REPRODUCTIVE PERFORMANCE AND OFFSPRING QUALITY IN () BROODSTOCK FED DIFFERENT DIETS Faculty of Agriculture Alexandria University El-Bermawi, N. Department of and Fish Production, Faculty of Agriculture (Saba Basha), Alexandria University. Egypt

Table 1. Chemical composition of formulated artificial diets.

Ingredient A1 A2 Abstract g/kg g/kg A study was conducted to determine the effect of the nutritional value on maturation of crayfish with the aim of Fish meal 250 250 establishing standard techniques for mass production of berried females producing high quality larvae. Three tested diets were composed of a mixture of fresh food items (trash fish, meals, 30% Artemia biomass), compared with two Prawn meal 100 180 types of artificial diets {35% (A1) and 40% (A2) protein}. Five crayfish females with an average individual weight of Soybean meal 250 270 60.88g were stocked in circular fiberglass tanks (each 1.5 m3 with capacity of 1000 L). The crayfish were fed the Wheat flour 240 240 experimental diets twice a day with daily amount calculated as 20% of total body weight. Gonadal maturation, egg hatching rate and fecundity were estimated. Offspring quality was evaluated through starvation test. The results of our Rice flour 152 62 study however showed that the crayfish fed diet A2 had a significant better hatching rate (95%) compared to the control. Vitamin& Mineral mix 3 3 No significant difference of hatching rate (90.9%) was observed for the crayfish fed diet A1. Survival of larvae under starvation conditions was significantly better when they originated from broodstock fed the control diet. No significant Corn Oil 5 5 effect of the different levels of n-3 HUFA in the diets on reproductive performance of crayfish broodstock. The results of Chemical composition (%) this study suggested that the sole use of the artificial feeds tested could not improve reproductive performance. Dry matter 88.59 90 On dry matter basis (%) Crude Protein 35.9 40.1 Table 3. Survival (%) of crayfish larvae under starvation conditions. Introduction Crude fat 6.47 10.3 Typical feeding practices for maturation of broodstock shrimp still rely on a wide range of Nitrogen free extract 42.3 33.6 Type of broodstock diet unprocessed or frozen marine , including squid, various mollusks (mussels, clams, oysters), Crud fiber 6 8 Exposure (h) fish roe, marine polychaetes, and (shrimp, , enriched Artemia biomass. Recently Control A1 A2 however, crustaceans have been excluded from shrimp maturation regimes due to the risk of disease Ash 9.3 8 0 100 100 100 transmission. Furthermore, no information was found concerning a reference fresh-food feeding regime which could serve as a control diet for shrimp broodstock nutrition studies. Nevertheless, 12 100 100 100 almost every attempt to completely replace fresh food with artificial diets, results in a decrease in 24 100 100 100 ovarian maturation, a reduced number of spawns and an inferior egg quality. In most situations, a 36 98.0±1.3 95.0±5.0 93.7±1.8 combination of fresh food and artificial diets gives better results than feeding regime that consist of fresh food only (Bray et al., 1990a,b; Galgani et al, 1989 ; Nascimento et al., 1991). Fresh food 48 91.0±2.8a 87.0±6.0b 87.6±1.8ab replacement levels are often higher than those applied in commercial facilities, which has had its 60 77.3±5.8 68.5±10 68.5±3.4 consequences for spawner survival, spawn frequency, fecundity, egg fertilization rate, hatch rate Results and discussion 72 66.2±6.4a 47.0±2.8b 46.7±2.6b and/or larval quality. Many authors have successfully used dry artificial broodstock diets at 50% of Diet plays an important role in crayfish broodstock condition (holdich, 2002). Broodstock nutrition is a b b the total feeding regime, even when bloodworm and Artemia biomass were replaced partially important for reproductive success because egg and larval production are strongly dependent on the 84 53.4±5.4 20.3±2.3 18.4±3.6 (Coutteau et al., 1998) or completely (Wouters et al., 2000a) in commercial-scale trials. diets offered (Bromage, 1995; Harrison, 1997; Garcia-Ulloa, 2000). The results presented in Table 2 96 47.2±5.0 0 0 Crustaceans like shrimp, crab and are also fed to shrimp spawners, but due to the risk of disease showed that the crayfish fed diet A2 had a significantly better hatching rate (95.3±1.7) compared to 108 34.5±8.1 0 0 transmission, they are used less frequently nowadays. Bloodworms (marine polychaetes Glycera other diets (control 88.3±2.2 and A1 diets 91.9±3.8). At the same time, no significant differences of dibranchiata and Americonuphis reseii) and Artemia biomass (ongrown Artemia) are used for diet. hatching rate was observed for the crayfish fed control and diet A1. Larvae survival % under 120 28.5±7.3 0 0 Supplementation Artemia biomass usually boosted with specific nutrients. It has been reported to starvation treatment was however significantly better when they originated from broodstock fed the stimulate ovarian maturation, increase spawn frequency and improve larval quality (Browdy et al., control diet (91.0±2.8%) compared to those diet A1 (87.0±6.0%) and diet A2 (87.6±1.8%). Diet 1989; Naessens et al., 1997; Wouters et al., 1999a). Artemia biomass can also be included into formulation is the most important factor for broodstock maturation. Artificial presented diets A1 and artificial broodstock diets as a freeze-dried meal to increase diet ingestion and stimulate ovarian A2 offer after all many advantage compared to fresh feed, including a reliable supply, minimal m a t u r a t i o n ( W o u t e r s et al. , 2000a). preparation time and known nutrient content. Moreover, they offer the opportunity to orally Table 4. Biochemical composition of crayfish tested diets and Larvae. administer drugs such as hormones or supplementary vitamins (Marsden et al., 1997). Millamena and Redclaw crayfish is presented and cultured in various tropical and subtropical zones (Ackerfors, Quinito (2000) reported that mud crab reproductive performance has been improved as a result of 1994). It breeds readily in captivity and develop directly from egg into juvenile phase without any using combination of natural. However, it has been shown that treatments based on a single fresh larval stages, which greatly facilitates their production. The main constrains to intensify astacid food are not optimal (Chamberlain, 1988; Bray et al., 1990a, b), for which the clam diet might not be crayfish culture are the unpredictable and usually poor survival and growth rates of stage 2 juveniles Type of broodstock diet Crayfish larvae considered an adequate control for comparison with artificial diets. The result of this study Nutrient during the first months of independent life (Taugbol and Skurdal, 1992; Celada et al., 1993; Saez- suggested that the sole use of the artificial feeds tested here could not improve crayfish reproductive control A1 A2 control A1 A2 Royuela et al., 1995). (Karplus et al, 2003) In intensive culture systems, including hatcheries and performance. A study by Mendoza et al. (1997) provides supportive evidence to the notion that the so- Protein (%) 61.7 35.9 40.1 39.3 31.1 33.1 nurseries, the animals are totally depending on supplemental food. Three factors should be taken into called squid factor might be a hormonally active substance. To identify the effective components in 20:5n-3 (EPA, mg.g) 1.6 5.5 4.6 3.9 5.0 4.1 considerations: food quality, food amount, and its availability to the cary-fish. Preferred food squid that were able to trigger secondary vitellogenesis, squid meal was separated into different 22:6n-3 (DHA, mg.g) 5.1 12.2 7.9 4.0 5.1 4.2 reduced aggressive activity more than did less preferred food in O. rusticus (Capelli and Hamilton, extracts by these authors. The results show that squid, apart from being a rich cholesterol source, 1984). Therefore to improve the understanding of the important role of nutrition in maturation of contains sexual steroids that trigger shrimp vitellogenesis. Similar findings are reported for the effect Σ n-3 HUFA (mg.g) 7.0 18.9 15.1 9.0 12.0 10.1 crayfish, a series of studies was conducted with the aim of establishing standard techniques for mass of mollusc extracts on M. japonicus vitellogenesis (Kanazawa, 1989, cited in Mendoza et al., 1997). production of berried females producing high quality larvae. In the present study, the nutritional value The observation that nutrient composition cannot always explain the success of natural food of diet composed of a mixture of fresh food items was compared with type of artificial diets. organisms made many researchers believe that reproductive hormones of these organisms contribute to the shrimp enocrinological cycle. This could be true in organisms that share the same hormones as Penaeid shrimp. Laufer et al., (1997) found that bloodworms contained methyl farnesoate, which is an ecdysone hormone that increase fecundity and hatch rate in cultured L. vannamei and enhanced ovarian development in other crustaceans (Laufer et al., 1998). Conclusion Lipid requirement is very important factor during broodstock maturation. It has been reported that It has been proven that the different protein levels in the broodstock diets resulted in a dietary highly unsaturated fatty acids (HUFA) and the n-3/n-6 ratio could improve the reproductive different protein level of larvae produced (Table 4) and this could apparently affect larval performance and hatchability of eggs in tiger shrimp (Millamena, 1989; Nguyenet al., 2009). survival under starvation condition. Materials and Methods Important differences do also exist among fresh food items. It is as such not easy to decide which The experiment was conducted in fiberglass tanks of 1.5 m3 using three PVC tubes (30cm). The tanks fatty acid profile would provide the best comparative tool (Roeland et al., 2001). Our study reveled were filled with sea water and tap water to depth of 50 cm and aerated. Salinity was maintained at 15±2 pot that, different levels of n-3HUFA in the diets showed no significant effect on the reproductive and temperature at 28±2 ºC. The water in each tank was replaced 100% daily with fresh mix water. performance (Table 4). Despite the essential fatty acids (EPA and DHA value in fresh food (control diet 1.6 and 5.1 mg.g) being lower than of artificial diets (A1 5.5, 12.2 and A2 4.6, 7.9 mg.g) broodstock Before test initiation, thirty crayfish females with an average individual wet weight of 60, 88 ±2 were fed fresh food produced stronger larvae (as determined by the starvation test). This might be due to acclimated to the laboratory condition and reared together in one experimental tank. They were fed with a the fact that the fresh food contained higher levels of other essential nutrients (e.g., protein level – mixture of fresh food (trash fish, shrimp meals, 30% Artemia biomass) until they spawned. After spawning, 61.7% for the control versus A1 35.9 and A2 40.1% diets), which apparently played a more crucial role References the crayfish were weighted. The spent spawners were randomly divided over six tanks and subjected to the during starvation. On the other hand because of using Artemia biomass it is usually boosted with Marsden, G.E., J.J. Guren, S.W. Hansford, and M.J. Burke. 1997. A moist artificial diet for dietary treatments. Fresh food mixture was used as the control treatment. The control diet was compared to specific nutrients improve larval quality (Browdy et al., 1989; Naessens et al., 1997; Wouters et al., prawn broodstock: its effect on the variable reproductive performance of wild caught two types of artificial diets, diet A1 and A2. Crude protein levels in diets were 35 and 40% (Table 1). The 2000; Royuela et al., 2007). Protein is the most critical ingredient in practical diets because it is . Aquaculture 149: 145-156. ingredients were ground and passed through a 0.5mm mesh sieve and then mixed thoroughly. Water was expensive and growth responses are affected (Cortes-Jacinto et al., 2003; Thompson et al., 2005; added to make stiff-like dough that was passed through a meat grinder to palletize the mixture. The resulting McClain and Romaire 2009) According to Harrison (1997), protein required in broodstock diets for Millamena O.M. 1989. Effect of fatty acid composition of broodstock diet on tissue fatty 2-mm pellets were dried in a forced-air oven at 40ºC for 8 h. Pellets were then packed in plastic bags and maturation and production of eggs is higher than the level required for grow out because gonad acid patterns and egg fertilization and hatching in pond reared Penaeus monodon refrigerated at 5ºC until used. Proximate composition of diets was determined according to AOAC (1995). maturation is a process of intense protein synthesis, mainly during vitellogenesis (Abdu et al., 2000). Fabricius broodstock . Asian Fish Sci. 22:127-134. Diets crude protein was determined using micro-kjeldahl method (APHA, 1989) and crud lipids were extracted with anhydrous ether in soxhlet extraction system (SoxtecAvanti 2050, Foss Tecator®, Copenhagu, Denmark) Millamena O.M. and e. Quinitio. 2000. The effects of diets on reproductive performance of following Bligh and Dyer (1959). Ash was determined by using a muffled furnace (AOAC, 1995), and crud eyestalk ablated and intact mud crab . Aquaculture 181:81-90. Table 2.Reproductive performance of crayfish broodstock fed various diets. fiber by the phenol-sulfuric acid method (Myklestad and Haug, 1972). The crayfish were fed the experimental Sokal R.R. and F.J. Rohlf. 1995. Biometry. The principles and practice of statistics in diets twice a day with the daily amount calculated as 20 % of total body weight. Survival and reproductive biological research. W.H. Freeman and company , San Francisco. 887p. output were observed daily and every three days respectively. Females that spawned were transferred to Type of food 1m3 aquaria for egg mass incubation and hatching. Offspring quality was evaluated through starvation test for Bray, W.A., Lawrence, A.L., Leung-Trujillo, J.R., 1990a. Reproductive performance of 120h . S u r v i v a l r a t e w a s e v a l u a t e d e v e r y 12h . Parameter ablated Penaeus stylirostris fed a soy lecithin supplement. J. World aquaculture Soc. 20: Control A1 A2 19A. Statistical analysis was performed through analysis of variances (Sokal and Rahlf, 1995). Bray, W.A., Lawrence, A.L. Lester, L.J., 1990b. Reproductive of eyestalk-ablated Penaeus Maturity (% of total females) 100 100 100 stylirostris fed various of total dietary lipid .J. World Aquaculture Soc. 21: 41-52. Galgani, M.L., Cuzon, G., Galgani, F., Goguenheim, J., 1989. Influnce du regime Spawning(% of total females) 100 100 100 alimentaire sur la reproduction en captivite de Penaeus indicus. Aquaculture 81:337-350. Nascmento, I.A., Leung-Trujillo, J.R., Lawernce, A.L., 1991. reproductive of ablated and Incubation time 10.0±0.8 8.5±0.9 9.0±1.0 unablated Penaeus schmitti in captivity using diets consisting of fresh-frozen natural and dried formulated feed. Aquaculture 99: 387-398.

Fertilization rate (%) 81.1±11.1. 82.1±11.4 83.7±12.9 Coutteau, P. Pinon, E., Balcazar, Y., 1998. Effect of a commercial maturation diet on broodstock performance of Penaeus vannami in a commercial hatchery. 1st Latin American Shrimp Culture Congress and Exhibition, Panama City. Abstract. b ab a Hatching rate (%) 88.3±2.2 91.9±3.8 95.3±1.7 Wouters, R., Zambrano, B., Mendoza, S., Espin, M., Lavens, P., 2000. Ensayos con una dieta artificial de maduracion basada enharina de Artemia Mundo Acuicola 6 (1); 17-20. Photoactive larvae (%of total) 98.5±0.3 97.8±3.0 97.5±1.5