Broodstock management

Management of broodstock normally encompasses those practices associated with typical pond production in grow-out ponds. However, in temperate climates, broodstock must be either imported or maintained in an indoor brood-stock holding facility. Because of the length of time these prawns must be overwintered, broodstock must be properly managed to ensure their health and survival. In the future, as efforts are made to domesticate and genetically improve M. rosenbergii, holding broodstock will become more commonplace in tropical environments as well. This will make good management practices, such as broodstock manipulation, universally important.

4.2.1 Broodstock manipulation, cryopreservation and hybridisation

In recent years, research has focused on better understanding ovarian and sperm development and their management for use in hatchery production. Santos (1998) had previously demonstrated that unilateral eyestalk ablation could be used to anticipate spawns, increase the number of mature females and diminish the time between each spawn. Young females (4 months after PL stocking) spawn about 20 days after eyestalk ablation and spawn again after about 30 days. Okumura & Aida (2001) evaluated eyestalk ablation on moult interval, ovarian development and haemolymph levels of ecdysteroid and vitellogenin in M. rosenbergii females held at 28°C and under 15L:9D photoperiod. They found that all destalked females exhibited a reproductive moult accompanied by ovarian development and egg laying compared to only 69 and 51% (with or without haemolymph sampling) in the controls. Vitellogenin levels followed a pattern similar to control reproductive females. Murmu et al. (2007) evaluated the effects of eyestalk ablation and feeds on growth, moulting and rematuration ofwild caught, spent females collected during the non-monsoon season in India and held under controlled conditions for 60 days. They found that ablated females showed the highest number of moults, shortest moult interval, highest gonadoso-matic index (GSI), lowest weight gain and produced the only ovigerous females.

Other researchers have evaluated hormonal effects on female reproduction. Tanboonteck et al. (2006) assessed the effects of two dosage levels (20 or 40 |ig per gram body weight) of 5-hydroxytryptamine (5-HT, serotonin) andoc-topamine on ovarian development of female M. rosenbergii, which were injected with 1 or 3 doses after they had spawned and eggs had been physically removed. They concluded that a single injection of 20 |ig of 5-HT per gram body weight at immature ovarian stage stimulates ovarian development. Earlier spawning in mated females could be chemically induced by injection of either 5-HT or octopamine after the pre-mating moult. Spawned embryos were normal and survived to hatching. Meeratana et al. (2006) also studied the effects of 5-HT on adult M. rosenbergii females at the ovarian I stage (spent) injected intramuscularly with various doses (1,5, 10, 20 or 50 |ig per gram body weight) on days 0, 5 or 10 and sacrificed on day 15. The lower doses (1 and 5 |ig per gram body weight) caused a significant increase in ovarian index compared to the controls. The ovaries of most of these low-dose treated prawns reached stage IV and contained synchronously mature oocytes. Controls mostly developed to stages II and III. Prawns given higher doses could reach stages III and IV, with lower percentages reaching stage IV as the dosage increased, and contained various types of oocytes. Based upon pre-treatment with the 5-HT

antagonist cyproheptadine and evaluation of effects of various organs (eyestalk containing optic lobes, supraoe-sophageal ganglion, thoracic ganglia and muscle strips) on ovarian maturation, they concluded that 5-HT did not induce ovarian maturation directly but through its action on the thoracic ganglion.

Development of male characteristics in juvenile prawns is controlled by the androgenic gland (Chapter 16). Because of this, the sex of males can be reversed to create neofe-males. When bred with females, they produce all males. Aflalo et al. (2006) developed a two step procedure in an attempt to develop commercial production of all-male populations of M. rosenbergii. In step one, they andrectomised (surgically removed the androgenic gland) PL25-60. But the low success rate of functional sex reversal resulted in 100% male progeny from only 1.3% of'neofemales'. They then added the second step where they took these progeny and andrectomised them earlier (PL20-30). In the second step, there was a significant increase in the number of sex-reversed animals (ovarian development) and shortening of time to maturation. Rungsin et al. (2006) evaluated production of all-male stock using neofemale technology from the Thai strain of M. rosenbergii. They determined that gonadal differentiation did not occur until both gonopore complexes and appendix masculinae appeared in males. Bilateral removal of androgenic glands from PL having gonopore complexes only resulted in 80.4% survival. Of those surviving, 30% developed into females and 27% matured. Mating of 12 neofemales with normal males resulted in allmale offspring in 8 crosses. Fecundity of neofemales was not different from that of normal females.

Cryopreservation of Macrobrachium sp. eggs and sperm is an important step in broodstock manipulation and potential hybridisation. In preparation for cryopreservation studies, Pillai &Mohanty (2003) developed a safe and rapid enzymatic digestion technique to individually separate advanced stage M. rosenbergii eggs (grey embryos with eyespot and heart beat) using the protease enzymes trypsin and collagenase. Treatment of eggs with a solution of collagenase (0.05%) and trypsin (0.25%) for 30 minutes yielded 100% separation. Using a solution of trypsin (0.25%) or collagenase (0.1%) alone or one with trypsin (0.05%) in combination with EDTA (400 mg/L) also provided 100% separation. In vitro hatching was also successful. Chow (1982) reported successful fertilisation using spermatophores stored at 2°C for 4 days without any cryoprotective agents. Chow etal. (1985) had preserved spermatophores in liquid nitrogen for a maximum of 31 days. Akarasanon et al. (2004) evaluated long-term cryopreservation of spermatophores of M. rosenbergii and found that preservation at -196°C with 20% ethylene glycol was suitable for up to 150 days based upon high sperm survival rates and high fertilising ability. Preservation at -20°C with 10 or 20% ethylene glycol or 10

or 20% glycerol provided a simple and efficient short-term storage for up to 10 days.

Hybridisation of Macrobrachium species has been attempted but with limited success. In a preliminary study, Graziani etal. (2001) usedleucilaminopeptidase and peptidase II to detect the presence of hybrids among four species of Macrobrachium. Among these species, the most genetically distinct were M. acanthurus and M. amazonicum, whilst the closest genetic distances were between M. carcinus and M. rosenbergii. Later, Graziani et al. (2003) attempted intra- and inter-specific crosses of M. rosenbergii and M. carcinus through both natural mating and artificial insemination. They were successful in producing larvae through both natural and artificial intraspecific crosses. Interspecific reciprocal crosses were only successful in producing hybrid zygotes through artificial insemination, but embryonic development did not progress beyond the gas-trula stage. They concluded that both behavioural and postzygotic reproductive isolation barriers exist preventing their hybridisation. Similarly, Salazar etal. (2005) evaluated the possibility of hybridisation between M. rosenbergii and M. acanthurus using controlled mating and artificial insemination. They compared the efficiency of both types of reproduction using intraspecific crosses for both species and found that the ethological reproductive isolation and the lack of viability of the hybrid zygotes constitute pre-and post-zygotic barriers that should preclude the natural hybridisation of these species. Fu et al. (2004) successfully produced viable F1 hybrids from crosses of female M. nip-ponense and male M. hainanense through artificial insemination. They achieved hatching rates of over 90% with about 20 to 60% developing to PL. F1 hybrids showed co-dominant expression of paternal and maternal alleles controlling the isozymes malate dehydrogenase and esterase.

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