**6. Binder effect on growth**

The relationship between the availability and composition of food and the growth rate of a given aquatic species is of a crucial importance for the optimization of the rearing conditions. In order to understand this relationship the main physiological processes of the organism (ingestion, assimilation, respiration, growth and reproduction) should be taken into account and evaluated by means of integrated models (Van der Meer, 2006). Theories of dynamic energy budgets (DEB) received great impetus in the last decades, against the previous prevalent static approach. Among the various models proposed, the Kooijman κrule DEB theory (Kooijman, 2001) is one of the most encompassing. As reported in Figure 9, this model assumes that a part of ingested food is assimilated, then it enters the reserves and therefore a part is spent for maintenance and growth (prioritary flux), while the rest is utilized for maturation and reproduction.

Being all DEB models relatively simple, they did not take into account species-specific physiological aspects of the single considered species, which therefore need to be considered each time. On the basis of these considerations and given that binders often account for a consistent part of a prepared pellet, the effect of these substances on the ingestion, digestion

Fig. 9. Schematic representation (modified from van der Meer, 2006) of the Koijman Dynamic Energy Budget model.

and assimilation processes of each reared species must be carefully taken into account in order to maximize the experimental feed efficiency.

#### **6.1 Fish**

A limiting factor in larvicolture of many fish and crustaceans is the availability and nutritional value of live food. Larvae of many species have been raised using *Artemia salina* naupli and rotifers. However, their nutritional quality may change according to the source and the time of harvest of cysts. Formulated feed is by far more attractive due to the possibility to adjust its composition and size to the nutritional needs of the species. Most commercial larval diets are microbound diets, where binders are used to stabilize feed pellets and ensure minimum leaching and disintegration (Hashim and Saat, 1992). Carrageenan containing pellets are suitable for the ayu, *Plecoglossus altivellis* (Teshima et al., 1982), but not for postlarval tongue sole (*Cynoglossus semilaevis*) (Liu et al., 2008). Both alginate and zein appear to be suitable for farming larvae of sea bass (*Dicentrarchus labrax*) (Person Le Ruyet et al., 1993) but not barramundi (*Lates calcalifer*) (Partridge & Southgate, 1999), although Lee et al. (1996) previously reported that *Lates calcifer* larvae were able to digest both alginate and gelatine bound diets. Sodiumcarboxymethilcellulose, sodium alginate and gelatin included in feed for postlarval tongue sole (*Cynoglossus semilaevis*) brought about a growth similar to controls (Liu et al., 2008). Recently, a microbound diet containing soy lecithin, wheat gluten, and alginate has been patented for the culture of larval fish and crustaceans (D'Abramo, 2003).

#### **6.2 Crustaceans**

Dietary binders have been tested for growth performance in several species, with often conflicting results. Wheat flour, whole cassava meal and dry molasses were effective binders and caused the highest weight gain in crayfish *Macrobrachium rosenbergii* post-larvae, in comparison to agar (Seixas Filho et al., 1997a), although agar containing pellets had the best texture (Seixas Filho et al., 1997b). Unfortunately, no comparison with a control group fed natural diet was provided in this study. In contrast, Kovalenko et al. (2002) report that growth of larvae fed alginate containing feed, was only 90% of that achieved for larvae fed natural diet (newly hatched nauplii of *Artemia*). Both lignosol and agar brought about a significant increase in weight in juvenile shrimp *Palaemonetes varians* and *Palaemon elegans* (Palma et al., 2008). Alginate, agar and pectin caused good growth performance in both *Cherax albidus* juveniles and adults with respect to control animals fed natural diet, with pectin giving the best results in both juveniles and adults (Volpe et al., 2008; Coccia et al., 2010).

#### **6.3 Molluscs**

18 Aquaculture

Assimilated food

Maturation Reproduction

Ingested food Faeces

Reserves

Fig. 9. Schematic representation (modified from van der Meer, 2006) of the Koijman

and assimilation processes of each reared species must be carefully taken into account in

A limiting factor in larvicolture of many fish and crustaceans is the availability and nutritional value of live food. Larvae of many species have been raised using *Artemia salina* naupli and rotifers. However, their nutritional quality may change according to the source and the time of harvest of cysts. Formulated feed is by far more attractive due to the possibility to adjust its composition and size to the nutritional needs of the species. Most commercial larval diets are microbound diets, where binders are used to stabilize feed pellets and ensure minimum leaching and disintegration (Hashim and Saat, 1992). Carrageenan containing pellets are suitable for the ayu, *Plecoglossus altivellis* (Teshima et al., 1982), but not for postlarval tongue sole (*Cynoglossus semilaevis*) (Liu et al., 2008). Both alginate and zein appear to be suitable for farming larvae of sea bass (*Dicentrarchus labrax*) (Person Le Ruyet et al., 1993) but not barramundi (*Lates calcalifer*) (Partridge & Southgate, 1999), although Lee et al. (1996) previously reported that *Lates calcifer* larvae were able to digest both alginate and gelatine bound diets. Sodiumcarboxymethilcellulose, sodium alginate and gelatin included in feed for postlarval tongue sole (*Cynoglossus semilaevis*) brought about a growth similar to controls (Liu et al., 2008). Recently, a microbound diet containing soy lecithin, wheat gluten, and alginate has been patented for the culture of

Dietary binders have been tested for growth performance in several species, with often conflicting results. Wheat flour, whole cassava meal and dry molasses were effective binders and caused the highest weight gain in crayfish *Macrobrachium rosenbergii* post-larvae, in comparison to agar (Seixas Filho et al., 1997a), although agar containing pellets had the best texture (Seixas Filho et al., 1997b). Unfortunately, no comparison with a control group fed natural diet was provided in this study. In contrast, Kovalenko et al. (2002) report that

Somatic maintenance Growth

order to maximize the experimental feed efficiency.

larval fish and crustaceans (D'Abramo, 2003).

**6.2 Crustaceans** 

Dynamic Energy Budget model.

**6.1 Fish** 

Alginate and gelatin have been tested on cephalopod growth. The presence of alginate as food binder in *Octopus maya* negatively affected growth rate and survival even when added at a very low percentage. Indeed, *Octopus maya* fed alginate-bound crab showed a survival rate of only 15% with respect to the control group fed with crabs (Rosas et al., 2008). Both *Octopus maya* and *Octopus vulgaris* fed diets containing alginate and gelatin as binders grew significantly less than the controls fed natural diets (Rosas et al., 2008; Valverde et al., 2008; Garcia et al., 2011).

Konjac glucomannan and xanthan gum were used together in different configurations (1:1; 1:3) in feed for abalone (*Haliotis discus hannai*). Although no differences in growth have been recorded between treatments, growth parameters were significantly higher in animals fed a natural diet (O'Mahoney et al., 2011).

#### **6.4 Sea urchins**

The rapid expansion of the culture of sea urchins has increased the demand for formulated feed, specifically developed taking into account the echinoids needs. It has been widely demonstrated as prepared diets increase both test growth and gonad yield with respect to natural food (Pearce et al., 2004; Otero-Villanueva et al., 2004; Schlosser et al., 2005; Azad et al., 2011) and also feed initially formulated for other species than echinoids proved to successfully support urchins' growth and gonad maturation (Fabbrocini & D'Adamo, 2010). On the other hand, these diets frequently lead to poor gonad quality in terms of texture, firmness, colour and taste (Pearce et al., 2002b; Schlosser et al., 2005), that means low marketability of the product. For these reasons research focused on the formulation of suitable feed for sea urchin must take into account the effects on both gonad yield and sensory quality of both the components of the experimental diets and the binders used to give consistence to the pellets. Numerous binders have been successfully employed in the formulation of feed for different sea urchin species. In many echinoid species gelatin based pellets lead to a greater urchin growth (Fernandez & Pergent, 1998; Daggett et al., 2005) and a higher gonad yield (Pearce et al., 2004; Barker et al., 2006; Phillips et al., 2009) with respect to urchin fed on fresh macroalgae; also a pellet based on gelatin strengthened with the enzyme transglutaminase (γ-glutamyltransferase) gave better results in terms of gonad index with respect to *Laminaria hyperboreus* diet in *Strongylocentrotus droebachiensis* (Mortensen et al., 2004). Similarly, a feed containing a mixed binder gelatin-sodium alginate supported juveniles growth of *Strongylocentrotus droebachiensis* better than fresh *Laminaria* (Kennedy et al., 2007). Also agar (Barker et al., 1998), sodium alginate (Akiyama et al., 2001; Pearce et al., 2002b), guar gum and corn starch (Pearce et al., 2002b) based pellets gave better results in terms of gonad indices with respect to a natural diet. Despite the fact that much of the research on sea urchin rearing in confined conditions has focused on the formulation of prepared feed, few published studies have examined the effects of the binder nature and concentration on urchins' gonad growth and sensory quality.

Akiyama et al (1997) tested the effectiveness as a binder of 30% sodium alginate and of 15% Curdlan™, a polysaccharide produced by the bacteria *Alcoligenes faecalis* var. *myxogenes* and commonly used in human foods. They found that gonad growth in *Pseudocentrotus depressus* was higher with alginate based feed, with respect to Curdlan™ based pellet. Pearce et al. (2002b) tested different experimental feed which differed only for the binder type (gelatin, guar gum, sodium alginate, and corn starch) and concentration (3% and 5%), in order to evaluate the binder effect on gonad growth of *Strongylocentrotus droebachiensis*. Even if gonad growth was faster with gelatin and alginate based pellets, at the end of the trial (12 weeks) gonad indices (GI) were similar for the various binders at the higher concentration (5%), while it was slightly lower with 3% guar gum and corn starch. Fabbrocini et al. (2011) tested the effectiveness of agar based pellets for gonad growth and gamete production in *Paracentrotus lividus*. Pellets made of commercial feed Classic© (hendrix) and 3% agar gave the same results of the Classic© alone, therefore agar did not hamper gonad growth; in addition, urchins fed on *Ulva*-agar pellets in a 4 weeks rearing trial progressed in the reproductive cycle as those fed on commercial feed, showing comparable gonad indices levels. Regarding gonad sensory quality, a prepared diet with a high content of gelatin gave good results in *Evechinus chloroticus* (Phillips et al., 2009). On the contrary, in *Strongylocentrotus droebachiensis* a gelatin-transglutaminase based diet gave poor results in terms of gonad taste (Mortensen et al., 2004), while the best results in terms of gonad colour were obtained with a starch-based pellet by Pearce et al. (2002b). All these considered, when choosing the binder it is crucial to take into account the urchin species to be reared and the objective to be reached: urchin growth, gonad yield and gamete production, or gonad sensory quality.

In Table 3 the effect of feed binders on growth are reported.

#### **7. Binder effect on feed digestibility**

Several reports indicate a relationship between binders and nutrient digestibility. Different techniques are used for evaluating feed digestibility (Khan et al., 2003) among which the total collection technique, although time-consuming and stressful for the animal, is the most reliable method. The feed is fed in known quantities to the animal and accurate records of feed intake and fecal output are kept. Thus, it is foreseeable that digestibility experiments in aquatic animals are prone to errors related to the difficulty of measuring the correct amount of feed ingested and the methods employed to collect feces (i.e., settlement, filtration) which result in leaching losses (Irvin & Tabrett, 2005). It has been shown that the settlement collection method results in an overestimation of protein digestibility by up to 6% when feces are collected once every 6 h rather than hourly post-feeding in shrimp (Smith & Tabrett, 2004). Alternatively, *in vitro* techniques may be used to provide a quick and low cost method to predict nutrient digestibility. The digestibility of several carbohydrate sources for juveniles of the spiny lobster *Jasus edwardsii* has been estimated by measuring their rates of hydrolysis *in vitro* using enzyme homogenates and postprandial haemolymph glucose concentrations following ingestion of semi-purified diets (Simon, 2009). Many different factors influence the efficiency of digestion. The impact of binder type on feed digestibility

the research on sea urchin rearing in confined conditions has focused on the formulation of prepared feed, few published studies have examined the effects of the binder nature and

Akiyama et al (1997) tested the effectiveness as a binder of 30% sodium alginate and of 15% Curdlan™, a polysaccharide produced by the bacteria *Alcoligenes faecalis* var. *myxogenes* and commonly used in human foods. They found that gonad growth in *Pseudocentrotus depressus* was higher with alginate based feed, with respect to Curdlan™ based pellet. Pearce et al. (2002b) tested different experimental feed which differed only for the binder type (gelatin, guar gum, sodium alginate, and corn starch) and concentration (3% and 5%), in order to evaluate the binder effect on gonad growth of *Strongylocentrotus droebachiensis*. Even if gonad growth was faster with gelatin and alginate based pellets, at the end of the trial (12 weeks) gonad indices (GI) were similar for the various binders at the higher concentration (5%), while it was slightly lower with 3% guar gum and corn starch. Fabbrocini et al. (2011) tested the effectiveness of agar based pellets for gonad growth and gamete production in *Paracentrotus lividus*. Pellets made of commercial feed Classic© (hendrix) and 3% agar gave the same results of the Classic© alone, therefore agar did not hamper gonad growth; in addition, urchins fed on *Ulva*-agar pellets in a 4 weeks rearing trial progressed in the reproductive cycle as those fed on commercial feed, showing comparable gonad indices levels. Regarding gonad sensory quality, a prepared diet with a high content of gelatin gave good results in *Evechinus chloroticus* (Phillips et al., 2009). On the contrary, in *Strongylocentrotus droebachiensis* a gelatin-transglutaminase based diet gave poor results in terms of gonad taste (Mortensen et al., 2004), while the best results in terms of gonad colour were obtained with a starch-based pellet by Pearce et al. (2002b). All these considered, when choosing the binder it is crucial to take into account the urchin species to be reared and the objective to be reached: urchin growth, gonad yield and gamete production, or gonad

Several reports indicate a relationship between binders and nutrient digestibility. Different techniques are used for evaluating feed digestibility (Khan et al., 2003) among which the total collection technique, although time-consuming and stressful for the animal, is the most reliable method. The feed is fed in known quantities to the animal and accurate records of feed intake and fecal output are kept. Thus, it is foreseeable that digestibility experiments in aquatic animals are prone to errors related to the difficulty of measuring the correct amount of feed ingested and the methods employed to collect feces (i.e., settlement, filtration) which result in leaching losses (Irvin & Tabrett, 2005). It has been shown that the settlement collection method results in an overestimation of protein digestibility by up to 6% when feces are collected once every 6 h rather than hourly post-feeding in shrimp (Smith & Tabrett, 2004). Alternatively, *in vitro* techniques may be used to provide a quick and low cost method to predict nutrient digestibility. The digestibility of several carbohydrate sources for juveniles of the spiny lobster *Jasus edwardsii* has been estimated by measuring their rates of hydrolysis *in vitro* using enzyme homogenates and postprandial haemolymph glucose concentrations following ingestion of semi-purified diets (Simon, 2009). Many different factors influence the efficiency of digestion. The impact of binder type on feed digestibility

concentration on urchins' gonad growth and sensory quality.

In Table 3 the effect of feed binders on growth are reported.

**7. Binder effect on feed digestibility** 

sensory quality.



Table 3. Effect of feed binders on growth

is another of a series of factors that must be taken into consideration when designing a formulated feed type that is intended to be successful. High concentrations of binder may in fact cause a reduction in diet digestibility. Measuring the apparent digestibility of binders is

*droebachiensis*  (adults)

(juveniles)

(adults)

(juveniles)

gelatin 10 *Octopus vulgaris* Negative Garcia et al.,

10 *Octopus vulgaris* Negative Valverde et al.,

*semilaevis*  (postlarvae)

*droebachiensis*  (adults)

*droebachiensis*  (juveniles)

*droebachiensis*  (juveniles)

*Strongylocentrotus droebachiensis*  (juveniles)

*Strongylocentrotus droebachiensis*  (juveniles)

is another of a series of factors that must be taken into consideration when designing a formulated feed type that is intended to be successful. High concentrations of binder may in fact cause a reduction in diet digestibility. Measuring the apparent digestibility of binders is

3; 5 *Strongylocentrotus* 

1 Cherax albidus

2 *Octopus Maya* Same as

*droebachiensis*  (adults)

*varians* (juveniles)

SPECIES GROWTH Reference

Positive

control

Same as control

Positive Pearce et al.,

Positive Palma et al.,

Positive Pearce et al.,

Positive Volpe et al.,

Positive Coccia et al.,

2002b

2008

2002b

2008

2010

2011

2008

2002b

2002b

al., 2004

Positive Pearce et al.,

Positive Pearce et al.,

Positive Daggett et al., 2005

Positive Mortensen et

Positive Kennedy et al., 2007

Rosas et al., 2008

Liu et al., 2008

BINDER % Binder

PROTEINS

MIXED BINDERS

transglutaminase

Gelatin+

Gelatin+ alginate

employed

Guar gum 3; 5 *Strongylocentrotus* 

Lignosol 2 *Palemonetes* 

2 *Palemon elegans*

Corn starch 3; 5 *Strongylocentrotus* 

Pectin 2.5 Cherax albidus

2 *Cynoglossus* 

5 *Strongylocentrotus* 

5 *Strongylocentrotus* 

? 0.13%

5 2

Table 3. Effect of feed binders on growth

necessary in order to better understand their bioavailability when incorporated in formulated diets and their potential influence on the digestibility of other major macroingredients such as protein. Unfortunately, the effect of binders on diet digestibility has been largely overlooked. Simon (2009) found that gelatin, but not agar or alginate (7-8% inclusion level), positively affected digestibility in the juveniles of the lobster *Jasus edwardsii*. Similarly, *Octopus maya* fed alginate containing pellets lost weight and died, suggesting that this binder negatively affects nutrient absorption, while gelatin promoted absorption of nutrients (Rosas et al., 2008). It seems that binders have detrimental effects on nutrient digestibility in fish, as they accelerate gastrointestinal transit time (Storebakken, 1985). The inclusion of guar gum to tilapia feed has nutrient digestibility coefficients lower than diets containing CMC, corn starch and wheat gluten (Fagbenro & Jauncey, 1995). This is in line with previous results showing that fish feed containing highly effective binders had negative effects on macronutrient digestibility probably due to physical effects, such as changes in viscoelastic properties (Storebakken & Austreng, 1987; Storebakken et al., 1998). Recently, the effect of two natural binders (fava and kidney beans) on protein and lipid digestibility in the Atlantic salmon *Salmo salar* showed no significant differences between the dietary treatments (Pratoomyot et al., 2011) and the addition of guar gum to the diet of rainbow trout positively affected digestibility when employed with plant meal (Brinker & Reiter, 2011). The pattern of food residence in the gut was found to vary little with diet or urchin size (38-42h) in *Psammechinus miliaris* (Otero-Villanueva et al., 2004). Lawrence et al. (1989) found that in *Paracentrotus lividus* gut passage-time and apparent dry matter digestibility were similar with agar-based feed containing either soybean or fish meals. Similar results were reported by Klinger et al. (1994) for *Litechinus variegatus*. No differences in absorption efficiency were recorded for *Evechinus chloroticus* fed on extruded or agarbased pellets (Barker et al., 1998). In *Pseudocentrotus depressus* feed efficiency was higher for alginate- based pellets than for kelp (Akiyama et al., 2001), while regarding the effect of different binders, alginate-based pellets showed a lower feed efficiency than Curdlan™ based ones (Akiyama et al., 1997).

#### **8. Binder effect on digestive enzymes**

It is commonly agreed that species exhibit a particular suite of digestive enzymes that reflect their different life history (Figueiredo & Anderson, 2009). Consequently, from the digestive enzyme profile it is possible to predict the ability of the species to use different nutrients. Since the digestive enzyme activity is high for those substrates that are more common in the diet (Moss et al., 2001) there is a common belief that the activity of digestive enzymes can be boosted by providing certain nutrients into the diet for an adequate amount of time.

It is well known that crustaceans adapt their digestive enzymatic profile and activity to the diet composition (Muhlia-Almazan et al., 2003; Johnston & Freeman, 2005; Pavasovic et al., 2007a), as a consequence of an enzymatic battery capable of hydrolyzing the wide variety of substrates that they encounter in their natural diet (Linton et al., 2009). Modifications in digestive enzyme activity in different penaeid shrimp larvae have been related to the quantity or quality of some component of the diet (Le-Vay et al., 1993; Rodriguez et al., 1994; Le Moullac et al., 1997; Lemos & Rodriguez, 1998; Brito et al., 2000). Protein composition of the diet increased protease activity in *Marsopenaeus japonicas* juveniles and *Litopenaeus vannamei* larvae (Van Wormhoudt et al., 1986; Le Moullac & Van Wormhoudt, 1994; Le Moullac et al., 1997). Some authors suggest that the increase in amylase activity found in

*Penaeus monodon* (Fang & Lee, 1992), and *Litopenaeus vannamei* (Brito et al., 2000), may be related to the low level of carbohydrate in the diet (Le Vay et al., 1993; Rodriguez et al., 1994; Kumlu & Jones, 1995; Lemos & Rodriguez, 1998). Protease, amylase and cellulase activities have been found to be positively correlated with dietary protein levels (Pavasovic et al., 2007b), and negatively correlated with high cellulose levels (Pavasovic et al., 2006). In contrast, in the white shrimp *Penaeus vannamei* dietary protein level did not influence total protease activity, although modulation of trypsin- and chymotrypsin-like activities has been documented (Muhlia-Almazan et al., 2003). Moreover, no significant difference was observed in protease activity of *Litopenaeus vannamei* (Rivas-Vega et al., 2006) and *Scylla serrata* subjected to different dietary treatments (Pavasovic et al., 2004). In *Cherax quadricarinatus* the digestive enzyme secretion is modified by the diet composition, such as the presence of animal- or plant-derived ingredients (Lopez-Lopez et al., 2005; Pavasovic et al., 2007a). In the mollusk *Babylonia areolate* a positive correlation between dietary lipid level and lipase activity has been reported (Zhou et al., 2007). Among teleosts, proteolytic activity has been found unresponsive to the protein content of the diet in *Pseudoplatystoma corruscans*  (Lundstedt et al., 2004) and in early weaned sea bass (*Dicentrarchus labrax*) larvae (Cahu & Zambonino-Infante, 1994), but not in *Colossoma macropomum,* where digestive protease increased along with dietary protein content (De Almeida et al., 2006). On the other hand, alkaline protease activity was higher in *Dentex dentex* fed a diet with less protein and more carbohydrates (Perez-Jimenez et al., 2009). Amylase activity was higher in *Pseudoplatystoma corruscans* fed diets containing 13-25% of starch and similarly, it could be adjusted through the starch level of the diet in *Colossoma macropomum* (De Almeida et al., 2006) and *Dentex dentex* (Perez-Jimenez et al., 2009). A positive correlation has been observed between lipase and dietary lipid levels in *Colossoma macropomum* (De Almeida et al., 2006) and *Lateolabrax japonicus* (Luo et al., 2010). In any case the emerging picture in teleosts is one of wide complexity and variability due to the different of habitats and life histories of such vertebrates.

Virtually no studies have been carried out with the main purpose of investigating the effect of feed binders on digestive enzyme activity. The effect of different binders on digestive enzymes profile has been studied by us in *Cherax albidus* (Volpe et al., submitted). In an attempt to understand if the presence of binders influenced the digestive enzymatic profile we undertook a screening of carbohydratases, lipases and proteases along the digestive tract of juvenile *Cherax albidus* under both natural diet (Coccia et al., 2011) and different experimental diets based on pellets containing different binders but the same proximate composition. We found that digestive enzyme activities did not statistically show significant differences in the digestive tract except for amylase that was significantly higher in the intestine compared to the gastric juice and hepatopancreas of animals fed pectin containing pellets for 12 weeks.
