**1.3.5 Fertility**

222 Aquaculture

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

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

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*

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

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

and possibly also male maturation (Carmona-Osalde, et al., 2004a).

(Reynolds, 2002); and 323 eggs in *P. leniusculus* (Celada et al., 2005).

may contribute to this species high survival rate (Nakata & Goshima, 2004).

under controlled conditions.

The ratio between total female length and egg counts (r2=0.6541) was positive, linear and significant (p≤0.01), defined by the equation y= 8.4126X - 216.4313. Average ovigerous female length was 54.4 mm (max = 71 mm; min = 37 mm) and average egg count per female was 240.9 (±S.D. 93.08) (max = 467; min = 77) (Figure 3).

Fig. 3. Ratio of egg count to total female length in *Procambarus acanthophorus* during study.

#### **1.3.6 Egg viability**

Average egg viability was 29.1% (± S.D. 31.7; n=66), with a maximum of 97.4% and a minimum of zero. Overall, females between 41 and 60 mm had the highest egg viability (97.4%). Those within the 46-50 mm size had an average viability greater than 40%, while those in the 66-70 mm had the lowest (2.9%) (Figure 4).

Fig. 4. Ratio of egg viability to total female length in *Procambarus acanthophorus* during study*.* 

Water temperature probably affected egg viability since when it surpassed 30 °C, the eggs detached from the female's abdomen and were soon infected by fungi.

#### **1.3.7 Female length at first sexual maturity**

The present reproductive biology results for the crayfish *Procambarus acanthophorus* show it to be apt for use in aquaculture systems. Based on specimens grown in captivity, it is known that it reproduces year around and females are sexually mature at 37 mm length (13 weeks) (Cervantes, 2008). This allows for constant reproduction and implementation of a continuous breeder replacement program to ensure high quality and genetically variable spawns. Under the study conditions, spawn viability was variable (29.1- 97.4%), although this could probably be kept above 44% by controlling environmental parameters (e.g. keeping water temperature below 30 °C) and thus providing a regular supply of juveniles for culture. In addition, the species did not exhibit aggressiveness or territoriality during the study. These results, in conjunction with previous studies indicating these species preference for feeds containing vegetable protein sources (Cervantes et al., 2007), confirm that the crayfish *P. acanthophorus* is a candidate for culture under controlled conditions, be it as a preservation strategy for commercial purposes.

#### **1.3.8 Embryology development**

Incubation of crayfish *P. acanthophorus* eggs has a duration period of three to four weeks, depending on temperature and embryonic development inside the egg. At the time of hatching, the larvae present physical characteristics and eating behavior similar to an adult (Cervantes, 2006; 2008). A detailed description of embryonic development until juvenile stage, suggests that the development lasts 21 to 27 days on average, where it can identify nine embryonic stages, four post-embryonic and one juvenile. Also was observed that embryonic development, presents 11 color changes of the eggs, which are however asynchronous the same ovigerous mass, so it is considered that the coloring of eggs during embryonic development is not a clear indicator the stage of development.

Under the laboratory conditions, fourteen embryonic development stages were identified for *P. acanthophorus* using the structure descriptions and nomenclature of Anderson (1982). The females produced fertile eggs which exhibited nine embryonic stages, with an average total elapsed time of 15±3 days. After embryonic stages, crayfish had four postembryonic stages and a final juvenile stage, which lasted an average of 10 days. Total elapsed time of development from fertilization through juvenile stage was on average 25 ± 3 days. Egg diameter and later embryo and juvenile length increased constantly until reaching a final length 600 times larger than initial diameter of a recently fertilized egg) (Figures 5 to 18).

224 Aquaculture

(97.4%). Those within the 46-50 mm size had an average viability greater than 40%, while

41 - 45 46 - 50 51 - 55 56 - 60 61 - 65 66 - 70 **Total female length (mm)**

Fig. 4. Ratio of egg viability to total female length in *Procambarus acanthophorus* during study*.* 

Water temperature probably affected egg viability since when it surpassed 30 °C, the eggs

The present reproductive biology results for the crayfish *Procambarus acanthophorus* show it to be apt for use in aquaculture systems. Based on specimens grown in captivity, it is known that it reproduces year around and females are sexually mature at 37 mm length (13 weeks) (Cervantes, 2008). This allows for constant reproduction and implementation of a continuous breeder replacement program to ensure high quality and genetically variable spawns. Under the study conditions, spawn viability was variable (29.1- 97.4%), although this could probably be kept above 44% by controlling environmental parameters (e.g. keeping water temperature below 30 °C) and thus providing a regular supply of juveniles for culture. In addition, the species did not exhibit aggressiveness or territoriality during the study. These results, in conjunction with previous studies indicating these species preference for feeds containing vegetable protein sources (Cervantes et al., 2007), confirm that the crayfish *P. acanthophorus* is a candidate for culture under controlled conditions, be it

Incubation of crayfish *P. acanthophorus* eggs has a duration period of three to four weeks, depending on temperature and embryonic development inside the egg. At the time of

detached from the female's abdomen and were soon infected by fungi.

**1.3.7 Female length at first sexual maturity** 

as a preservation strategy for commercial purposes.

**1.3.8 Embryology development** 

y = 138.26 - 1.84X R² = 0.1132

those in the 66-70 mm had the lowest (2.9%) (Figure 4).

0

10

20

30

**Egg viability (%)**

40

50

Fig. 5. Stage 1 (days 1-2; 0-11% development). - a) The recently fertilized eggs have a spherical shape and large quantities of mucus are present evidence of recent spawning. b) Egg mass has uniform light beige color which corresponds to the vitellus. By day 2 the vitellus has divided and small drops appear, probably the beginning of scission. It is observed the egg capsule (EC) and egg stolon (ES).

Fig. 6. Stage 2 (days 3-4; 11-22%). - a) Cellular division continues and the vitellus is completely divided into small drops. A region of greater cell accumulation (CA) is observed which corresponds to the zone where the blastopore will form. b) The egg mass begins to change color (yellow-olive green).

Fig. 7. Stage 3 (days 5-6; 22-33%). - a) Cell division continues and a group of cells begins to form on the egg ventral surface, corresponding to the germinal disk (GD). Starting on day 6, the cell layer begins to expand and form a depression, corresponding to the gastrula. b) The gastrula's ventral plate sinks in to form a groove, which is how the blastopore (Bl) appears; the forward portion of the caudal papilla starts to develop. The blastopore then closes and the rear portion of the caudal papilla appears. d) The egg mass changes in color from dark beige to a translucent olive green.

Fig. 8. Stage 4 (days 7-8; 33-44%). - a) An embryo with rudimentary anterior appendages (RA) is evident. Outlines of the frontal lobules and antennules, antennae and mandible can be distinguished. b) On day 8 the heart beat (HB) and embryo contractions can be. c) The egg mass slowly changes in color from olive green to brown.

Fig. 9. Stage 5 (days 9-10; 44-55%).- a) Primordial eyes or ocular lobules (OL) appear as two elevations in front of the body, a transversal groove appears in the vitellus, crossing the middle of the egg, and a long, thin, anterior-curved caudal papilla is present. The embryo contracts more frequently and the vitellus is clearly visible. b) The egg mass has a translucent, light yellow color and the ocular spots appear as black dots.

Fig. 7. Stage 3 (days 5-6; 22-33%). - a) Cell division continues and a group of cells begins to form on the egg ventral surface, corresponding to the germinal disk (GD). Starting on day 6, the cell layer begins to expand and form a depression, corresponding to the gastrula. b) The gastrula's ventral plate sinks in to form a groove, which is how the blastopore (Bl) appears; the forward portion of the caudal papilla starts to develop. The blastopore then closes and the rear portion of the caudal papilla appears. d) The egg mass changes in color from dark

Fig. 8. Stage 4 (days 7-8; 33-44%). - a) An embryo with rudimentary anterior appendages (RA) is evident. Outlines of the frontal lobules and antennules, antennae and mandible can be distinguished. b) On day 8 the heart beat (HB) and embryo contractions can be. c) The

contracts more frequently and the vitellus is clearly visible. b) The egg mass has a

translucent, light yellow color and the ocular spots appear as black dots.

Fig. 9. Stage 5 (days 9-10; 44-55%).- a) Primordial eyes or ocular lobules (OL) appear as two elevations in front of the body, a transversal groove appears in the vitellus, crossing the middle of the egg, and a long, thin, anterior-curved caudal papilla is present. The embryo

egg mass slowly changes in color from olive green to brown.

beige to a translucent olive green.

Fig. 10. Stage 6 (days 11-12; 55-66%). - a) OL are well-defined on the anterior body, while the abdominal somites and periopods remain rudimentary, although the chelae are visible. b) The caudal papilla is folded after and covered by the periopods, almost reaching the head. c) The egg mass is heterogeneously colored, with tones varying from light brown, to olive green and khaki.

Fig. 11. Stage 7 (days 13-14; 66-77%). - a) Eyespots (Es) are visible, and deep grooves cross through the vitellus along the dorsal medial line. b) The heart (H) can be seen to beat strongly and regularly, Embryo interior is clear and more complex, the periopods are elongated and thin, and a small rostrum appears between the eyes. c) Egg mass coloring is heterogeneous, varying from olive green to bright orange.

Fig. 12. Stage 8 (days 15-17; 77-88%). - a) The embryo occupies approximately three quarters of the egg ventral surface. b) The thoracic appendages are more developed and the chelae are totally formed. The eyes are sessile and elongate. c) The egg mass has taken on a translucent bright orange color. d) The embryo and eyes are clearly visible.

Fig. 13. Stage 9 (day18; 88-99%). - a) Shortly before hatching, the embryo appears compressed inside the egg such that the appendages seem flat and overlapped; there is no space remaining in the chorion. The chelae have grown in front of the eye base, the rostrum (Ro) is visible between the eyes and a groove sagittally crosses half the embryo. b) Egg mass color is bright yellow, rudimentary appendages are visible on the translucent embryos and the eyes are clearly identifiable.

Fig. 14. Stage 10. Hatching (day 18; Development 100%). - The hatching process lasts an average of 10 to 15 min, from when the chorion breaks to when the embryo is completely free. a, b) It begins with the first fissure (Fi1) in the surface of the chorion barely visible, fissure (Fi2) of the chorion covers the folds of the eyes and maxillae; c) notable fissure (Fi3) accompanied by total rupture of the chorion (TRC). d) Breaking the chorion, the periopods are the first to get out. e) Expulsion of the embryo (EE) to the outside of the chorion. f) External appearance of hatchings, births shown asynchronism. This is considered a critical phase in embryo survival since exiting the chorion requires considerable energy expenditure.

Fig. 13. Stage 9 (day18; 88-99%). - a) Shortly before hatching, the embryo appears

the eyes are clearly identifiable.

expenditure.

compressed inside the egg such that the appendages seem flat and overlapped; there is no space remaining in the chorion. The chelae have grown in front of the eye base, the rostrum (Ro) is visible between the eyes and a groove sagittally crosses half the embryo. b) Egg mass color is bright yellow, rudimentary appendages are visible on the translucent embryos and

Fig. 14. Stage 10. Hatching (day 18; Development 100%). - The hatching process lasts an average of 10 to 15 min, from when the chorion breaks to when the embryo is completely free. a, b) It begins with the first fissure (Fi1) in the surface of the chorion barely visible, fissure (Fi2) of the chorion covers the folds of the eyes and maxillae; c) notable fissure (Fi3) accompanied by total rupture of the chorion (TRC). d) Breaking the chorion, the periopods are the first to get out. e) Expulsion of the embryo (EE) to the outside of the chorion. f) External appearance of hatchings, births shown asynchronism. This is considered a critical

phase in embryo survival since exiting the chorion requires considerable energy

Fig. 15. Post-embryonic stage I (days 18-19). - a) The cephalothorax is formed by a yellow, elongated dorsal hump containing the remaining vitellus, which supplies nutrients to the organism during the following post-embryonic stages; the rest of the body is translucent. The eyes are round and sessile (ES) and contain dark pigment on about one quarter of the overall surface. The antennae and antennules are caudally curved and have sensory villi. b) The partially developed telsons and uropods are fused together in the membranous ligaments that attach the organisms to the chorion interior. c) The hatched organisms remain attached to the mother's pleura.

Fig. 16. Post-embryonic stage II (days 20-21).- a) The eyes are pedunculate (PE) with dark pigment, the dorsal hump which corresponds to the yolk remaining (YR) that nourishes the organism is smaller than in the previous stage (almost half its original size) and the cephalothorax has almost reached it final anatomy. Red dots begin to cover the entire body, the beginning of chromatophore pigmentation. b) The telson and uropods (TUS) appear to be separate with bristles at the ends. c) The organisms remain attached to the mother's pleura.

Fig. 17. Post-embryonic stage III (days 22-23). - a) The number and size of chromatophores increases over the entire body, but peduncles eyes not have dark pigment. Yolk Reserves (YR) are still present in the vitellus but almost exhausted, no exogenous feeding activity is observed. b) Telson and uropods are larger-well defined, this last are divided in endopodites and exopodites (TEnEx) which are still immobile and short but well-defined. c) Independent locomotion does not yet occur and the organisms remain attached to the mother's periopods.

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 Parastacidae family).

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

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 & Sandeman, 1991). In stark contrast to all the above species, *Austropotamobius pallipes* (Lereboullet, 1858), a cold water species, requires over seven months to complete embryo development (Holdich & Lowery, 1988).

During the embryonic development trial, selected live eggs were kept in Petri dishes to evaluate survival. Artificially incubated fertile eggs were found to remain viable after development stage eight. This means that egg lots could be artificially incubated to reduce development period and synchronize times in mass production settings and/or if only small lots of reproductive-age females are available. The present study is the first report of embryonic development and artificial incubation of eggs in *P. acanthophorus*. It constitutes a significant contribution to the biology of this and other decapods crustaceans with potential for use in alternative, sustainable aquaculture systems.
