**1. Introduction**

216 Aquaculture

Takeuchi T.; Wang Q.; Furuita H.; Hirota T.; Ishida S & Hayasawa H. (2003). Development

Takeuchi, T.; Ohkuma, N.; Ishida, S.; Ishizuka, W.; Tomita, M.; Hayasawa, H. & Miyakawa,

Tamaru C.S.; Lee C, & Ako H. (1993). Improving the larval rearing of stripped mullet (*Mugil* 

Taylor R. G.; Whittington J. A.; Grier, H.J. & Crabtree R. E. (2000). Age, growth, maturation,

Tringali, M.D. & T. M. Bert (1996). The genetic stock structure of common snook

Tsukamoto, K. (1993). Quality of fish for release. In: Kitajima, C. *Healthy Fry for Release, and Their Production Technique*, pp. 102–113 Koseisya Koseikaku, Tokyo. Tucker, J.W.J.; Landau, M.P. & Falkner, B.E. (1985). Culinary value and composition of wild

Van Der Meeren, T. (1991). Algae as first food for cod larvae *Gadus morhua* L. : filter feeding

Vasquez-Yeomans L.& Carrillo-Barrios-Gomez E & Sosa-Cordero E. (1990).The effect of the

Watanabe, T. (1993). Importance of docosahexaenoic acid in marine larval fish. *Journal of* 

Webb J.F. ( 1999). Larvae in fish development and evolution. In: B.K. Hall & M.H. Wake. *The* 

Werner R.G & Blaxter J.H.S (1981) The effect of prey density on mortality, growth and food

Wyatt, T. (1972). Some effects of food density on the growth and behaviour of plaice larvae.

Yanes-Roca, C.; Rhody, N.; Nystrom, M.; Main, K. (2009). Effects of Fatty acid composition

Yufera M.; Pascual E & Fernandez-Diaz C. (1999). A highly efficient microencapsulated food

Zambonino Infante, J.L.; Cahu, C.L. & Peres, A. (1997) Partial substitution of di- and

or ingestion by accident? *Journal of Fish Biology* 39, 225–237

*tenuis*, larvae. *Environmental Biology of Fisheries* 29, 193-200.

*Conseil international pour l'Exploration de la Mer* 178, 405-408.

(*Centropomus undecimalis*). Aquaculture 287, pp 335-340

development. *Journal of Nutrition* 127, 608–614.

for rearing early larvae of marine fish. *Aquaculture* 177, 249-256.

*World Aquaculture Society* 24, 495-501.

*Marine Biology* 14, 210–216

*Science* 69, 547-554.

*Aquaculture* 119, 167-174.

45, 327–337

984.(Abstract)

200.(Abstract)

Press]

Proceedings of a US-Asia Workshop

of microparticle diets for Japanese flounder *Paralichthys olivaceus* larvae. *Fisheries* 

H., (1998). Development of micro-particle diet for marine fish larvae 1st International Symposium on Nutrition and Feeding of Fish, Abstract only 193p. Tamaru C.S.; Murashige R & Lee C.S. (1994). The paradox of using background

phytoplankton during the larval culture of striped mullet, *Mugil cephalus* L.

*cephalus*) by manipulating quantity and quality of the rotifer. *Brachiounus plicatillis*.

and protandric sex reversal in common snook, *Centropomus undecimalis*, from the east and west coast of South Florida. *Fisheries Bulletin* 98, 612-624.(Abstract). Toledo, J.D.; Golez, S.N.; Doi, M. & Ohno, A. (1997). Food selection of early grouper,

*Epinephelus coioides*, larvae reared by the semi-intensive method. *Suisan Zoshoku* 

(*Centropomus undecimalis*). *Canadian Journal of Fishery and Aquatic Science.* 53, 974-

and captive common snook, *Centropomus undecimalis*. *Florida Scientist* 49, 196-

nanoflagellate *Tetraselmis suecica* on the growth and survival of grunion, *Leuresthes* 

*Origin and Evolution of Larval Forms* , pp. 109-158. San Diego, CA, USA: Academic

consumption in larval herring (*Clupea harengus L*.). *Rapports et Proces-verbaux du* 

and spawning season patterns on egg quality and larval survival in common snook

tripeptides for native proteins in sea bass diet improves *Dicentrarchus labrax* larval

#### **1.1 Ecological importance**

The crayfish are a group of crustaceans that habit in different environments in the world, both in lotic systems as lentic, in addition to caverns, which makes them cosmopolitan organisms with a wide range of tolerance to environmental conditions. Over 600 crayfish species are known to exist in the worldwide, with at least 100 species in Australia and about 300 in the Americas (Holdich, 1993), mostly (85%) in North and Central America (Rojas, 1998). Species in Mexico include one in the *Orconectes* genus, 10 in the *Cambarellus* genus and 44 in the *Procambarus* genus, the latter also distributed in Belize, Honduras and the United States (Villalobos, 1948; Hobbs, 1984; Rojas, 1998; López, 2006). The genus *Procambarus* habits in temporary water bodies, during the dry season can be seen in small holes in the soil, similar to the anteaters, which conduct to tunnels and chambers with sufficient moisture for the crayfish to survive to the drying (López, 2008). The crayfish have been adapted in various ways, according to environmental conditions that occur in the places they want to colonize. The first adaptation is their ability to spend a lot of time, even months, faced with the lack of water and breathe atmospheric oxygen (Huner, 1995) in some cavemen environments it has been recorded that these organisms exhibit a diminution in their effective breathing rate as a response to the decrease in the concentration of oxygen, and undersupply of food (Mejía, 2010). Despite their abundance, less than a dozen crayfish species are cultivated worldwide and only two species constitute sizable commercial fisheries (Huner, 1994). Crayfish have a high potential for use in aquaculture systems because they are at the bottom of the trophic chain, feeding largely on carrion and detritus, for that they are therefore considered fundamental for maintaining ecological balance in natural ecosystems (Rojas, 1998), and is possible their maintained in control conditions. The global diversity of crayfish allows to establish a productive activity associated with diverse environments, even in reduced environments with eutrophication, sulfate-reducing bacteria and that there is a proliferation of algae (Sánchez et al., 2009).

### **1.2 Aquaculture importance**

Crayfish like other decapods crustaceans, have some biological characteristics that make them potentially important species for aquaculture, among which include: adaptation to conditions of captivity and handling; accept artificial feeds of different origins (shrimp, fish aquatic plants, vegetables), can even be fed diets with vegetable protein (75%); have a relatively short life cycle (two years or less). First studies indicates that these organisms can breed in captivity at early age (about four months), and first spawning had high survival rates (> 75%), with reproduction all year, and some females had more than one spawning per year.

The physiological characteristics of crayfish allow them to adapt to extreme climatic variations, diversifying their potential habitats, ensuring reproduction and contributing to progeny survival under adverse conditions. This occurs under natural or artificial conditions, making them promising organisms for use in aquaculture systems (Gutiérrez-Yurrita, 1994; Rodríguez-Almaraz & Mendoza-Alfaro, 1999). Of the crayfish species which can be cultivated in subtropical environments, *Procambarus acanthophorus* stands out for its biological attributes, such as high number of progeny per spawn, resistance to a wide range of environmental and water quality conditions, and successful performance in captivity (Arrignon, 1985; Cervantes, 2008; Cervantes-Santiago et al., 2010a).

#### **1.3 Advances in laboratory research for aquaculture facilities**

#### **1.3.1 Environmental requirements of the species**

Advances in laboratory research indicate that crayfish *P. acanthophorus* has a high potential for being used in aquaculture, for that reason different trials were done to determinate the best biotechnology for semi intensive and intensive culture conditions in monoculture and polyculture facilities.

Studies under laboratory conditions had showed that crayfish *P. acanthophorus* (Villalobos, 1948; 1993), can be quickly adapted to conditions of captivity, despite coming from natural environments. In the study the organisms were maintained at an average temperature of 26 ± 20C and photoperiods between 12 and 14 h light: dark, oxygen concentrations from 0.5 to 5 mgL-1, indicating that they may like other crustaceans endure low contents O2. The pH tolerance of the species ranges from 6 to 9, the ammonium concentration is about 0.5 mlL-1, and hardness greater than 200 mlL-1 as calcium carbonates.

#### **1.3.2 Feeding**

Even though it is known that crayfish accept balanced food for aquaculture species as shrimp. Laboratory experiment with 20 formulated diets containing different protein (200, 250, 300, 350 and 400 gkg-1) and lipid (60, 80, 100 and 120 gkg-1) levels (Table 1) on growth and survival in juvenile crayfish (*P. acanthophorus*) during 12-week nutritional trial, indicate


Table 1. Formulation and proximate composition of experimental diets containing different protein/lipid ratios fed *P. acanthophorus*.

natural ecosystems (Rojas, 1998), and is possible their maintained in control conditions. The global diversity of crayfish allows to establish a productive activity associated with diverse environments, even in reduced environments with eutrophication, sulfate-reducing bacteria

Crayfish like other decapods crustaceans, have some biological characteristics that make them potentially important species for aquaculture, among which include: adaptation to conditions of captivity and handling; accept artificial feeds of different origins (shrimp, fish aquatic plants, vegetables), can even be fed diets with vegetable protein (75%); have a relatively short life cycle (two years or less). First studies indicates that these organisms can breed in captivity at early age (about four months), and first spawning had high survival rates (> 75%), with reproduction all year, and some females had more than one spawning

The physiological characteristics of crayfish allow them to adapt to extreme climatic variations, diversifying their potential habitats, ensuring reproduction and contributing to progeny survival under adverse conditions. This occurs under natural or artificial conditions, making them promising organisms for use in aquaculture systems (Gutiérrez-Yurrita, 1994; Rodríguez-Almaraz & Mendoza-Alfaro, 1999). Of the crayfish species which can be cultivated in subtropical environments, *Procambarus acanthophorus* stands out for its biological attributes, such as high number of progeny per spawn, resistance to a wide range of environmental and water quality conditions, and successful performance in captivity

Advances in laboratory research indicate that crayfish *P. acanthophorus* has a high potential for being used in aquaculture, for that reason different trials were done to determinate the best biotechnology for semi intensive and intensive culture conditions in monoculture and

Studies under laboratory conditions had showed that crayfish *P. acanthophorus* (Villalobos, 1948; 1993), can be quickly adapted to conditions of captivity, despite coming from natural environments. In the study the organisms were maintained at an average temperature of 26 ± 20C and photoperiods between 12 and 14 h light: dark, oxygen concentrations from 0.5 to 5 mgL-1, indicating that they may like other crustaceans endure low contents O2. The pH tolerance of the species ranges from 6 to 9, the ammonium concentration is about 0.5 mlL-1,

Even though it is known that crayfish accept balanced food for aquaculture species as shrimp. Laboratory experiment with 20 formulated diets containing different protein (200, 250, 300, 350 and 400 gkg-1) and lipid (60, 80, 100 and 120 gkg-1) levels (Table 1) on growth and survival in juvenile crayfish (*P. acanthophorus*) during 12-week nutritional trial, indicate

and that there is a proliferation of algae (Sánchez et al., 2009).

(Arrignon, 1985; Cervantes, 2008; Cervantes-Santiago et al., 2010a).

**1.3 Advances in laboratory research for aquaculture facilities** 

**1.3.1 Environmental requirements of the species** 

and hardness greater than 200 mlL-1 as calcium carbonates.

**1.2 Aquaculture importance** 

per year.

polyculture facilities.

**1.3.2 Feeding** 

that the protein requirement for young *P. acanthophorus* is in a range between 210 and 280 gkg-1, without observing a specific requirement of lipids, the results also suggest that in culture, it is possible to use foods with a maximum of 279 gkg-1 of protein and 60 gkg-1 lipid for better growth in crayfish, which can use up to 75% protein of vegetal source and only 25% from animal source, with growth performance and uptake efficient (Cervantes, 2006 ) (Table 1).

The study results indicate that nutritional diets can be used with protein content between 211 and 232 gkg-1 to feed growing crayfish in order to minimize feed costs, indicating that these organisms consume protein from vegetal source and assimilated efficiently (1.09:1 FCR) regardless of sex. This was verified by the assessment of carcass composition of crayfish fed with 20 experimental diets and by sex, where females were found to store more lipids (%) and body caloric energy (MJ/100 g) without significant differences in relation with the values reported for males. Other important information obtained during the nutritional study was the detection of ovigerous females in treatments 200/120, 250/60 and 400/120, but could not be attributed to the generation of egg protein or lipids tested, which suggests that in general all the diets allowed the bodies to cover their energy requirement for basic functions, but also could reproduce and promote sexual maturation.

The crayfish survival ranged from 66% to 86%, without differences between treatments. Because of the lowest survival recorded in the juvenile fed with the 250/12 gkg-1 diet, although this treatment yielded the most efficient parameters (WG, SGR and DWG), a correlation analysis was performed between weight gain and survival to determine a possible influence of mortality on growth. The weight gain survival ratio was not significant (r2 > 0.037; P > 0.1365), indicating that crayfish growth was only affected for the experimental diets.

#### **1.3.3 Life cycle and reproduction**

A vital aspect to consider when determining an organism culture potential, is its reproductive capacity under controlled conditions, which in turn depends on its ability to adapt to the culture system, feed and water quality. Factors reported to significantly affect crayfish reproductive capacity include water temperature, photoperiod, and sex ratio (Yeh & Rouse, 1995; Carmona-Osalde et al., 2002; 2004a, 2004b). The results in laboratory conditions demonstrate that the crayfish *P. acanthophorus* is a candidate for aquaculture production in a closed cycle since it effectively reproduces in captivity. During the time that organisms captured from the wild remained in captivity, it was observed mating and reproduction, from which ovigerous females were obtained, indicating that breeding in captivity could be obtained. Under the study conditions, P*. acanthophorus* exhibited the peak of the reproductive activity during November and December, when average water temperature was 25 °C. The lowest reproductive activity occurred in February and March, when wide variations in water temperature (25 ± 5°C) may have affected organism metabolism and consequently their reproductive cycle (Figure 1).

In a similar researches, Rodríguez-Serna et al. (2000) reported that reproduction in *P. llamasi* occurs year around, although, in contrast to *P. acanthophorus*, this species has three spawning peaks between November and June. Its maximum activity is in May and June and its minimum in August and October, when temperatures above 26.7 °C negatively affect its reproductive efficiency. Temperature is clearly a limiting factor for reproduction in

that the protein requirement for young *P. acanthophorus* is in a range between 210 and 280 gkg-1, without observing a specific requirement of lipids, the results also suggest that in culture, it is possible to use foods with a maximum of 279 gkg-1 of protein and 60 gkg-1 lipid for better growth in crayfish, which can use up to 75% protein of vegetal source and only 25% from animal source, with growth performance and uptake efficient (Cervantes, 2006 )

The study results indicate that nutritional diets can be used with protein content between 211 and 232 gkg-1 to feed growing crayfish in order to minimize feed costs, indicating that these organisms consume protein from vegetal source and assimilated efficiently (1.09:1 FCR) regardless of sex. This was verified by the assessment of carcass composition of crayfish fed with 20 experimental diets and by sex, where females were found to store more lipids (%) and body caloric energy (MJ/100 g) without significant differences in relation with the values reported for males. Other important information obtained during the nutritional study was the detection of ovigerous females in treatments 200/120, 250/60 and 400/120, but could not be attributed to the generation of egg protein or lipids tested, which suggests that in general all the diets allowed the bodies to cover their energy requirement

The crayfish survival ranged from 66% to 86%, without differences between treatments. Because of the lowest survival recorded in the juvenile fed with the 250/12 gkg-1 diet, although this treatment yielded the most efficient parameters (WG, SGR and DWG), a correlation analysis was performed between weight gain and survival to determine a possible influence of mortality on growth. The weight gain survival ratio was not significant (r2 > 0.037; P > 0.1365), indicating that crayfish growth was only affected for the

A vital aspect to consider when determining an organism culture potential, is its reproductive capacity under controlled conditions, which in turn depends on its ability to adapt to the culture system, feed and water quality. Factors reported to significantly affect crayfish reproductive capacity include water temperature, photoperiod, and sex ratio (Yeh & Rouse, 1995; Carmona-Osalde et al., 2002; 2004a, 2004b). The results in laboratory conditions demonstrate that the crayfish *P. acanthophorus* is a candidate for aquaculture production in a closed cycle since it effectively reproduces in captivity. During the time that organisms captured from the wild remained in captivity, it was observed mating and reproduction, from which ovigerous females were obtained, indicating that breeding in captivity could be obtained. Under the study conditions, P*. acanthophorus* exhibited the peak of the reproductive activity during November and December, when average water temperature was 25 °C. The lowest reproductive activity occurred in February and March, when wide variations in water temperature (25 ± 5°C) may have affected organism

In a similar researches, Rodríguez-Serna et al. (2000) reported that reproduction in *P. llamasi* occurs year around, although, in contrast to *P. acanthophorus*, this species has three spawning peaks between November and June. Its maximum activity is in May and June and its minimum in August and October, when temperatures above 26.7 °C negatively affect its reproductive efficiency. Temperature is clearly a limiting factor for reproduction in

for basic functions, but also could reproduce and promote sexual maturation.

metabolism and consequently their reproductive cycle (Figure 1).

(Table 1).

experimental diets.

**1.3.3 Life cycle and reproduction** 

Fig. 1. Proportion of ovigerous females versus water temperature (°C) during a ten-month period.

*P. llamasi*, with optimum spawning at 21 °C, although breeder length and degree of female sexual maturity can also affect spawning (Carmona-Osalde et al., 2004a).

In the study, a total of 92 ovigerous females were recorded from the 192 placed in the reproduction tanks during the study. ANOVA showed no significant variation for the number of ovigerous females at the three sex ratios used in the treatments, with 30 ovigerous females at 1:1, 40 at 1:3 and 22 at 1:5 male: female ratios, respectively (Figure 2).

Fig. 2. Number of ovigerous females per sex ratio treatment

The results indicate that a higher quantity of gravid females can be obtained if sex ratio of 1:3 (male: female) is using during reproduction, which indicates that the ratio male: female is a variable that can affect the mass production juveniles. This is higher than the 1M: 1F ratio reported for optimum reproductive efficiency in peneid shrimp (Martínez, 1999), but lower than the 1M: 5F ratio reported for *Cherax quadricarinatus* (Yeh & Rouse, 1995). In the crayfish *Astacus astacus*, a 1M: 3F ratio is inefficient since all females do not reproduce at this ratio, for that reason a 1M: 2F ratio is recommended for best results (Taugbol & Skurdal, 1990). In *P. llamasi*, higher densities resulted in higher mating and ovigerous female rates during reproduction, meaning density significantly influenced female sexual maturation and possibly also male maturation (Carmona-Osalde, et al., 2004a).

Other interesting result is that mating was first observed when females reached an average length of 30 mm, although the first viable spawns did not occur until females reached 37 mm total length. This is therefore considered the female length at first sexual maturity for *P. acanthophorus*. This is similar to *P. llamasi* females, which exhibit initial reproductive behavior at 30 mm but have viable spawns only between 40 and 60 mm length. This indicates that crayfish mature and reproduce at an early age, an advantage for production under controlled conditions.

Aspects of reproductive biology such as fertility vary by species, ranging from as little as five eggs in *Astacus pachypus* up to 960 in *Cherax destructor* (Lee & Wickins, 1992). Egg counts for *P. acanthophorus* females in the present study ranged from 77 to 467 per female, with an average of 240.9. The ranges are similar with reported average fertility ranges for other crayfish species: 100-700 eggs in *P. clarkii* (Lee & Wickins, 1992; McClain & Romaire, 2007); 200-700 eggs in *P. llamasi* (Rodríguez-Serna et al., 2000); 300-400 eggs in *P. zonangulus* (Reynolds, 2002); and 323 eggs in *P. leniusculus* (Celada et al., 2005).

Egg count per female in crayfish depends on organism age and length. For instance, egg production in *A. leptodactylus* varies according to female length, with organisms measuring 47 to 76 mm producing between 200 and 400 eggs (average = 305.9), and 72 mm long females producing a average maximum of 588 eggs (Köksal, 1988; Mustafa et al., 2004). In the present study, total female length in *P. acanthophorus* had a linear, positive and significant (p≤0.01) relationship (r2=0.654) with egg count. This is similar to the positive relationship between cephalothorax length and egg count reported for *Austropotamobius pallipes* where females with a 25 mm minimum carapace length produced a maximum of 80 eggs (Brewis & Bowler, 1985). The number of eggs per spawn can also depend on egg size. The crab *Cambaroides japonicus* produces only 22 to 75 eggs per spawn but these are 2.13 to 2.50 mm in diameter; this is significantly larger than mean egg size in other crustaceans and may contribute to this species high survival rate (Nakata & Goshima, 2004).

Independent of egg count, not all eggs hatch. This can be caused by biotic and/or abiotic factors that lead to losses during embryo growth. Temperature was the main factor causing egg loss in the present study since at higher temperatures fungi began to grow on the egg masses, affecting water quality in the hatchery units. Rodríguez-Serna et al. (2000) reported a similar incident in *P. llamasi* which seriously affected egg survival. Water quality and stability are clearly key elements during crayfish incubation since this is apparently a phase when pathogenic microorganisms attack eggs. Maximum egg viability in the present study was 97.4% in 56 mm long females. Embryo survival varied according to female length but did not exhibit a clear pattern; it was highest (42%) in 56-60 mm females and almost absent in 66-70 mm females. This decrease may be the result of female age since egg quality, and therefore viability, generally decreases as age increases, although this cannot be emphatically stated in the present case because the breeders were collected from the wild and their ages were therefore not exactly known. Two suggestions arise from the above results for commercial production of *P. acanthophorus*. First, breeders should be between 41 and 60 mm in length to ensure the highest possible egg and viable progeny counts. Second, adequate female nutritional condition and genetic quality need to be ensured since these are expressed in progeny quality and survival, perhaps by maintaining well-fed breeder stocks and employing constant selection to improve genetic quality.

### **1.3.4 Physicochemical parameters**

Water chemical and quality parameters during the crayfish reproduction trial were within tolerance ranges for organisms of the same sex (Malone & Burden, 1988; McClain & Romaire, 2007; Cervantes-Santiago et al., 2010b): temperature, 23.8±2.2 °C; dissolved oxygen, 5.7±0.18 mg L-1; total hardness, 110 mg L-1CaCO3; pH, 8.67±0.13; ammonium, 0.18±0.10 mg L-1; N-nitrite, 0.25±0.20 mg L-1; and N-nitrate, 32.5±20.6 mg L-1.
