**1.3.9 Culture**

230 Aquaculture

Fig. 18. Post-embryonic stage IV (days 24-25).- a) Fully development eyes or mature eyes (ME), pigmentation spots (PS) dispersed throughout the body, pleopods (Pl) are visible; the vitellus is exhausted but exogenous feeding has not begun. b) Telson elongated and uropods compound (protop, endopod, exopod) with bristles and sensorial filaments at the ends; but independent movement does not yet occur. Three pairs of pleopods (Pl) are visible on the abdominal somites, and c) the organisms remain attached to the mother's pleopods.

Descriptions were based on the external morphological changes observed in the embryos during ontogeny because the most important ontogonic events during development in *P. acanthophorus* occur in the embryos while still in the chorion. These define the different stages and morphological changes in ways similar to those reported by Montemayor et al. (2010) in *P. regiomontanus* (Villalobos, 1954) (Cambarideae family); Sandeman & Sandeman (1991) in *C. destructor*, and García-Guerrero et al. (2003) in *C. quadricarinatus* (both of the

Embryonic development in some crustaceans is highly dependent on water temperature (Bottrell, 1975; Herzig, 1983). In the present study, the crayfish *P. acanthophorus* embryos developed at an average temperature of 23.8 ± 2.2 °C during January-March. This coincides with Cervantes (2008), who reported that this species can reproduce year round under laboratory conditions as long as average temperature is kept at 25 °C. Auvergne (1982) stated that optimum temperature for each life stage in crustaceans is species dependent; for instance, *Astacus astacus* (Linnaeus, 1758) has a range of between 18 to 20 °C whereas *Procambarus clarkii* (Girard, 1852) requires a range of 22 to 26 °C. In further examples, Sandeman & Sandeman (1991) reported satisfactory development in *C. destructor* eggs incubated at 19 °C; García-Guerrero et al. (2003) described successful embryo development in *C. quadricarinatus* at 26°C; and García-Guerrero & Hendrickx (2009) reported proper

As embryo development progressed, egg length increased; initial egg diameter was 1.3 mm and total length of juvenile organisms was 6±1 mm. This development trajectory differs from the 16 stages (22 days) reported for *P. regiomontanus*, with eight embryonic stages, eight post-embryonic stages and an average juvenile size of 2 cm (Montemayor et al., 2010). However, both these *Procambarus* species have embryonic development periods near the 30 day average for cambarid crustaceans. *Procambarus clarkii* completes its embryonic development in an average of three to four weeks (McClain & Romaire, 2007), and *P. llamasi* completes it in 27 to 30 days (Rodríguez-Serna et al., 2000). These contrast with the longer development periods of other species. *C. quadricarinatus* has a 42-day development period with ten embryonic stages, two post-embryonic stages and a juvenile stage (García-Guerrero et al., 2003), while C. destructor has a 40-day development period with an unknown number of embryonic stages, at least two post-embryonic stages and a juvenile stage (Sandeman &

development in fertilized *Macrobranchium americanum* eggs at 24 °C.

Parastacidae family).

#### **1.3.9.1 Optimum density for growth**

Advances in knowledge for the commercial culture of crayfish have shown that in this specie territorial habits are not present, allowing stocking densities as high as 100 orgm-2 without affecting the growth density and survival, which is considered an advantage in production. In addition, laboratory studies indicate that it is possible to keep under recirculation systems the biculture of crayfish and tilapia at densities between 30-50 orgm-2, where the main species is tilapia, and crayfish support sustainable use of water.

#### *1.3.9.1.1 Response parameters*

A experimental trial was done when different densities of culture where evaluated, survival (%), showed no significant differences between treatments and remained in a range between 67 and 85%, with the increase of survival were crayfish were kept at densities of 50 and 90 orgm-2, while the lowest occurred where the crayfish were kept at a density of 60 orgm-2, without a positive relation between survival and density (Table 3).


T1=50, T2=60, T3=70, T4=80, T5=90, T6=100 (org/m2)

Treat.= Treatments (org/m2); SUP= Survival; IW= Initial Weight; FW= Final Weight; IWG= Individual weight gain; SGR= Specific growth rate; IFC= Individual food consumed and FCR= Feed Conversion rate

Table 3. Response parameters ± s.d of the crayfish was culture at different densities.

With respect to growth, the final weight (FW g), weight gain (WG%) and specific growth rate (SGR%/day) for all treatments was similar. The organisms in treatment 1, had a greater weight gain, during the first 30 days of culture maintained a growth rate similar to that of crayfish of the other treatments, then increased its rate of growth, although not differ significantly from other organisms under study, it was considered that the density did not significantly affect growth or survival of the crayfish. The juvenile kept it in treatment 5 and 6 had the lowest growth without significant differences between treatments (95% confidence).

#### **1.3.9.2 Biculture**

Actually research on the feasibility of polyculture systems including fish and crustaceans, has produced inconsistent conclusions, for that reason is difficult to determinate the potential of polyculture in recirculation system for sustainable aquaculture. In a study with red claw crayfish *C. quadricarinatus* and tilapia *Oreochromis niloticus* in a polyculture system in earthen ponds, Brummett & Alon (1994) reported positive results for the crayfish growth and survival; whereas using the same species combination Rouse & Kahn (1998) reported that competition for feed and space between species negatively affected survival in *C. quadricarinatus*. An alternative to the above system is the use of recirculation systems in crustacean/fish polyculture because water quality and feed supply can be controlled, and shelters can be provided for crustaceans to prevent territorial competition, allowing in both species a survive and grow properly (Karplus et al., 2001). Polyculture is particularly appealing since it makes extremely efficient use of resources and can increase production. It can be quite viable for producers as long as appropriate species are identified in terms of biology and market demand. Using a polyculture with common carp, grass carp, silver carp, tilapia, mullet and Malaysian prawn, Cohen et al. (1983) reported efficient water use and an increase in production from 3.5 to 11 tons/ha/year. In a polyculture system for carp (*Cyprinus carpio*) and crayfish (*Cambarellus montezumae*) growth in artificial ponds in which feeding was focused mainly on the carp, Auró et al. (2000) reported that these species could coexist and use the food in the system, making it a viable system.

Competition is increasingly high for good quality water sources for productive activities such as aquaculture. There is also a need to optimize space during production and to promote productive and sustainable alternative activities in rural areas. Under these conditions, the most adequate option for polyculture systems is to use recirculation systems, ensuring that the species in the system do not compete for resources and have established market niches (Kazmierczak & Caffey, 1995). With the objective to determine the feasibility of a crayfish *P. acanthophorus*/ tilapia *O. niloticus* polyculture and monoculture using a water recirculation system, as aquaculture sustainable alternative, a experimental research in outdoor facilities was conducted. Six plastic tanks (3 m diameter x 1.2 m depth) in a recirculation system with a biological and sand filter were used. During 90-day experimental period, three treatments were evaluated with two replicates per treatment in a completely random design. T1: crayfish monoculture (1.02±0.2 g); T2 polyculture: crayfish (1.04±0.2 g) and tilapia (2.99±0.1 g); T3: tilapia monoculture (3.45±0.6 g).

Survival in the T2 crayfish was significantly lower (34.7%) compared to that in the T1 crayfish (72%). In contrast, the tilapia in both T2 and T3 had similar survival (>95%) and growth rates (83-86 g) with no apparent effect from the presence of the crayfish (Table 4).

With respect to growth, the final weight (FW g), weight gain (WG%) and specific growth rate (SGR%/day) for all treatments was similar. The organisms in treatment 1, had a greater weight gain, during the first 30 days of culture maintained a growth rate similar to that of crayfish of the other treatments, then increased its rate of growth, although not differ significantly from other organisms under study, it was considered that the density did not significantly affect growth or survival of the crayfish. The juvenile kept it in treatment 5 and 6 had the lowest growth without significant differences between treatments (95%

Actually research on the feasibility of polyculture systems including fish and crustaceans, has produced inconsistent conclusions, for that reason is difficult to determinate the potential of polyculture in recirculation system for sustainable aquaculture. In a study with red claw crayfish *C. quadricarinatus* and tilapia *Oreochromis niloticus* in a polyculture system in earthen ponds, Brummett & Alon (1994) reported positive results for the crayfish growth and survival; whereas using the same species combination Rouse & Kahn (1998) reported that competition for feed and space between species negatively affected survival in *C. quadricarinatus*. An alternative to the above system is the use of recirculation systems in crustacean/fish polyculture because water quality and feed supply can be controlled, and shelters can be provided for crustaceans to prevent territorial competition, allowing in both species a survive and grow properly (Karplus et al., 2001). Polyculture is particularly appealing since it makes extremely efficient use of resources and can increase production. It can be quite viable for producers as long as appropriate species are identified in terms of biology and market demand. Using a polyculture with common carp, grass carp, silver carp, tilapia, mullet and Malaysian prawn, Cohen et al. (1983) reported efficient water use and an increase in production from 3.5 to 11 tons/ha/year. In a polyculture system for carp (*Cyprinus carpio*) and crayfish (*Cambarellus montezumae*) growth in artificial ponds in which feeding was focused mainly on the carp, Auró et al. (2000) reported that these species could

Competition is increasingly high for good quality water sources for productive activities such as aquaculture. There is also a need to optimize space during production and to promote productive and sustainable alternative activities in rural areas. Under these conditions, the most adequate option for polyculture systems is to use recirculation systems, ensuring that the species in the system do not compete for resources and have established market niches (Kazmierczak & Caffey, 1995). With the objective to determine the feasibility of a crayfish *P. acanthophorus*/ tilapia *O. niloticus* polyculture and monoculture using a water recirculation system, as aquaculture sustainable alternative, a experimental research in outdoor facilities was conducted. Six plastic tanks (3 m diameter x 1.2 m depth) in a recirculation system with a biological and sand filter were used. During 90-day experimental period, three treatments were evaluated with two replicates per treatment in a completely random design. T1: crayfish monoculture (1.02±0.2 g); T2 polyculture: crayfish

Survival in the T2 crayfish was significantly lower (34.7%) compared to that in the T1 crayfish (72%). In contrast, the tilapia in both T2 and T3 had similar survival (>95%) and growth rates (83-86 g) with no apparent effect from the presence of the crayfish (Table 4).

coexist and use the food in the system, making it a viable system.

(1.04±0.2 g) and tilapia (2.99±0.1 g); T3: tilapia monoculture (3.45±0.6 g).

confidence).

**1.3.9.2 Biculture** 


1Values in the same column with the same superscript are not statistically different (p>0.05)

S% = survival rate; IW = initial weight; FW = final weight; IWG = individual weight gain; WG% = percentage weight gain; SGR = specific growth rate.

Table 4. Growth and efficiency parameters in crayfish and tilapia monocultures and crayfish/tilapia polyculture in a water recirculation system1.

The lower crayfish survival rate in T2 had no apparent effect on growth, as might be expected due to the density effect, since IWG and WF in T2 (0.033±0g; 3.9±0.3g, respectively) were significantly lower than in T1 (0.042±0g; 4.8±0.4g, respectively). Tilapia growth in T2 and T3 followed a steeply-sloped exponential curve whereas crayfish growth was constant but with a lesser slope, reflecting their lower growth rate (Figure 19).

Water quality parameter values during the trial were within the ranges tolerated by tilapia fingerlings and crayfish culture. Dissolved oxygen (DO) concentration was 3.8 mgL-1 throughout the experimental period, lower than the 5 mg/L recommended for optimum growth in *P. acanthophorus* (Cervantes-Santiago et al., 2007). This level coincides with the >3 mgL-1 DO level recommended for *P. clarkii* (Huner, 1994), and suggests that *P. acanthophorus* can adapt to environmental variations during cultivation. Like crayfish, tilapia can also tolerate low DO levels (<2 mgL-1), although levels greater than 3 mgL-1 are recommended for good growth (El-Sayed & Abdel-Fattah, 2006). This DO level also favors proper functioning of the biological filter in the recirculation system and prevents the death of nitrifying bacteria (Yousef et al., 2003). Increased temperature during the experimental period improved growth in the tilapia and crayfish, although efficient growth in *P. acanthophorus* is reported to occur at temperatures <28°C. Optimum growth in tilapia occurs at 28°C, even though the species can tolerate a range of 15-35°C. Apparently, environmental parameters are no impediment to polyculture of Nile tilapia and *P. acanthophorus*. Higher temperatures (23-33°C) have also been reported to increase growth in a polyculture of *C. quadricarinatus* and tilapia (Rouse & Kahn, 1998). In addition, the temperature tolerance exhibited by *P. acanthophorus* coincides with overall temperature tolerance (10-38 °C) among *Procambarus* genus crayfish (Holdich, 2002). Of course, individual species have specific optimum temperature ranges for growth; for instance, *P. clarkii* prefer temperatures from 22- 30°C (Holdich, 2002) with optimal levels around to 20-25°C (Huner & Gaude, 2001). This tolerance for a wide range of environmental temperatures highlights the potential for crayfish cultivation in commercial systems.

Water pH levels were adequate for proper growth in both tilapia and crayfish, although both can tolerate pH from 3.5 to 12, another advantage for polyculture of these species (Huner, 1994; El-Sayed & Abdel-Fattah, 2006). Ammonium, N-nitrite and N-nitrate (mgL-1) values were below sublethal and lethal levels for the two cultured species (Huner, 1994;

T1: Crayfish, T2: Crayfish/Tilapia, T3: Tilapia

Fig. 19. Weight gain in crayfish and tilapia monocultures and crayfish/tilapia polyculture in a water recirculation system.

El-Sayed & Abdel-Fattah, 2006; Cervantes, 2008), indicating proper biological filter functioning.

This is the first report of a crayfish *P. acanthophorus* / Nile tilapia *O. niloticus* polyculture. The feasibility of this species pair in polyculture as a productive alternative when a recirculation system to increase water efficiency use was evaluated, because both tilapia and crayfish are readily available, and the *Procambarus* genus is diverse around the world, similar than tilapia. The polyculture system was successful in that both species developed properly during the experimental period, although survival among the *P. acanthophorus* (37.4%) was significantly lower than in the crayfish monoculture (72%), and crayfish IWG and FW were lower in the polyculture than in the monoculture. Presence of the tilapia in the same tanks was apparently the main potential cause of this overall lower growth performance in the polyculture since all other variables were within proper ranges for crayfish. There may have been interspecies competition for space and/or feed, or predation by the tilapia of the crayfish during molting partially due to a substantial size difference between species. The latter possibility was not evaluated in the present study since the same initial stocking sizes were used in all treatments: average initial stocking sizes were 1.03±0.77 cm (crayfish) and 3.15±1.04 cm (tilapia), giving a clear growth advantage to the tilapia. The tilapia did not necessarily need to be antagonistic for them to prey on the much smaller crayfish in T2. Different types and quantities of shelters could help to protect the crayfish from the larger tilapia, and manipulating initial stocking size of the different species might help to reduce predation.

Auró et al. (2000) highlighted the feasibility of crayfish/fish polyculture in a study using carp *Cyprinus carpio* and crayfish *Cambarellus montezumae* in artificial ponds. The species coexisted and had enough food resources at densities of up to 50 Org m-3 for both species under good water quality conditions. The crayfish *P. acanthophorus* has excellent potential for use in aquaculture systems, because it can be culture to high densities, tolerates handling, adapts to variable environmental conditions and accepts different kinds of artificial diets in captivity, although it does not reach sizes as large as other crustaceans, such as *C. quadricarinatus* (Cervantes, 2008; Cruz-Ordoñez, 2009). Its use in sustainable rural aquaculture production systems is promising under monoculture, and possibly under polyculture conditions after further research into optimum initial stocking size, densities and crayfish shelter type and quantity. Greater production of alternative protein sources using aquaculture in rural areas is an important step towards increasing food availability and diversity.

In addition to the above, the biculture system may be associated to aquaponic production of herbs (cilantro, basil and aquaponic green fodder) with satisfactory results.

#### **1.3.9.3 Genetic improvement**

234 Aquaculture

Fig. 19. Weight gain in crayfish and tilapia monocultures and crayfish/tilapia polyculture in

El-Sayed & Abdel-Fattah, 2006; Cervantes, 2008), indicating proper biological filter

This is the first report of a crayfish *P. acanthophorus* / Nile tilapia *O. niloticus* polyculture. The feasibility of this species pair in polyculture as a productive alternative when a recirculation system to increase water efficiency use was evaluated, because both tilapia and crayfish are readily available, and the *Procambarus* genus is diverse around the world, similar than tilapia. The polyculture system was successful in that both species developed properly during the experimental period, although survival among the *P. acanthophorus* (37.4%) was significantly lower than in the crayfish monoculture (72%), and crayfish IWG and FW were lower in the polyculture than in the monoculture. Presence of the tilapia in the same tanks was apparently the main potential cause of this overall lower growth performance in the polyculture since all other variables were within proper ranges for crayfish. There may have been interspecies competition for space and/or feed, or predation by the tilapia of the crayfish during molting partially due to a substantial size difference between species. The latter possibility was not evaluated in the present study since the same

T1: Crayfish, T2: Crayfish/Tilapia, T3: Tilapia

a water recirculation system.

functioning.

An experiment was designed to estimate genetic variability of growth as heritability (h2) of the weight at different ages. For which was captured 2135 organisms (4.1g ± 1.79) from its natural medium (G0) of which 10% heavier were selected (i = 1,755) for each sex: 140 females (5.62g ± 1.97) and 48 males (6.02g ± 1.9) to form the progenitors of the line selection (LS), while the control line (LC) consisted of organisms take it at random. These organisms were maintained for reproduction in two rectangular fiberglass tanks of 2.4 m2 and 0.15 m deep (one tank per line), and a density of 49 orgm-2, and relation females: males (3:1). Biweekly organisms were reviewed to identify gravid females From these organisms 30 fullsib families per line (LC and LS) were obtained (F1), and grown individually for five months, tracking them individually in a recirculation system consisting of 60 rectangular plastic tubs (54 cm x 37 cm x 22 cm) distributed in three levels with mechanical and biological filtration under laboratory conditions and fed 2 times day with balanced feed for shrimp (35% protein). Once the organisms in lines arrived at three months old, and due to differential mortality in the families and in order to reduce the effect of the environment, the density was standardized to 11 organisms per family (55 orgm-2) to continue growth in the same. Heritability for growth in broad sense (h2) and in each age, for both, the control (LC) and selection line (LS) was estimated from variance components (ANOVA method REML) using a full-sib design from the formulas described by Roff (1997). Growth between the lines in F1 was also compared for each age.

Table 4 shows estimates of heritability from full-sib design for F1. The value of h2 estimates for LC was 0.48 initially and decreased until the fourth month of age, not to suffer variations due to the rearrangement of the population in the third month compared to LS which began


Table 4. Estimates of heritability (h2) ± E.S in F1 at the different ages (months), in selection line (LS) and control line (LC)

with a value of 1.1 reducing the third month (0.27) have to rise again in the fourth month (0.58). Due to the differential survival and in order to reduce the effect of common environment which is reported in previous studies with crustaceans (Benzie et al., 1997; Hetzel et al., 2000; Pérez-Rostro & Ibarra, 2003), the density of families in the third month were standardized, leaving 11 organisms per family in F1.

Estimates of heritability (combined males and females) in F1 at the end of the trial was similar for both lines (0.27 ± 0.11 and 0.34 ± 0.12, LC and LS, respectively), coinciding with results obtained by Pérez-Rostro & Ibarra (2003) in white shrimp *Litopenaeus vannamei*, where values of 0.20 were obtained at 17 weeks of culture as well and those of Cameron et al. (2004) in the red claw crayfish *C. quadricarinatus*, where a decrease in the value of heritability of 0.38 for the first year to 0.13 after 4 years.

When growth gain between the lines was compared, it was seen that LS was significantly heavier (9.6%) than the control line (Figure 20).

Fig. 20. Weight gain between selection line (LS) and control line (LC) during the five months of culture.

The values of heritability indicate that the species has a positive response to selection, so they can continue to implement a screening program to improve the growth of the species and thus promote commercial cultivation.
