Effect of polyunsaturated fatty acids on the fecundity of the Amazon river prawn Macrobrachium amazonicum (Heller, 1862)
Abstract
Effects of the ratio of dietary fatty acids, namely n-3 (mainly long chain polyunsaturated – LC-PUFA) to n-6 PUFA on the fecundity of Macrobrachium amazonicum were evaluated. In T1, the diet had equal and low levels of dietary n-3 and n-6 fatty acids (6 mg g−1). In T2, the concentration of n-3 (6 mg g−1) was a half of the concentration of the n-6 (12 mg g−1), and in T3, the diet had equal and high concentrations of n-3 and n-6 (12 mg g−1). Females with ovaries in stages I, III and V were collected. Higher gonadosomatic index (GSI) (6.89%) was observed in females in ovarian stage V than at other ovarian stages; however, the hepatosomatic index (HIS) showed high values in all females with ovaries in the stage III. A ratio of 1:2 n-3:n-6 fatty acids increased the GSI of mature females and the number of eggs spawned. Raising the level of both n-3 and n-6 fatty acids from ~0.6% to ~1.2% of the diet did not produce any effect on the GSI or on fecundity, suggesting that the ratio is more important than the absolute value of these two families of fatty acids.
Introduction
The Amazon River prawn, Macrobrachium amazonicum, is a native species of South America, which has been commercially exploited by artisanal fisheries (Odinetz-Collart 1987; New 2005). It is well accepted by consumers of all social classes (Moraes-Riodades & Valenti 2001) and presents great potential for aquaculture. However, the lack of specific technology for farming this species deters its culture on a commercial scale. During the current decade, a considerable research focus on the aquaculture of this species has been carried out. Preliminary results have showed that M. amazonicum is robust, polytrophic, disease-resistant and may be raised in intensive systems in all culture phases (Moraes-Valenti & Valenti 2010). However, low fecundity is a major constraint to culturing this species. The number of larvae hatched varies widely from 21 to 2600 per female and increases with female size (Guest 1979; Lobão, Rojas & Valenti 1986; Da Silva, Sampaio & Santos 2004). Therefore, hatcheries need to maintain a large broodstock to provide the quantity of larvae to stock rearing tanks, which increases hatchery production costs.
The dietary regime impacts reproductive performance in crustaceans (Harrison 1990). Das, Saad, Ang, Law and Harmin (1996), Cavalli, Lavens and Sorgeloos (1999) and Venkataramani, Rajagopalsamy and Ravi (2002) have demonstrated that maternal diets affect the reproduction of freshwater prawns. However, information about specific nutritional requirements for prawn broodstock is limited (D'Abramo & New 2010) even though it could affect egg and larval quality. There are few papers on reproductive female nutrition (de Caluwé, Lavens & Sorgeloos 1995; Das et al. 1996; Cavalli et al. 1999; Cavalli, Menchaert, Lavens & Sorgeloos 2000; Cavalli, Lavens & Sorgeloos 2001a; Cavalli, Montakan, Lavens & Sorgeloos 2001b, Cavalli, Tamtin, Lavens, Sorgeloos, Nelis & Leenheer 2001c, Venkataramani et al. 2002; Cavalli, Batista, Lavens, Sorgeloos, Nelis & Leenheer 2003; Murmu, Sahu, Mallik, Reddy & Kohli 2007) and all of them focus on an Asiatic species, the giant river prawn Macrobrachium rosenbergii. Therefore, studies on diets for freshwater prawn broodstock are necessary.
Lipids play a major role in many reproductive processes of crustaceans, and studies have shown that the levels and composition of dietary lipids greatly affect ovarian maturation and reproductive performance. (Harrison 1990, 1997). However, the dietary lipid level can be low if it provides sufficient levels of essential fatty acids; using a 2:1 cod liver oil/corn oil semi-purified diet. Sheen and D'Abramo (1991) found weight gain in juvenile M. rosenbergii to be highest at 6%. Among them, the polyunsaturated fatty acids (PUFA) are particularly important. The content of PUFA in artificial and natural diets impacts survival, growth, feed conversion, fecundity, egg hatchability, molting and osmotic stress tolerance in crustaceans (Millamena, Bombeo, Jumalon & Simpson 1988; D'Abramo & Sheen 1993; Breet & Müller-Navarra 1997; Cavalli et al. 1999). An increase of n-3 and n-6 PUFA in broodstock diets enhanced fecundity and egg hatchability in the freshwater prawn M. rosenbergii (Cavalli et al. 1999). However, D'Abramo (1997) stated that the ratio of n-3 and n-6 fatty acids may be more important than their absolute value to crustacean nutrition.
The effect of dietary PUFA in M. amazonicum is totally unknown. Considering the above rationale, the present study was designed to evaluate the effects of the ratio of dietary n-3 and n-6 PUFA on the fecundity of M. amazonicum.
Materials and methods
Experimental diets
Three diets, formulated to be isonitrogenous and isolipidic and to differ only by the levels of n-3 (mainly long chain PUFA – LC-PUFA) and n-6 PUFA hereafter referred to in our article simply as n-3 and n-6 diets, were tested (Table 1). As there is no standard reference diet for M. amazonicum, the general diet composition was adapted from the several diet formulae presented by New (2002) for feeding M. rosenbergii. The diets were formulated using practical ingredients, with the exception of the premix, which was a proprietary brand. Variation in fatty acid content was obtained by changes in the amounts of sunflower oil, fish oil and hydrogenated plant oil. The hydrogenated plant oil used was a mixture of vegetable oils that had been hydrogenated. In treatment T1, the diet showed equal and low levels of n-3 and n-6 (6 mg g−1 of diet = 0.6%) fatty acids. In treatment T2, the concentration of n-3 fatty acids (6 mg g−1 of diet = 0.6%) was a half of the concentration of n-6 fatty acids (12 mg g−1 of diet = 1.2%), and for treatment T3, the diet had equal and high concentrations of n-3 and n-6 fatty acids (12 mg g−1 of diet = 1.2%). These treatments were chosen based on the fatty acid profile of M. rosenbergii (Tidwell, Webster, Coyle, Daniels & D'Abramo 1998; Cavalli et al. 2001b), the unique species of the genus, for which such information is available.
| Diet T1 | Diet T2 | Diet T3 | |
|---|---|---|---|
| Ingredients | |||
| Corn graina | 10.00 | 10.00 | 10.00 |
| Wheat meala | 16.00 | 16.00 | 16.00 |
| Rice meala | 4.35 | 4.35 | 4.35 |
| Soybean meala | 38.00 | 38.00 | 38.00 |
| Fish mealb | 25.50 | 25.50 | 25.50 |
| Sugar cane (attractant)c | 0.40 | 0.40 | 0.40 |
| Sunflower oila | – | 3.50 | 2.00 |
| Fish oilb | 1.00 | 1.50 | 3.00 |
| Hydrogenated plant oil mixturea | 4.00 | – | – |
| Vitamin and mineral supplementd | 0.75 | 0.75 | 0.75 |
| Calculated proximate composition (%) | |||
| Moisture | 9.8 | 9.8 | 9.8 |
| Crude protein | 32.2 | 32.2 | 32.2 |
| Total lipid | 7.9 | 7.9 | 7.9 |
| Nitrogen-free extract (NFE) | 35.1 | 35.1 | 35.1 |
| Crude fibre | 4.0 | 4.0 | 4.0 |
| Ash | 11.0 | 11.0 | 11.0 |
| Gross energy (kcal/kg) | 3664.1 | 3664.5 | 3664.5 |
- a Commercial Brazilian feedstuff ingredients
- b Brazilian herring meal and oil.
- c By-product from the Brazilian alcohol and sugar industry.
- d Agromix AC 50—vitamin and mineral supplement, containing (per kilogram mix): vitamin A = 176 000 I.U.; vitamin D3 = 40 000 I.U.; vitamin E = 500 mg; vitamin K3 = 36 mg; vitamin B12 = 560 mg; niacin = 700 mg; biotin = 3 mg; pantothenic acid = 500 mg; folic acid = 30 mg; choline = 20 mg; iron = 1100 mg; copper = 300 mg; manganese = 1800 mg; zinc = 1200 mg; iodine = 24 mg; selenium = 3 mg; methionine = 20 mg; calcium = 176 mg; phosphorus = 68 g; sodium = 23 g; chlorine = 36 g; B.H.T. = 1 g.
Diet ingredients were individually crushed and mixed using a kitchen mixer. Then, the mixture were homogenized with 45% (mass.volume−1) water and extruded through a 5 mm diameter diet. The pellets were air dried for 24 h, packed and stored at −10°C until use. Moisture, crude protein and crude fibre levels were determined following the procedures of AOAC (Association of Official Analytical Chemists) (2005). Total lipids levels were determined using the method of Folch, Ascoli, Lees, Meath and Lebaron (1951), and modified by Bligh and Dyer (1959). Fatty acid composition (Table 2) was determined using method of Hartman and Lago (1973) and Horwitz (2005), by a Varian model 3900 gas chromatograph system FID and a CP SIL 88 column.
| Fatty acids | T1 (0.6 n3/0.6 n-6) | T2 (0.6 n-3/1.2 n-6) | T3 (1.2 n-3/1.2 n-6) |
|---|---|---|---|
| 14:0 | 0.6 ± 0.1 | 0.6 ± 0.1 | 0.8 ± 0.1 |
| 16:0 | 8.1 ± 0.2 | 7.5 ± 0.2 | 5.0 ± 0.3 |
| 16:1 | 1.0 ± 0.0 | 1.0 ± 0.1 | 1.6 ± 0.0 |
| 18:0 | 1.4 ± 0.1 | 1.0 ± 0.0 | 1.1 ± 0.1 |
| 18:1 n-9 | 4.7 ± 1.1 | 5.8 ± 1.6 | 6.1 ± 2.0 |
| 18:2 n-6 | 5.1 ± 0.6 | 10.3 ± 1.6 | 11.4 ± 2.0 |
| 18:3 n-3 | 0.2 ± 0.1 | 0.2 ± 0.1 | 0.3 ± 0.1 |
| 20:1 n-9 | 0.2 ± 0.0 | 0.3 ± 0.1 | 0.5 ± 0.0 |
| 20:4 n-6 | 1.0 ± 0.3 | 1.4 ± 0.1 | 0.8 ± 0.2 |
| 20:5 n-3 (EPA) | 3.2 ± 0.3 | 3.6 ± 0.0 | 5.3 ± 0.1 |
| 21:5 n-3 | 0.4 ± 0.2 | 0.1 ± 0.0 | 0.5 ± 0.0 |
| 22:02 | 1.3 ± 0.2 | 0.9 ± 0.3 | 1.2 ± 0.1 |
| 22:6 n-3 (DHA) | 1.9 ± 0.2 | 1.7 ± 0.2 | 4.9 ± 0.3 |
| Σ Unsaturated | 10.2 ± 1.1 | 9.1 ± 1.2 | 7.0 ± 2.0 |
| Σ Monounsaturated | 1.0 ± 0.0 | 1.0 ± 0.1 | 1.6 ± 0.0 |
| Σ(n-6) | 6.1 ± 0.9 | 11.7 ± 1.7 | 12.2 ± 2.2 |
| Σ(n-3) | 5.7 ± 0.8 | 5.6 ± 0.1 | 11 ± 0.5 |
| (n-3)/(n-6) | 0.93 | 0.47 | 0.90 |
- EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
Experimental animals and general procedures
The animals used in this study were reared in the broodstock ponds of the Crustacean Sector, Aquaculture Center at São Paulo State University, SP, Brazil. They were the second generation produced from wild broodstock collected in the State of Pará, Brazil (01°14′30″S 48°19′52″W). Seventy-two M. amazonicum females with ovaries at the final maturation phase were selected, measured and weighed. The mean total length (from tip of the rostrum to the end of telson) and mass were 80 ± 15 mm and 9.5 ± 1.8 g respectively. The animals were randomly transferred to 12 tanks (experimental units), six individuals per tank, stocked in individual chambers. Each experimental unit comprised a 1 m3 tank, divided into six chambers by hapa nets, provided with aeration and a 10 L biological filter (Shawn, Alston & Sampaio 2010). Water continuously circulated through the biological filter to maintain low levels of ammonia and nitrite. Temperature was maintained at about 29°C by heaters; photoperiod was provided by natural sunlight and was about 13L:11D (light:dark).
Females were fed treatment diets in excess (~5% body mass/d) at 17:00 hours. Every morning, faeces, molted exoskeletons and uneaten feed were siphoned out. The experimental period was 60 days, but the first 5 days were considered as an acclimation period to the beginning of a new ovarian maturation cycle. Water quality was determined by measuring pH with a Micronal B374 pH-metre (Micronal S/A, São Paulo, SP, Brazil), dissolved oxygen with a YSI Model 55 polarographic oxygen metre (Yellow Springs Instruments Company, Yellow Springs, OH, USA) and ammonia-N according to the methods of Solorzano (1969), using a Hach Model DR-2000 spectrophotometer (Hatch Company, Ames, IA, USA).
Gonadal development and molting were monitored daily. Maturation stages were classified based on ovarian size and colour as observed through the carapace according to a scale adapted from Chang and Shih (1995) to M. amazonicum. Four females from each treatment (one from each replicate), whose ovaries had reached maturation stage I were weighed, measured and killed in ice with thermal shock. The ovaries and hepatopancreas were collected and weighed on a Mettler-Toledo AT21 analytical balance (Mettler-Toledo Incorporation, Im Langacher, Greifensee, Switzerland) to the nearest 1 μg. The same procedure was carried out for females at maturation stages III and V. Gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated as the percentage of gonad and hepatopancreas to total body mass respectively.
Fecundity was determined on four females fed each diet (one per replicate). No mortalities occurred during the experiment. These females were observed daily, and after three ovulation cycles and following a pre-nuptial molt, a male GC2 morphotype (total mass 11.0 ± 1.3 g) was introduced into the compartment. Each male was of the same morphotype, weight and size, and each female in the various treatments received one male (1:1) spermatophore during natural copulation. After copulation and spawning, the male was removed to avoid any possible aggression. Three days after spawning, eggs were manually removed from the female pleopods, and the females were weighed individually, whereas the eggs were packed in aluminium foil and dried in an oven at 100°C for 12 h. Then, they were transferred to desiccators and weighed on a Mettler-Toledo AT21 analytical balance to the nearest 1 μg. Three independent samples of around 80 eggs of each egg clutch were weighed and the eggs counted. Then, the mean egg dry mass was determined for each sample and for each female (mean of the three samples). The individual absolute fecundity was estimated by dividing the egg clutch dry mass by the mean egg dry mass. The relative fecundity was obtained from the ratio of individual absolute fecundity to total female wet mass.
Statistical analyses
This experiment was set up according to a completely randomized design. GSI and HSI data were assessed by analysis of variance (anova, F-test) using a split plot design, with the diets T1, T2 and T3 as whole plots, and maturation stages (I, III, V) as small plots. Fecundity and egg size data were subjected to one-way anova (F-test) with three treatments (diets) and four replicates (females). When F-values indicated significance (P < 0.05), means were compared using Tukey′s test (Sokal & Rohlf 1995). Differences were considered significant at P < 0.05. All data were analysed using SAS version 8.1 software (SAS Institute, Inc., Cary, NC, USA).
Results
Water quality variables were very similar in all experimental units and therefore, culture conditions were considered the same. They were: temperature 28.5 ± 0.5°C, pH 8.2 ± 0.1, dissolved oxygen 6.5 ± 0.5 mg L−1 and total ammonia nitrogen 2.5 ± 1.5 mg L−1. Analysis confirmed that the levels of n-3 and n-6 fatty acids and the n-3:n-6 ratios in the diets were as formulated (Table 2).
There was significant (P < 0.05) interaction between fatty acid ratio and ovarian stages for GSI (Table 3). GSI was significantly higher (P < 0.05) in females at maturation stage V subjected to treatment T2 (0.6 n-3/1.2 n-6), whereas females with ovaries at stage I and III did not differ among the different fatty acids ratios. There was no interaction between fatty acid ratio and ovarian stages for HSI. For all treatments, the HSI significantly (P < 0.05) increased from ovarian maturation stage I to III and decreased again at stage V. There was no significant (P < 0.05) difference between stages I and V.
| Variable | Diets | Maturation stages | ||
|---|---|---|---|---|
| I | III | V | ||
| GSI | T1 (0.6 n-3/0.6 n-6) | 0.55 ± 0.2cA | 2.15 ± 0.4bA | 5.59 ± 0.1aB |
| T2 (0.6 n-3/1.2 n-6) | 0.52 ± 0.1cA | 1.98 ± 0.1bA | 6.89 ± 0.3aA | |
| T3 (1.2 n-3/1.2 n-6) | 0.62 ± 0.2cA | 1.79 ± 0.3bA | 5.49 ± 0.4aB | |
| HSI | T1 (0.6 n-3/0.6 n-6) | 2.90 ± 0.8bA | 3.88 ± 1.2aA | 3.12 ± 0.3bA |
| T2 (0.6 n-3/1.2 n-6) | 4.01 ± 0.7bA | 5.05 ± 1.2aA | 2.62 ± 1.0bA | |
| T3 (1.2 n-3/1.2 n-6) | 3.89 ± 0.7bA | 4.77 ± 1.2aA | 3.17 ± 0.5bA | |
The fatty acid ratio significantly affected (P < 0.05) absolute and relative fecundity, but not egg mass (Table 4). Females fed the T2 (0.6 n-3/1.2 n-6) diet presented the highest fecundity, whereas no significant difference (P < 0.05) was observed between treatments T1 (0.6 n-3/0.6 n-6) and T3 (1.2 n-3/1.2 n-6).
| Diets | Total body wet weight (g) | Absolute fecundity (eggs/female) | Relative fecundity (eggs/g female) | Individual egg dry weight (μg.103) |
|---|---|---|---|---|
| T1 (0.6 n-3/0.6 n-6) | 10.7 ± 0.7a | 5314 ± 306b | 533 ± 36b | 75 ± 50a |
| T2 (0.6 n-3/1.2 n-6) | 11.2 ± 0.4a | 7269 ± 482a | 635 ± 56a | 68 ± 30a |
| T3 (1.2 n-3/1.2 n6 PUFA | 11.1 ± 0.3a | 5806 ± 310b | 525 ± 34b | 69 ± 30a a |
Discussion
The dietary proportion of PUFA affected M. amazonicum fecundity. A ratio of 1:2 n-3:n-6 fatty acids induced the highest GSI of mature females and number of eggs spawned. Raising the level of both n-3 and n-6 fatty acids from ~0.6% to ~1.2% of diet did not produce any effect on GSI and fecundity, suggesting that the ratio is more important than the absolute value of these two families of fatty acids. It is well known that desaturase enzymes have higher affinity for long-chain PUFA n-3 fatty acids than for equivalent chain length n-6 PUFA series (Harrison 1990). Probably, the additional amount of long-chain n-3 PUFA present in the 1.2 n-3/1.2 n-6 diet competed with n-6 PUFA for available desaturase enzymes and impaired the conversion of 18:2 (linoleic acid) to other PUFA family metabolites. Hence, although a dietary level of 1.2% n-6 PUFA appears to increase ovary development and fecundity in M. amazonicum, the excess of n-3 long chain PUFA may break this effect. Conversely, Cavalli et al. (1999) observed that an increase of both n-3 and n-6 PUFA groups from ~0.5 to ~1.4% of diet increased GSI and fecundity in M. rosenbergii. This suggests that the metabolism of PUFA is species-specific for freshwater prawns. It may be an evolutionary response to different food availability in the environment where each species evolved.
The dietary levels and the proportion of PUFA did not affect ovaries at stages I and III, but affected ovaries at stage V. This suggests that n-3/n-6 fatty acids may not act on all the processes of ovarian development, but only on the final deposition of yolk. Studies carried out in wild M. rosenbergii females showed that the increase in ovarian mass during gonadal maturation is due to lipid and yolk protein accumulation in the interior of oocyte cells (Fang-Yi & Ching-Fong 1997). Thus, the highest GSI observed at stage V of gonadal maturation of M. amazonicum might reflect lipid and protein accumulation in reproductive cells. It has been suggested that some lipids may be transferred from the hepatopancreas to the ovary during gonadal maturation in Crustacea, but studies on the contents of lipids in hepatopancreas, haemolymph and ovaries did not confirm this hypothesis (Harrison 1990). Studies carried out with Penaeus monodon females showed that most of the energy accumulated in the hepatopancreas was really used for spawning and was not stored in the eggs (Millamena & Pascual 1990). In the present study, the decrease of HSI from maturation stage III to V may be due to the mobilization of lipids (or other material) from the hepatopancreas to the ovaries or due to the use of lipids to provide energy for increasing biosynthesis during the late maturation phase (or both). On the other hand, when we compared the behaviour of the ovary and hepatopancreas in females in stage V, we did not observe statistical differences indicating an inverse relationship between GSI and HIS. This result may be attributed to a comparatively higher coefficient of variation combined with a small sample size. So, we can conclude that the levels and ratio of PUFA did not affect HIS in relation to the production of yolk or nutritional reserves.
Katre (1977) has emphasized that the size of an individual egg may depend on the total number of oocytes competing to share the yolk deposition. Such an effect was observed in M. rosenbergii, in which egg size decreased as relative fecundity increased (Cavalli et al. 1999). Conversely, our study with M. amazonicum shows that the 0.6 n-3/1.2 n-6 diet increased the number of eggs, but did not significantly affect individual egg size. Thus, the yolk increased proportionally to the egg number. Egg clutch is restricted by space in the ovaries and in the abdominal brood chamber. Although the total yolk available to eggs can be increased by improving the diet, the space in the ovaries remains the same. Therefore, as fecundity increases, egg size would be expected to decrease. However, this spatial limitation was not confirmed in our study (Table 4) and further research on this aspect is necessary.
The fecundity obtained in the present work was much higher than the values reported in the literature, which are normally lower than 2500 eggs per female (Kensley & Walker 1982; Lobão et al. 1986; Scaico 1992; Da Silva et al. 2004). This may be due to our use of large and well-nourished females. However, the diet used to feed females in the present experiment had a relatively low level of protein (32%). Increased M. rosenbergii fecundity was obtained when dietary protein was raised from 30% to 40% (Das et al. 1996). It is possible that the fecundity of M. amazonicum would increase if a higher level of protein containing a suitable amino acid profile was used. The development of a suitable diet for M. amazonicum maturation associated with the use of large females for broodstock will be important to decrease the cost of obtaining larvae in commercial hatcheries for this species.
M. amazonicum presents low fecundity compared with the giant river prawn M. rosenbergii, which spawns around 80 000 to 100 000 eggs (New 2002). However, the Oriental river prawn Macrobrachium nipponense, a small but very important freshwater prawn farmed in China, only produces ~5000 eggs (Kutty & Weimin 2010). This value is similar to the mean fecundity observed in M. amazonicum in the present study. Hatching rate increases with female size in M. amazonicum, and values close to 60–80% for large females were previously reported (Lobão et al. 1986; Scaico 1992). Therefore, 4000–5500 larvae can be expected to be produced by each 10–11 g female feeding inert diet. These values can be used to determine the minimum number of females necessary for a broodstock population, which is very important for planning the broodstock facilities to optimize costs.
Further research is necessary to identify the physiological mechanisms by which dietary PUFA acts on egg production in Macrobrachium species. In addition, it is essential to know the effect on egg and larval viability, as well as to determine the best absolute values of these fatty acids to improve good quality larval production.
Acknowledgments
Sincere thanks are due to Dr Maria Elizabete Macedo Viegas, São Paulo University, USP, Brazil, for her assistance in the formulation of the diets, and to Dr Euclides Braga Malheiros, São Paulo State University, UNESP, Brazil, for his assistance in statistical analysis. The Brazilian Council for Science and Technology (CNPq) supported this study through grants to the first (Proc. 133456/2001-3) and last author (Proc. 500574/2003-0).
