus magdalenae larvae, Artemia salina and balanced feed with increasing inclusion levels (35 to 55% crude protein (CP)). Larvae 22 h post-hatching (HPH), with an average initial weight and length of 3.07 ± 0.69 mg and 6.069 ± 0.22 mm, respectively, were seeded at a density of 17 larvae L − 1 in tanks with an effective volume of 60 L. The main zootechnical parameters, water quality and enzyme activity were analyzed with respect to the experimental diets. Signiﬁcantly, the best diet was that of 45% CP when compared with the other CP levels used. Speciﬁc serum enzyme activity was found from hatching, with ﬂuctuating values, the speciﬁc pepsin-like activity started at 111 HPH. B. moorei larvae can receive a dry diet from 124 HPH and their requirement corresponds to 48.5% CP.


Introduction
The constant production of quality larvae and juveniles in Neotropical species is a limiting factor for the development of aquaculture programs, as well as for studies related to the actions of preservation and recovery of these species in their environments [1,2].In fact, the larviculture of Neotropical species is mainly carried out in semi-intensive systems [3] with rather poor results due to the availability of food and predation [4,5].Similarly, larvae with altricial development [6] and exacerbated cannibalism have difficulty accepting dry diets, as is the case of Brycon moorei [7,8], a species endemic to Colombia and currently classified as vulnerable (A2d), due to factors such as the rapid loss of its natural environment [9].Therefore, it is necessary to find new cultivation strategies and feeding techniques [10,11] and to rely on ontogenetic studies to understand the changes in structures related to feeding and digestive capacities that help to determine the type of nutrients that are best assimilated by the larvae [12,13].In this sense, the evaluation of the presence and level of digestive enzyme activity can be used as an indicator for several productive parameters in fish larvae [14].
Little is known about the nutritional requirements in fish larvae [15], due to difficulties associated with size, variability, rapid changes, growth rates, among others [16].Although there are some reports on enzyme activity in marine [17] and freshwater [2] fish larvae, it has been shown that enzyme activity and its modulation may be related to dietary items [18].In this regard, there are no known reports on the influence of diets on zootechnical performance and enzyme activity in larvae and juveniles of B. moorei.Therefore, the aim of this work was to understand the effect of the feeding protocol, the influence of diets on zootechnical parameters and the activity of some enzymes in this species.

Location
The experimental phase was carried out at the Doradal Fish Farming Station (Estación Piscícola Doradal) located at coordinates 5 • 52 59.5 N and 74 • 47 03.6 W; 165 km from Medellín, in the village of Doradal, in the Municipality of Sonsón, Antioquia, Colombia; at an altitude of 150 m.a.s.l.; average relative humidity of 75% and a temperature between 24 • C and 35 • C.

Environmental Parameters and Larval Collection
For larval collection and management, protocols were followed as proposed by David-Ruales and colleagues [9][10][11].Larvae with an average initial weight and length of 2.97 ± 0.23 mg and 6.19 ± 0.029 mm, respectively, were kept in a recirculation system (RS) with 30 rectangular experimental units (EU) of 65 L effective volume.The RS maintained environmental parameters within comfort ranges for the species (Table 1); water quality monitoring was performed daily at 7:00 am, with a Hachh FF2 Kit and a YSI multiparameter probe (Professional plus).The inlet flow rate of each tank was controlled by flowmeters (Parker MR) and maintained at 3.5 L min −1 .

Feeding Protocol Design
The feeding protocol for B. moorei was carried out for a period of 15 days in the RS, using 1 day post-hatching (DPH) larvae; the fish were fed 5 times a day as follows: during the first 3 days with recently hatched Prochilodus magdalenae forage larvae (FL), from day 4 Artemia salina nauplii (A) were used and, from day 6, a dry diet (C) was offered with increasing inclusions of crude protein (CP) (35, 40, 45, 45, 50 and 55%); completing the transition to C from the thirteenth day onwards.All food items were offered ad libitum for a period of 5 min per EU; Table 2 shows the modified strategy of David and Castañeda [11].Tanks were cleaned depending on the accumulation of residues or variations in water quality factors, mainly nitrogenous; each diet trial was performed in quintuplicate in order to sustain the number of larvae per number of samples for each treatment.
Table 3 displays the ingredients and the centesimal composition for each crude protein level; the diets were formulated based on the work of de Lazo (2000) [19] with a (CP) energy ratio of approximately 1:10.The product was developed in the Nutrition Laboratory of the Aquaculture Institute of the Universidad de los Llanos.Proximate and energy analyses were carried out in the Nutrition Laboratory of the Corporación Universitaria Lasallista, under the guidelines of the "Association of Official Agricultural Chemists" [20].

Zootechnical Parameters
For the growth analyses, 10 larvae were randomly sampled (wet weight) before offering the first daily feed ration [21,22].For each sample the following were recorded: weight (mg), length (mm), weight gain (WG-mg); length gain (LG-mm), daily weight gain (DWG-mg), feed conversion (FC), specific growth rate (SGR-%), condition factor (K), survival (S%) and consumption (C mg/day/larva).The records and calculations of the zootechnical parameters were based on the work of several authors [23][24][25].I, Intake = feed intake (mg)/day/larva, where BW1 and BW2 represent the initial and final body weights of each fish (in mg), T1 and T2 represent the times corresponding to those weights and Ni and Nf represent the initial and final numbers of fish, respectively.

Enzyme Extraction and Total Soluble Protein Dosing
For the analyses of each experiment, 1 g of larvae and/or juveniles (wet weight) was collected before offering the first daily feed ration.At time 0, and until the fifth day posthatching, the complete larvae were arranged in perforated plastic conical cylinder tubes, assigned a code and immediately placed in liquid nitrogen, where they were preserved until the enzymatic assays.After the fifth day, samples were collected on a daily basis [26].Larvae and tissues were homogenized in ultrapure cold water (1:10 weight/volume with Ultra-Turrax IKT25, AMEX ® homogenizer); total soluble protein was obtained according to the protocol of Lowry and colleagues (1951) [27].

Enzyme Activity Assays
To measure trypsin activity, Na-Benzoyl-dl-arginine-4-p-nitroanilide (BAPNA) (Sigma-Aldrich St. Louis, MO, USA) was used as a substrate in a final concentration of 1 mM in 50 mM TRIS-HCL buffer, pH 8.2, following the protocol of Erlanger and colleagues [28].To measure chymotrypsin, the substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAPNA) (Sigma-Aldrich St. Louis, MO, USA) was used, at a final concentration of 1 nM, in 100 mM phosphate buffer, pH 7.8, according to the protocol of DelMar and colleagues [29].For both enzymes, the reading was carried out at an absorbance of 410 nm.A unit of enzyme activity (U) was defined as the amount of enzyme catalyzing 1 µmol of substrate min −1 [29].
To determine amylase activity, the substrate used was 2% (m/v) starch in 100 mM acetate buffer, pH 7.5, following the protocol of Noelting and colleagues [30].One unit of amylase activity (U) was defined as the amount of 1 µmol of glucose formed per min −1 determined from the standard glucose curve.
Pepsin-like activity, with 2% (w/v) bovine hemoglobin as a substrate, at a final concentration of citrate-phosphate substrate-buffer 100 mM, pH 3.0, was determined according to the protocol of Anson [31].An enzyme unit (U) was defined as the amount of enzyme responsible for altering absorbance by 0.001 Optical Density Units (ODU/min).Readings were carried out in a microplate spectrophotometer (TECAN-Infinite pro, San Diego, CA, USA); all enzyme assays were performed in the Biochemistry laboratory of the Federal University of Santa Catarina (UFSC), Brazil.

Statistical Analysis
A completely randomized experimental design was applied, with 5 treatments and 6 replicates per treatment.To determine the optimal protein level, the polynomial regression model was used, reflected in the equation Y = B 0 + B 1 t + B 2 t 2 + E, where: Y is the answer, B 0 is the model intercept, B 1 the linear regression coefficient associated with the treatment t, B 2 corresponds to the quadratic regression coefficient associated with the treatment t 2 and E to the experimental error [32].The data of the treatments in terms of their zootechnical variables were tested for homoscedasticity by Levene's test, and normality by the Shapiro-Wilk test, then analyzed by one-way ANOVA, followed by Tukey's test.If normality was not found in the data, significant differences were determined using the nonparametric Kruskal-Wallis and Mann-Whirney U tests.Percentage data were transformed by Arc-Sin.The 5% significance level was used for all tests (p < 0.05).The software used was Statgraphics Centurion V7, licensed to the Corporación Universitaria Lasallista.

Results
The feeding protocol (P) led to satisfactory results for the management of the species, with a weight gain of 3258.7% and a length gain of 328% in 15 days.Table 4 shows the zootechnical variables that were analyzed in relation to the level of protein inclusion.Comparing the different diets, for most parameters, the 45% CP diet was significantly the most effective; there were no significant differences in any of the diets for K and survival; thus the DWG was 1.54 times higher than the 55% diet and 2.87 times higher than the 40% one.The best feed conversion was found in the 55% diet, with a value of 0.5 ± 0.07; there was no significant difference with the 45% CP diet (0.6 ± 0.03), but there were significant differences with the 35%, 40% and 50% CP diets.
Protein requirements in the early development of B. moorei were estimated at a value of approximately 48.5% CP; the polynomial model presented a coefficient of determination of 75%. Figure 1 shows the results.
The lowest value for all diets was found at 71 HPH, when the item offered was forage larvae (FL) (0.025 ± 0.0038 mU mg of protein −1 ).The specific activity for chymotrypsin was also present from hatching (12.1923 ± 1.66 mU mg of protein −1 ).The maximum value found was 12.23 ± 0.41 mU mg of protein −1 at 111 HPH, when larvae were fed with Artemia salina (A).The lowest value was reached at 243 HPH (0.6264 ± 0.03 mU mg of protein −1 ) and corresponds to the time of feeding with Dd for the 40% CP diet.The ratio between trypsin and chymotrypsin (T:C) decreased from 0.96 to 0.02 when the dietary item was changed from FL to A, that is, between 0 and 123 HPH, maintaining low values until the end of the experiment, with the lowest value for the 50% CP diet being 0.029-T:C and the highest value for the 45% CP diet, being 0.061-T:C, without any clear trend.Figure 2 shows the T:C ratio with respect to the HPH and the feeding protocol.
there was no significant difference with the 45% CP diet (0.6 ± 0.03), but there were significant differences with the 35%, 40% and 50% CP diets.
Protein requirements in the early development of B. moorei were estimated at a value of approximately 48.5% CP; the polynomial model presented a coefficient of determination of 75%. Figure 1  The lowest value for all diets was found at 71 HPH, when the item offered was forage larvae (FL) (0.025 ± 0.0038 mU mg of protein −1 ).The specific activity for chymotrypsin was also present from hatching (12.1923±1.66mU mg of protein −1 ).The maximum value found was 12.23 ± 0.41 mU mg of protein −1 at 111 HPH, when larvae were fed with Artemia salina (A).The lowest value was reached at 243 HPH (0.6264 ± 0.03 mU mg of protein −1 ) and corresponds to the time of feeding with Dd for the 40% CP diet.The ratio between trypsin and chymotrypsin (T:C) decreased from 0.96 to 0.02 when the dietary item was changed from FL to A, that is, between 0 and 123 HPH, maintaining low values until the end of the experiment, with the lowest value for the 50% CP diet being 0.029-T:C and the highest value for the 45% CP diet, being 0.061-T:C, without any clear trend.Figure 2 shows the T:C ratio with respect to the HPH and the feeding protocol.As for specific amylase activity, this was also present from the time of hatching (0.1895 ± 0.01mU mg of protein −1 ).The highest values are observed at the time of transition to the dry diet (A + Dd) (2.59 ± 0.09 mU mg of protein −1 ), specifically, at 195 HPH for all diets and then at 219 HPH, only when Dd (2.09 ± 0.16 mU mg of protein −1 ) is offered, for the 35% CP diet.The lowest values are observed for the 55% CP diet (0.0 mU mg of protein −1 ) at 219 and 267 HPH, respectively.Figure 3 shows the variations in enzymatic activity of these three enzymes.As for specific amylase activity, this was also present from the time of hatching (0.1895 ± 0.01 mU mg of protein −1 ).The highest values are observed at the time of transition to the dry diet (A + Dd) (2.59 ± 0.09 mU mg of protein −1 ), specifically, at 195 HPH for all diets and then at 219 HPH, only when Dd (2.09 ± 0.16 mU mg of protein −1 ) is offered, for the 35% CP diet.The lowest values are observed for the 55% CP diet (0.0 mU mg of protein −1 ) at 219 and 267 HPH, respectively.Figure 3 shows the variations in enzymatic activity of these three enzymes.As for the specific pepsin-like activity, it can be seen that it was not initiated from the moment of hatching.Its activity starts to develop at 111 HPH, with A as the food item and with the lowest value recorded (0.00267 ± 0.0002 mU mg of protein −1 ).This increases slightly up to 195 HPH (0.03798 ± 0.0007 mU mg of protein −1 ) across the different diets.As for the specific pepsin-like activity, it can be seen that it was not initiated from the moment of hatching.Its activity starts to develop at 111 HPH, with A as the food item and with the lowest value recorded (0.00267 ± 0.0002 mU mg of protein −1 ).This increases slightly up to 195 HPH (0.03798 ± 0.0007 mU mg of protein −1 ) across the different diets.When the transition is made to a dry diet (A + DD) from 219 HPH, there is a strong tendency for an increase in specific activity, with the highest values at 243 HPH for the 50% (0.9998 ± 0.17 mU mg of protein −1 ) and 55% CP (0.9471 ± 0.03 mU mg of protein −1 ) diets (Figure 4).When the transition is made to a dry diet (A + DD) from 219 HPH, there is a strong tendency for an increase in specific activity, with the highest values at 243 HPH for the 50% (0.9998 ± 0.17 mU mg of protein −1 ) and 55% CP (0.9471 ± 0.03 mU mg of protein −1 ) diets (Figure 4).

Discussion
The larval state is the stage with the biggest transformations in the lives of fish [33]; these changes can be modulated in nature by environmental influences and ethological factors [34,35], but in closed systems, these factors can be controlled through feeding protocols that respond to the needs of the species; in this case, P. magdalenae larvae were used for several reasons.First of all, it is a species that cohabits with B. moorei, it belongs to a low trophic level (detritivorous) and in terms of association by fishery, it represents over 80% of the catches in the Magdalena river basin, forming the food base of species with superior habits [36].The second reason is based on the success reported by several authors of the use of P. magdalenae larvae in first feeding protocols for fish of the genus Brycon [11,37,38].The third reason can be found in the allometric growth ratios with respect to the maximum mouth opening for B. moorei [13], which imply for the species a differentiation in the size of the prey from larger to smaller proportions with respect to its development.The successful use of Artemia salina nauplii has been extensively tested as a first feeding strategy in freshwater [39] fish larvae, including B. moorei [7,11,40].Finally, the design of dry diets must be in accordance with the capability of the larvae to assimilate them and with the quality of the ingredients in meeting the requirements of the species; it is understood that, in early stages of development, these needs are high [41].In the present study, all diets were formulated with high quality ingredients in order to meet the needs of the species, relating their use to the time of development of the larvae's digestive system.

Discussion
The larval state is the stage with the biggest transformations in the lives of fish [33]; these changes can be modulated in nature by environmental influences and ethological factors [34,35], but in closed systems, these factors can be controlled through feeding protocols that respond to the needs of the species; in this case, P. magdalenae larvae were used for several reasons.First of all, it is a species that cohabits with B. moorei, it belongs to a low trophic level (detritivorous) and in terms of association by fishery, it represents over 80% of the catches in the Magdalena river basin, forming the food base of species with superior habits [36].The second reason is based on the success reported by several authors of the use of P. magdalenae larvae in first feeding protocols for fish of the genus Brycon [11,37,38].The third reason can be found in the allometric growth ratios with respect to the maximum mouth opening for B. moorei [13], which imply for the species a differentiation in the size of the prey from larger to smaller proportions with respect to its development.The successful use of Artemia salina nauplii has been extensively tested as a first feeding strategy in freshwater [39] fish larvae, including B. moorei [7,11,40].Finally, the design of dry diets must be in accordance with the capability of the larvae to assimilate them and with the quality of the ingredients in meeting the requirements of the species; it is understood that, in early stages of development, these needs are high [41].In the present study, all diets were formulated with high quality ingredients in order to meet the needs of the species, relating their use to the time of development of the larvae's digestive system.
For B. moorei, in the present study, better survival and weight gain values were found (43% and 221.44 ± 64.56 mg, respectively), compared to those reported for the same species [11]; these differences can be explained by the variations in the feeding protocol.
Although the survival value may be low compared to other Neotropical species, it is known that the family Bryconidae, and specifically B. moorei, shows the highest level of cannibalism recorded for freshwater species [7,8,38,42].Therefore, it is important to establish a feeding protocol that is in line with the nature of the species; in this regard, in B. sinuensis [37] and Brycon siebenthalae [43], these findings are also reported.
However, the production system can also affect these results.In general, larviculture of Neotropical species is mainly carried out in semi-intensive systems [3], but with low survival rates due to food availability and predation [4,5].Nevertheless, recently, the focus has shifted to studies on recirculation for Neotropical freshwater species [26,[44][45][46][47].In this respect, the economic viability of intensive systems has been demonstrated for the species Piaractus mesopotamicus [4,48] and for B. moorei [11].It has been shown that cofeeding strategies and gradual transition to a dry diet leads to better results than traditional production systems [49][50][51][52][53][54], and it is recognized that substituting live feed with commercial rations before day six generally leads to lower survival rates [52].This study reports results consistent with those for species with indirect development.
Given that protein is the main nutrient for development in fish [12,55] and that determining amino acid requirements in larvae is complex [56], for better results, it is recommended that nutritional, physiological and management strategies be combined [15,55,57].For example, in Piaractus mesopotamicus larvae, positive effects were found by managing a 12-day transition time to a dry diet [48], with a CP level of 38%, fed to larvae over 10 mg [58].The results of the present study indicate a transition from day 6, with full Dd from day 13, reflecting positive effects with a 45% CP diet.
The main proteases responsible for protein hydrolysis in the intestine of fish are trypsin and chymotrypsin.Although their importance is recognized, there is still uncertainty regarding the specific contribution of each enzyme to the process of protein metabolism [59]; these enzymes, including amylase, may be present in the early stages of ontogenetic development, as has been described in several species [26,45,60].In B. moorei these enzymes are present from hatching; the same behavior was observed in larvae of the species Centropomus undecimalis [61], Centropomus nigrescens [62], Centropomus viridis [63], Pseudoplatystoma punctifer [46] and Pseudoplatystoma fasciatum [64].Each case, including the species being studied, showed peaks of activity which are specific to each species and may be the result of predetermined genetic factors [65], specific substrate requirements or variations in diet [66,67], feeding protocol [68], as well as physical factors such as temperature and species-specific differences, among others [18,[69][70][71][72][73][74][75].Fish are known to exhibit plasticity in the production of digestive enzymes in response to the type of diet [72,73], therefore, larvae could modulate enzyme production, reducing metabolic expenditure [16].Furthermore, in larvae that do not have a stomach at the beginning of their ontogeny, the enzymes trypsin and chymotrypsin [14,66,73] are responsible for protein hydrolysis.Other authors state that these enzymes play an important role in the process of chorion rupture at hatching and in the cleavage of yolk proteins [74,75] or, in the case of amylase, it may be related to the presence of glycogen reserves in the yolk sac [76], which could explain the findings of this study.In the present study, no trends between variations in serum enzyme activity and protein inclusion levels were found in this regard; the contribution of each enzyme to protein hydrolysis in the fish intestine is still unclear and subject to debate [59,77].However, with respect to the T:C ratio, a strong tendency to decrease was observed relative to the feeding protocol, specifically when switching from FL to A; it has been determined for other species that, when this ratio drops, it coincides with low growth rates [78,79].
This study showed a gradual increase in pepsin-like specific activity after five DPH and, with large variations in relation to protein inclusion from day eight onwards, indicating the presence of gastric glands and therefore a functional stomach; a morphological feature that could suggest that larvae of B. moorei are able to digest the formulated diet.Similar results have been found in other freshwater species with incomplete ontogenetic development, but with large differences in the time of gastric gland development [22,[80][81][82][83][84]; in B. moorei the glandular stomach appears at 122 HPH [85] and, in this regard, several features associated with the maturity of this organ are considered.Some authors consider that certain species can develop a stomach early, as is the case in the species Solea senegalensis, without implying functionality [86]; in the species Paralichthys olivaceus, a storage function is suggested first, prior to acid proteolytic activity [87].Other authors point out that, although the gastric glands may indicate acid digestion, it may take some time before they are actually functional [88].It is therefore suggested that a developed stomach implies functional gastric glands, in accordance with the increase of pepsin-like enzyme activity, which is when the transition to formulated diets can be initiated.

Conclusions
In B. moorei, a gradual transition to a dry diet is essential.Therefore, applying the feeding protocol as described above improves the results in terms of zootechnical parameters.The serum enzyme activity started at hatching and no relationship was found between the peaks of enzyme activity with respect to the inclusion of the protein level in the diet.However, a tendency to decrease was observed in the T:C ratio with respect to the feeding protocol.Also, the development and maturity of the digestive system must be taken into account, which, together with the onset of acid digestion, allows the larvae of this species to be fed from 123 HPH onwards, with a minimum of CP of 45% and a maximum of 50%, with larvae having a minimum total length of 11.38 mm and a minimum weight of 15.28 mg.

Figure 1 .
Figure 1.Mean comparison test using the polynomial regression model for the different CP levels (35 to 55% CP).Asterisk denotes a coefficient of determination of 75%.

Figure 1 .
Figure 1.Mean comparison test using the polynomial regression model for the different CP levels (35 to 55% CP).Asterisk denotes a coefficient of determination of 75%.

Figure 2 .
Figure 2. Trypsin:chymotrypsin ratio (T:C) in relation to the inclusion levels in the experimental diets (35 to 55% CP), the hours post-hatching (HPH) and the food item: forage larva (FL), Artemia salina nauplii (A); A + dry diet (Dd) and dry diet (Dd).

Figure 4 .
Figure 4. Specific pepsin-like activity (mU mg of protein −1 ) in relation to CP inclusion levels in the experimental diets, the number of hours post-hatching and the food item: Artemia salina nauplii (A); A + dry diet (Dd) and dry diet (Dd); 111/HPE time at which pepsin-like activity becomes apparent.

Figure 4 .
Figure 4. Specific pepsin-like activity (mU mg of protein −1 ) in relation to CP inclusion levels in the experimental diets, the number of hours post-hatching and the food item: Artemia salina nauplii (A); A + dry diet (Dd) and dry diet (Dd); 111/HPE time at which pepsin-like activity becomes apparent.

Fishes
Author Contributions: Methodology, C.D.-R., D.M.F. and F.C.-L.; formal analysis, C.D.-R., D.M.F. and F.C.-L.; investigation, C.D.-R.; resources, C.D.-R.and D.M.F.; writing-original draft preparation, C.D.-R.and D.M.F.; writing-review and editing, C.D.-R., D.M.F. and F.C.-L.; funding acquisition, C.D.-R.and D.M.F.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Research Center of the Universidad de los Llanos, as part of the doctoral training project entitled: Early development of the species Brycon moorei-Steindachner, 1878 (Dorada del Magdalena): embryonic, morphometric, histological and histochemical aspects, productive parameters and digestive enzyme activity in relation to diet, through rector resolution No.0005 de 2017.Institutional Review Board Statement: The maintenance, handling and experiments conducted on fish during this study were carried out in strict accordance with the guidelines of the Experimental Animal Welfare Ethics Committee of the Universidad de los Llanos (Acta n • 04 legal document of 2 October 2017).

Table 3 .
Dietary composition and proximate analysis of the experimental diets used in the experiment.

Table 4 .
Effect of protein levels on zootechnical parameters of the feeding protocol (n = 105 per CP level), different letters (a-c) indicate significant differences (p < 0.05).