Influence of dietary fatty acid composition on enzymatic activity and digestive histology in silver catfish (Rhamdia quelen)

Dietary fatty acids may influence the development of the digestive tract and initial activation of digestive enzymes in fish. The objective of this study was to evaluate the effect of the diet lipid profile on ontogeny and activity of digestive enzymes in silver catfish post larvae fed practical diets. Five diets were tested, replacing broiler liver by soybean protein concentrate (SPC) or fish meal (FM): Standard, 15SPC, 30SPC, 15FM and 30FM. The enzymatic activity of fish was analyzed from 32 Hours after fertilization (Haf). Were analysed acid protease, trypsin, chymotrypsin, lipase, amylase and maltase. The development of the digestive system was analyzed from first feeding up to 28 days by histological techniques. The enzymatic activity presented peaks for the post-larvae fed with the 15SPC diet and fish fed with diet 15FM showed best growth. The development of the digestive system did not suffer damage from the diets offered. Post-larvae fed the 30SPC diet showed reduced development of the digestive tract. The diet 15FM provide good lipid profile for silver catfish post larvae.


Introduction
Digestive enzymes act according to the diet composition and the dietary habits of fish (Mente et al., 2017). The main essential fatty acids that should be present in early fish diets are 22: 6n-3, 20: 5n-3 and 20: 4n-6, and their ratio should be adequate for proper fish development (Sargent et al., 1999;El Kertaoui et al., 2019).
The enzymes involved in the digestion of luminal protein (trypsin, chymotrypsin), lipids (lipases) and carbohydrates (amylases and maltases) are present in larvae before the first feeding (Lazo et al., 2011;Alveal et al., 2019). The development of the digestive tract, the identification of digestive enzymes and analysis of their activities provide valuable information about the nutritional status of larvae and post-larvae of fish (Yúfera & Darias, 2007;Silveira et al., 2013;Alveal et al., 2019). The first gastric glands can be detected a few days or weeks after hatching and their numbers progressively increase by covering the epithelium of the stomach (Zambonino Infante et al., 2008;Alveal et al., 2019).
Fish larvae under go morphological, anatomical and functional changes during development and the digestive tract is still immature at hatching (Teles et al., 2017). The stomach in some species is not differentiated during the first days of life (Teles et al., 2017). The development of these organs and gastric glands appears at different ages for the various species of fish (Zambonino Infante and Cahu, 2007;Teles et al., 2017;Alveal et al., 2019). In the catfish, the digestive tract is formed 48 hours after hatching at 24ºC. At 16Haf, differentiation of the digestive tract is complete, presenting oropharyngeal cavity, esophagus, liver, pancreas, stomach and intestine (Silveira et al., 2013).
Fish meal made from waste of silver catfish (FM) tested in the feeding of juveniles, is considered as an alternative sustainable for this species (Rossato et al., 2018). Fish meal has an adequate percentage of fatty acids that is essential for early fish development (Sargent et al., 1999, Seong et al., 2019. According to Vizcaíno et al. (2014) the rising costs of fish meal worldwide promote great research effort aimed to find alternative and renewable ingredients for aquafeeds. The use of available food waste as an alternative protein source for producing fish feed has been suggested as a means of tackling the problem of sourcing safe and sustainable feed (Mo et al., 2018).
Soy protein concentrate is usually the main source of post larvae of fish feed protein. But it has many anti-nitritional factors  that inhibit enzyme activity and low palatability (Tacon and Akiyama, 1997). Consequently, it is one of the major causes of the low survival of post larvae of fish. But SPC is the most abundant raw material compared to animal meal. The soybean meal was tested of Piaia and Radünz Neto (1997) and SPC by Fontinelli and Radünz Neto (2007).
But in proportions and quantities that did not favor the development of the animals. For carnivorous fish, some studies revelated that the fish were able to tolerate less than 50% of SPC replacement in the feed (Mohd Faudzi et al., 2018). Research, Society andDevelopment, v. 10, n. 3, e26010312530, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.12530 Therefore, in this study we evaluated percentage of inclusion of this source for post-larvae of silver catfish.
The objective of this study was to evaluate the effect of the diet lipid profile on ontogeny and activity of digestive enzymes in silver catfish post larvae fed practical diets.
The first food was supplied at 108Haf when the larvae began to consume exogenous food, considered post-larvae. The animals were fed every 2 hours (h) between 08h and 20h in amounts exceeding its intake capacity. Housed in a recirculation system composed of 35 experimental units and fed for 28 days. Each unit consists of two plastic containers provided with side screen to prevent the exit of the post-larvae. With individual supply and flow, connected in thermoregulated water recirculation system. 240 post-larvae were used per experimental unit (45 animals per liter). Five treatments were tested with seven replicates. Biometric evaluation was performed on 10 post-larvae per experimental unit for seven days in order to monitor growth. Samples were weighed on a digital scale (0.001 g) without return. The animals were sedated with benzocaine (50 mg L-¹) (AVMA, 2007), weighed, and measured in order to obtain the Weight of the whole fish (mg).
Analysis of the development of the digestive system, through histology, was performed through samples of five postlarvae per treatment in each period. Samples of all treatments and periods were collected in biometrics every week. Samples collected every 7 days after starting feeding. In the following periods: 108; 240; 408; and 600Haf. After statistical analysis of the weights, only the post-larvae of the treatments that presented higher and lower weight at the end of the experimental period were chosen.The samples of the treatments with the highest (15FM) and the lowest (30SPC) were analyzed at 21 days. When for the small number of animals due to collections for enzymatic analysis and cases of cannibalism, the experimental units of the animals that consumed the 30SPC diet did not present enough animals and the treatment was terminated. The samples were fixed in Bouin solution for 12 hours and then stored in 70% alcohol until the analyzes were carried out. Later they were dehydrated in ethanol, diaphanized in xylol and embedded in paraffin, according to the methodology described by Cargnin-Ferreira and Sarasquete (2008). The histological sections were cut with a thickness of 4 μm in a rotary microton and stained with hematoxylin-eosin. Histological histories of the post-larvae were made through serial cuts for later analysis of the ontogenetic development of the organs of the digestive system (stomach, intestine, pancreas and liver). They were evaluated in light photomicroscope (Zeiss, Primo Star model). All post-larvae collected were euthanized by benzocaine overdose.
The experimental design was completely randomized. Initially, all data obtained were submitted to the normality test of Shapiro-Wilk, considered normal distribution of those data that presented p > 0.05. Subsequently ANOVA and test of comparison of means for the different components were performed. The minimum level of significance used in the statistical analysis was 5% (p <0.05).
The animals fed the 15FM diet presented higher weight (> 350 mg) than the 30FM treatment (> 150 mg) at the end of the experiment (Figure 1). In this study at 600Haf the post-larvae fed the 30SPC diet did not present a representative number to continue the experiment, due to collections for analysis, cannibalism cases and mortality. The activity of protease, trypsin, lipase and maltase enzymes increased from 72Haf ( Table 2). The body protein of all treatments remained in similar patterns, with peaks in the different treatments in the periods tested. Acid protease was elevated for animals consuming the 15FM (> 120 µmol tyrosine / min / mg protein), followed by the 30FM and 15FM, the standard showed similar values (> 80). The trypsin activity showed a peak for the 15SPC in the period 168Haf (> 25 µmol TAME / min / mg protein) (Figure 2). The other treatments showed that trypsin activity remained below 20 µmol TAME / min / mg protein.
Chymotrypsin activity had a peak for 15SPC in the 168Haf. The activity was higher than 25.000 µmol BTEE / min / mg protein while the other treatments the activity in all periods were below 20.000 µmol BTEE / min / mg protein (Figure 2). The  Treatments: Standard, adapted from Coldebella et al. 2011 (15, 30, 30, 30, 30 and 30%) of the liver for soybean protein concentrate (SPC). Haf: Hours after fertilization. In the histological sections was observed reduced development of the animals fed the 30SPC ( Figure 3A and 3C) in relation to the animals fed the 15FM ( Figure 3B and 3D). As a consequence, the development of the organs linked to the digestion of food is similarly lower in the animals fed the 30SPC (Figure 3). In relation to the development of intestinal villi, Research, Society andDevelopment, v. 10, n. 3, e26010312530, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.12530 no alterations were observed between the different treatments tested. When we compare the treatments, the intestinal villi and stomach crypts are proportionally smaller in the animals that consumed the 30SPC ( Figure 3E, 3F and 3G).
The diet presented an adequate profile in fatty acids, especially the 15FM diet, higher values of arachidonic acid (20: 4 n-6) and the sum of polyunsaturated fatty acids and n-6. Studies have shown that essential fatty acids (EFAs) such as docosahexaenoic acid (DHA, C22: 6 n-3) and eicosapentaenoic acid (EPA, C20: 5 n-3) are important in the nutrition of fish larvae (Mousavi-Sabet et al., 2013;El Kertaoui et al., 2019). According to the NRC (2011), freshwater fish strictly demand only linoleic acid (LA) and linolenic acid (ALA) fatty acids, unlike marine and stenoaline fish, in which diet it is indispensable to include highly unsaturated fatty acids (Segura et al., 2017). The diet 15FM presented higher values of arachidonic acid (ARA) in the composition. In marine fish larvae, studies have indicated that ARA levels may be important for tolerance to stress, pigmentation, growth and survival (Park et al., 2006). Freshwater fish have a greater ability to convert C18:0 fatty acid to highly unsaturated fatty acids (Asil et al., 2017). Arachidonic acid supplementation in diet can improve growth performance and enhance physiological characteristics of freshwater fish (Ji et al., 2011;Asil et al., 2017). This may have contributed to the greater development of the post-larvae fed the diets that presented the highest percentage of this fatty acid. Fatty acids are essential mainly in this phase of fish life, since they contribute to the formation of membranes and hormones (Izquierdo and Koven, 2011), consequently increasing growth and reducing mortality.
The inclusion of 30SPC in diets for post-larvae of silver catfish inhibits the development of the animals. According to Kumar et al., (2017) total replacement of soybean meal to replace fish meal in practical diets for Silurus glanis induced negative influences on growth and feed utilization. But for silver catfish, the inclusion of 15SPC in the diet provided positive performance. The nutritional value of soybeans is limited by the presence of several antinutritional factors such as trypsin and chymotrypsin protease inhibitors that affect the digestion and physiology of animals (Zhou et al., 2017). Soy products may contain non-starch polysaccharides that are not digested by fish, may have detrimental effects on nutrient performance and digestibility and on fish health (Krogdahl et al., 2010, Serra et al., 2019. These adverse effects are associated with the viscous nature of non-starch polysaccharides and their interaction with intestinal epithelium, mucus and microbiota, which result in physiological and inflammatory imbalances , Serra et al., 2019. The soybeans, in the form of protein concentrate had less of these factors. These, even in small amounts may have contributed to reduce the development of the animals as observed for those fed on the 30SPC diet. The fish that consumed the 15SPC presented similar development to that found for the animals that consumed the 15FM. Tolerance regarding the amount of SPC added to feed is closely related to the dietary habits and nutritional requirements of each species (NRC, 2011). From this, that total liver replacement is not advisable, as the inclusion of 30SPC in the diet of catfish post-larvae.
The activity of the main enzymes (acid protease, trypsin and chymotrypsin) at 32Haf were reduced. The enzymatic activity in larvae was low up to 32Haf. Then the sharp rise and fall to new 96Haf, when the food transition end. This behavior was also observed by Silveira et al. (2013). The yolk is the source of energy for the larvae to develop, and at the same time a substrate for the differentiation and growth of new structures (Portella et al., 2012). This is the first food to be metabolized with the help of existing enzymes, the main digestive enzymes produced by the pancreas being trypsin, amylase and lipase that can be detected even before opening the mouth (Gao et al., 2016;Cui et al., 2017). The activity of the acid protease had a gradual increase from 32Haf to 166Haf (Table 2). Values expressed as mean ± standard error of the mean. * Total body protein (mg / g), acid protease (μg tyrosine / min / mg prot), trypsin (umolTAME / min / mg prot), chymotrypsin (umol BTEE / min / mg prot), lipase min), amylase (IU / mg protein / min), maltase (IU / mg protein / min). Haf: hours after fertilization. Source: research data. For Salminus brasiliensis larvae, acid protease activity was detected on the third day after hatching and then increased up to the seventh day, which indicates functionality of the stomach (Vega-Orellana, Fracalossi & Sugai, 2006). The trypsin activity remained linear up to 72Haf and from this began to reduce. According to Conceição, Aragão and Rønnestad, (2011) is considered the key factor in the activation of pancreatic enzymes. For Salminus brasiliensis larvae, the activity of intestinal proteinase (trypsin and chymotrypsin) were detected 12 h after hatching before the start of exogenous feeding (Vega-Orellana, Fracalossi & Sugai, 2006). The activity of trypsin and chymotrypsin decreased from 72Haf and the activity of the acid protease, amylase and maltase increased until the beginning of exogenous feeding. Trypsin and chymotrypsin in newly hatched larvae are involved in the cleavage of proteins contained in the yolk  and are the first enzymes to act more actively on the same. Lipase increased from 40Haf to 84Haf. This enzyme is linked to the digestion of calf lipids contained in vitellogenin which are broken down by lipases and contribute to the development of the embryo (Mommsen & Korsgaard, 2008). It is an enzyme of pancreatic origin that acts in the intestine acting on the lipids present in the chyme, releasing fatty acids and glycerol, which play important energetic, structural and hormonal functions in the organism (Moura et al., 2012).
After the beginning of the exogenous feeding (108Hpf), a peak of enzymatic activity occurred and later it stabilized.
This peak occurred mainly for acid protease for all diets, two hours after the first feeding. For the other enzymes the peak occurred more markedly for the 15SPC diet (trypsin, chymotrypsin and amylase at 168Hpf, lipase and maltase at 192Hpf).
This behavior occurred as a way of adaptation of the post-larvae to the diet so that there was an improvement of the digestibility and absorption of the nutrients. Also observed by Mitra et al. (2017) that concludes that these fluctuations of the enzymatic activities in featherback larvae reflect the ability of the fish to adapt with the diet during ontogenetic shift.
According to Silveira et al. (2013), pepsin activity was low up to 32Haf, increasing and decreasing in the transition to exogenous feed. In the post-larvae that received the 30SPC diet, trypsin activity decreased from 120 to 312Haf. The same behavior observed for the amylase that at 144Haf had expressive decrease with recovery at 216Haf. The reduced enzymatic activity in the initial period of development may have contributed to the low growth presented by the animals that consumed this diet. Afterwards all the enzymes have stabilized, which may indicate an adaptation of the post-larvae to the food provided.
For most fish species, digestive capacity becomes more efficient after hatching, when there is an abundance of gastric glands, pepsin-like enzymes and hydrochloric acid, which perform the first hydrolysis of proteins, thus facilitating the subsequent action of the enzymes alkaline, such as trypsin and chymotrypsin (Portella et al., 2014).
The decline in specific activity of both enzymes throughout the larval development of the catfish could be explained by the normal increase in tissue proteins (Zambonino Infante et al., 2008;Mitra et al., 2017). Reflecting anatomical and physiological changes in fish post-larvae, but not corresponding to the reduction in the amount of digestive enzymes (Zambonino Infante et al., 2008). As well as the progressive transformation of larval alkaline digestion, characterized primarily by pancreatic proteases, such as trypsin and chymotrypsin, to a juvenile acid digestion mode (Babaei et al., 2011;Mitra et al., 2017).
The development of the digestive system occurred in the same way in the 15FM and 30SPC diet animals. Slower development was observed in smaller animals due to the lower digestibility of SPC in relation to fish meal (FM) confirmed in studies carried out by Hien et al. (2017). According to Drew et al. (2007) the protein quality of the ingredient depends fundamentally on its digestibility. In the 15FM diet, the addition of fish meal contributed to improve the palatability of the rations. According to Liu et al. (2012), the animal meal significantly improves palatability. Diets with total liver replacement by SPC, fish did not develop as much as those fed the diet with partial replacement (15SPC). Low consumption, low palatability and cases of cannibalism were observed. In addition to the reduced amount of antinutritional compounds (Fontinelli & Radünz Neto, 2007;Sá et al., 2013) present in SPC, they may interfere in the general development of post-larvae.
The post-larvae fed the 30SPC diet showed reduced development in relation to the animals that consumed the other diets. The histopathological analysis of the post-larvae independent of the different diets, did not reveal morphological alterations in the tissues and internal organs. No inflammatory process was observed in the individuals of both diets. Intestinal villi were normal.
According to Lazo et al. (2011), the major histological changes in the intestinal mucosa include decreased enterocyte height and number and size of epithelial folds. Further studies should be conducted to further clarify these effects.

Conclusion
The results of the present study show that silver catfish consumed the 15FM diet, presented highest amount of fatty acids C20:4 n-6 and C22:6 n-3, lower incidence of peaks in the production of digestive enzymes.