Growth performance, muscle biochemical constituents ...

Aquaculture Nutrition 2016

doi: 10.1111/anu.12443

..........................................................................................

1 2 1

1

1

3

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India; 2 Marine Planktonology & Aquaculture Laboratory, Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India; 3 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

This study was conducted to evaluate the influence of herbs such as Ocimum sanctum, Phyllanthus amarus and Solanum trilobatum on the freshwater prawn, Macrobrachium rosenbergii, postlarvae (PL). These herbs were powdered and incorporated at 5% with the basal diet separately and in a combination. The M. rosenbergii was fed with these feeds for a period of 90 days. Results indicated that significant (P < 0.05) improvements were observed in the survival, nutritional indices (weight gain, specific growth rate and protein efficiency ratio), proximate muscle biochemical constituents (total protein, amino acid, carbohydrate and lipid), profiles of essential amino acids and fatty acids of prawns fed with herbincorporated feeds. Among these herbs supplemented, P. amarus produced significantly (P < 0.05) better performance followed by S. trilobatum and O. sanctum. However, prawns fed with these herbs combination showed an insignificant (P > 0.05) improvement in survival and growth performance when compared to individual supplementation. Hence, this study suggests that the herbs (O. sanctum, P. amarus and S. trilobatum) can be used as a supplementary feed for a sustainable development of freshwater prawn culture. KEY WORDS:

amino acid, fatty acid, herbs, Macrobrachium rosenbergii, protein

Received 6 October 2015; accepted 29 January 2016 Correspondence: T. Muralisankar, Department of Zoology, School of Life sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India. E-mail address: [email protected]

..............................................................................................

ª 2016 John Wiley & Sons Ltd

Next to fish culture, farming of crustacean (shrimps, prawns, crabs, crayfish, lobsters, etc.) has made a significant progress during the last few decades in many parts of the world. According to FAO (2015), the global production of farmed crustacean was 6.7 metric tons in 2013. The culture of freshwater prawn, Macrobrachium rosenbergii, has great attention among crustaceans due to its better environment tolerance, faster growth, larger size, delicious meat quality and high market value. According to Marine Products Export Development Authority (MPEDA) report, India has produced 3332 metric tons of M. rosenbergii during the year 2012–2013 (FAO 2014). Feed is a major input in aquaculture systems which supply all essential nutrients (protein, lipid, carbohydrate, vitamins and minerals) to cultivable species for regulating growth, physiological process, reproduction and resistance against diseases. The herbs and its products play a vital role in shrimp and prawn culture. The active compounds of herbs, such as alkaloids, flavonoids, pigments, terpenoids, starch, steroids and essential oils have the ability to promote growth, body biochemical constituents in freshwater prawns (Bhavan et al. 2011, 2012). The herbs used in the culture of shrimps and prawns can improve the immunity, antimicrobial activity, appetite and provide antistress characteristics due to the presence of active compounds (Citarasu 2010; Bhavan et al. 2011, 2012; Poongodi et al. 2012; Shanthi et al. 2012; Radhakrishnan et al. 2013). This is an eco-friendly practice which will reduce the side effects caused by synthetic compounds that are applied as growth promoters and disease management.

The medicinal herb, Ocimum sanctum is commonly known as Tulsi or holy basil in India. Traditionally, fresh juice or decoction of O. sanctum leaves is used to promote health and treat various disorders as advocated in Ayurveda, the Indian System of Medicine. Some investigations have shown that the extracts of O. sanctum possess significant antioxidant and antistress properties (Devi et al. 2000). The Solanum trilobatum is an important Indian medicinal plant used in traditional medicine for many centuries (Mohan et al. 1998). In India, the leaves of S. trilobatum have been used in food preparations. In traditional medicine, it is used to treat common cold, asthma, cough, dyspnoea, chronic febrile infections and parturition. The constituents of this plant include sobatum, b-solamarine, solaine, solasodine, glycoalkaloid, diosgenin and tomatidine. This plant possesses a broad spectrum of antibiotic, antibacterial and anticancer activities (Mohan & Devi 1996). The herb, Phyllanthus amarus, is commonly used in Central and Southern regions of India and is found in other countries including China, Philippines, Cuba, Nigeria and Guam (Oluwafemi & Debiri 2008). It is traditionally used in the treatment for malaria-related symptoms, jaundice, diarrhoea, diabetes, flu, kidney ailments, chronic dysentery, frequent menstruation, ringworm, ulcers, genitourinary tract infections diseases, etc. The present study was conducted to investigate the potential effects of herbs, such as O. sanctum, P. amarus and S. trilobatum, on the growth, muscle meat qualities such as biochemical compositions (protein, amino acid, carbohydrate and lipid), carcass quality in terms of minerals (sodium and potassium), profiles of amino acids and fatty acids of M. rosenbergii postlarvae (PL).

Macrobrachium rosenbergii PL (age PL 4) were purchased from Happy Bay Aqua Nova Hatchery, Mugaiyur, Kancheepuram District of Tamil Nadu, India. About 1000 prawn PL were acclimatized to the laboratory condition in a cement tank (1000 L) filled with tap water (temperature, 28 1.34 °C; pH, 7.47 1.42; TDS, 1200.00 167.00 mg L 1; DO2, 7.20 0.54 mg L 1; BOD, 15.00 2.56 mg L 1; COD, 75.00 4.00 mg L 1) for 2 weeks before the commencement of the experiments. During acclimatization, prawns were fed with boiled egg albumin, live Artemia nauplii (Artemia salina) and commercially available scampi feed. Water was changed every morning in order to

maintain a healthy environment for the prawn apart from providing artificial aeration. This ensured a sufficient oxygen supply for the prawn and an environment devoid of accumulated metabolic wastes like faeces and mounts.

The five different feeds were composed with locally available feed ingredients (Table 1). Soy bean meal, green gram, ground nut oil cake, tapioca flour and eggs were purchased from the local merchants. Cod liver oil and vitamin B complex capsules (Pfizer Ltd., Mumbai, India) were purchased from local pharmacies. Further, the medicinal herbs such as O. sanctum (OS), S. trilobatum (ST) and P. amarus (PA) were collected from Mottankurichi village, Dharmapuri District, Tamil Nadu, India. These herb leaves were shade-dried and powdered separately using mixer grinder. The feeds were prepared by mixing the dry ingredients for 15 min. The mixture was then steam-cooked at 90–100 °C for 15 min and allowed to cool at room temperature. Subsequently, the 5% of chosen herbs powder was added individually (each herb for each diet) and a mixture [all the three herbs were mixed at equal proportion (16.67 g kg 1 of each herb)]. Cod liver oil, vitamin B complex and egg albumin were also added and mixed thoroughly. The prepared dough was pelletized in a hand pelletizer (Pigeon manufactures, Kolkata, India) fixed with 3-mm-diameter mesh. The pellets were collected and dried at room temperature until they reached a constant weight. Feed without the addition of herbs was also prepared with basal ingredient (BI) and served as control. The proximate biochemical compositions such as protein, carbohydrate, lipid, moisture and ash contents of feeds were analysed according to the standard procedures of AOAC (1995) (Table 1).

The feed leaching was analysed at specific time intervals of 2, 4, 6 and 8 h. One gram of feed sample in triplicate was placed in a glass bowl containing 100 mL of water. The sample containing water was filtered in No. 30 blotting silk cloth, and the residue was dried in a hot air oven at 65 °C until a constant weight was reached. The mean weight before immersion and after drying was used to calculate the percentage of dry matter loss, which is the measure of the water stability of the pellets for the corresponding time intervals. Percentage dry matter leaching was calculated by the following equation:

..............................................................................................

Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

Table 1 Composition of formulated feeds (g kg 1) Ingredients

Composition

Soy bean meal Green gram Ground nut oil cake Tapioca flour Egg albumin Cod liver oil Vitamin mix1 Herbal powder (OS/ST/PA/M)2 Proximate compositions (g kg Protein Carbohydrate Fibre Lipid Ash Moisture (%) Gross energy (kJ g 1)

390 250 240 60 30 20 10 50 1

described by Tekinay & Davies (2001). The feed conversion ratio (FCR) and protein efficiency ratio (PER) were calculated by the method described by Zhou et al. (2007). S (%) = final prawn number/initial prawn number 9 100 WG (g) = final weight SGR (%/day 1) = lnw2

initial weight lnw1/t

9 100,

dry basis)

where w2 and w1 are final weight and initial weight, respectively and t is time in days.

400.60 292.50 52.60 191.0 63.30 6.0 13.80

FCR = feed consumed dry weight/weight gain (wet weight) PER = weight gain/protein intake 9 100

1

Becosules capsules (manufactured by Pfizer), each capsule contains thiamine mononitrate 1P 10 mg; riboflavin 1P10 mg; pyridoxine hydrochloride 1P 3 mg; vitamin B12 (as tablets 1 : 100) 1P 15mcg niacinamide 1P100 mg; calcium pantothenate 1P 50 mg; folic acid 1P 1.5 mg; biotin USP 100 mcg; ascorbic acid 1P 150m. 2 OS, O. sanctum; ST, S. trilobatum; PA, P. amarus; M, herbs mixed at equal proportions.

Dry weight leaching (%) = dry weight (g) at t0 weight (g) at t1 9 100,

dry

where t0 is initial weight (before immersion) and t1 is final weight (after immersion).

In this study, five groups of M. rosenbergii PL (age PL 20) ranging from 1.1 to 1.7 cm in length and 0.13 to 0.18 g in weight were assigned in triplicate for 90 days. One group served as control fed with basal diet (BI) only, and the remaining four groups were fed with respective herb-supplemented diets [BI+OS, BI+ST, BI+PA and BI+M (herbs mixture)]. The experimental prawns were fed with these feeds at 10% of body weight. Each group consisted of 30 PL in an aquarium maintained with 30 L of tap water. The 40% water medium of each aquarium was renewed daily at early morning by siphoning method without any disturbance to the prawns and aerated adequately. The unfed feeds, faeces and other metabolic wastes were removed on daily basis while renewing rearing water.

The nutritional indices such as survival (S), weight gain (WG) and specific growth rate (SGR) were calculated as

..............................................................................................


At the end of 90-day feeding experiment, prawns were sacrificed and muscle tissues from abdominal segments were immediately processed for analysing the proximate composition. The content of total protein in the ethanolprecipitated sample was estimated following the method of Lowry et al. (1951). Total carbohydrates present in TCAextracted sample were estimated by the method of Roe (1955). Total lipid was extracted with chloroform–methanol mixture following the method of Folch et al. (1957) and was estimated according to Barnes & Black-Stock (1973). Amino acids were extracted using sodium tungstate and H2SO4 and assayed by the method of Moore & Stein (1948). The preweighed wet tissue samples were dried at 40 °C in hot air oven until reached a constant weight to measure the moisture content. The dried tissue sample was subjected to 600 °C for 4 h in a muffle furnace to measure the ash content following the method of APHA (2005).

Contents of minerals such as sodium (Na+) and potassium (K+) were estimated in the muscle tissues of PL by the simple flame photometric method of Jeffery et al. (1989) by using Elico CL 220 flame photometer. The values were calculated by adopting the following formula: Na+ (or) K+ content = sample reading/standard reading 9 standard concentration/sample concentration 9 purity of NaCl or KCl 9 molecular weight of NaCl or KCl/dilution factor.

The profile of amino acid analyses in formulated feeds and experimental PL was analysed using high-performance thinlayer chromatography (HPTLC) by the method of Hess & Sherma (2004). The powdered samples of each 250 mg of feed/prawns were dried (80 °C for 3 h), digested with 6M aqueous hydrochloric acid and dried under vacuum. The powdered sample was dissolved in distilled water, and 5 lL of the sample was loaded in 8-mm-thick precoated silica gel 60F254 TLC plate (20 cm 9 15 cm) and processed in CAMAG-LINOMAT 5 instrument. The plate was developed with butanone–ammonia–pyridine–water (3.9:1:3.4:2.6 mL) mobile phase. The plate was sprayed with ninhydrin reagent prepared in propan-2-ol and dried. The developed plate was documented using photo-documentation chamber (CAMAG-REPROSTAR 3) at UV 254-nm and UV 366nm lights. Finally, the plate was scanned at 500 nm using CAMAG-TLC SCANNER 3. The peak area of the sample was compared with standard amino acids and quantified. Four groups of standard amino acids were also analysed. Group-I: proline, serine, asparagine, glutamine and methionine; Group-II: aspartic acid, glutamic acid, alanine, valine and phenyl alanine; Group-III: lysine, glycine, threonine, isoleucine and tyrosine; and Group-IV: arginine, cystine, histidine, leucine and tryptophan.

The fatty acids in formulated feeds and the PL fed with the feeds were determined following the gas chromatographic (GC) method of Nichols et al. (1993). Fatty acids were obtained from lipids by saponification using NaOH dissolved in the methanol–water mixture (hydrolysis with alkali) in feeds/prawns samples. They were converted into fatty acid methyl ester using HCl and methanol mixture, which can be easily identified by gas chromatography. The fatty acid methyl ester was separated using a mixture of hexane and anhydrous diethyl ether. For the organic phase, aqueous NaOH was used as base wash and the upper organic layer was separated. Two microlitres of the sample was injected and analysed using Chemito 8610 gas chromatography (Chemito Instruments Pvt. Ltd., Mumbai, India), with BPX70 capillary column and flame ionization detector. Nitrogen was used as the carrier gas. The chromatogram was used for the calculation. Standard fatty acids were analysed simultaneously. Based on the retention time and peak area of the standard fatty acids, each fatty acid in the unknown sample was identified.

Triplicate samples were used in all experiments. Data from each treatment were subject to one-way analysis of variance (ANOVA) using SPSS (version 11.5; IBM, Chicago, IL, USA), followed by Duncan’s multiple range test (DMRT) to compare the differences between treatments, where significant differences (P < 0.05) were observed.

The analysed proximate compositions such as protein, amino acid, carbohydrate, fibre and ash contents were found to be 400.60, 292.50, 52.60, 191.10 and 63.30 g kg 1, respectively. Also, 6% of moisture and 13.80 kJ g 1 of gross energy were recorded in the basal feed (Table 1). Further, six essential amino acids (lysine, arginine, cystine, isoleucine, threonine and phenylalanine) and three nonessential amino acids (asparagine, alanine and glutamic acid) were detected in basal feed. Among these, all essential amino acids were found to be higher when compared to non-essential amino acids. In fatty acid profile of basal feed, five saturated fatty acids (myristic acid, palmitic acid, stearic acid, behenic acid and lignoceric acid) and six unsaturated fatty acids [oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] were noted. Among these fatty acids, the unsaturated fatty acids were found to be higher compared to saturated fatty acids (Table 2).

In the present study, the leaching percentage of experimental diets was ranged from 18.3 to 18.8%. Diet 3 (18.3%) showed lowest leaching followed by diet 2 (18.50%), diet 1 (18.60%) and diet 4 (18.60) when compared to control diet (18.180%). However, there was no significant (P > 0.05) difference found between the diets (Table 3).

The survival, growth rates such as length, weight, weight gain, specific growth rate and protein efficiency ratio were significantly (P < 0.05) improved in the prawns fed with herb-incorporated feed when compared to control feed-fed prawns. Among the experimental prawns, P. amarus-incor-

..............................................................................................


porated feed-fed prawns group showed significantly better elevation when compared to other herb-supplemented feedfed prawn groups. However, feed conversion ratio was significantly (P < 0.05) lower in herb-incorporated feed-fed prawn groups when compared to control (Table 4).

Table 2 Amino acid and fatty acid composition of basal feed (control diet) Basal feed1

Composition Amino acid (g kg dry basis)

Essential 1

Non-essential

Fatty acid (% per 2 lL methylated fatty acid)

Saturated

Unsaturated

1

composed with supplementation).

basal

1.68 0.18 3.20 0.32 1.24 0.11 0.60 0.01 1.60 0.08 1.36 0.34 1.20 0.09 0.60 0.01 0.60 0.02 0.855 0.017

Lysine Arginine Cystine Isoleucine Threonine Phenylalanine Asparagine Alanine Glutamic acid Myristic acid (14 : 0) Palmitic acid (16 : 0) Stearic acid (18 : 0) Behenic acid (22 : 0) Lignoceric acid (24 : 0) Oleic acid (18:1n-9) Linoleic acid (18:2n-6) Linolenic acid (18:3n-3) Arachidonic acid (20:4n-6) EPA (20:5n-3) DHA (22:6n-3)

ingredients

only

17.33 2.110 10.80 2.000

In this study, similar amounts of nutritive levels were observed in basal diet (Table 1). However, the muscle biochemical constituents such as protein, amino acid, carbohydrate, lipid and ash levels were significantly (P < 0.05) elevated in herb-incorporated feed-fed prawn PL when compared to the control. Among these herbs, P. amarus produced significantly (P < 0.01) better performance followed by S. trilobatum, O. sanctum and herbs mixture. However, the moisture content of experimental PL was significantly lower when compared to control PL (Table 4).

The estimated whole-body mineral contents such as Na+ and K+ were significantly elevated (P < 0.05) in herbincorporated feed-fed prawn PL when compared to control. Among these herbs, P. amarus-supplemented feed-fed prawn PL showed a significant improvement followed by S. trilobatum, O. sanctum and then the herbs mixture (Table 4).

0.965 0.018 0.747 0.032 27.84 2.010 14.56 1.920 1.170 0.092 0.572 0.010 9.347 1.012 5.690 0.197 (without

herb-

In this study, six essential amino acids such as lysine, arginine, cystine, isoleucine, threonine and phenylalanine and five non-essential amino acids such as asparagine, alanine, glutamic acid, tyrosine and glycine were observed in the muscle of M. rosenbergii PL. The essential amino acids such as lysine, cystine, isoleucine and phenylalanine and non-essential amino acids such as alanine and glutamic acid were significantly (P < 0.05) higher in herb-supplemented feed-fed prawn PL when compared to control. In contrast, arginine was found to be significantly (P < 0.05)

Table 3 Leaching (%) of experimental diets in different hours 2h

4h

6h

8h

Diet

Initial (g)

Final (g)

Leaching (%)

Final (g)

Leaching (%)

Final (g)

Leaching (%)

Final (g)

Leaching (%)

Control (BI) Diet 1 (BI+OS) Diet 2 (BI+ST) Diet 3 (BI+PA) Diet 4 (BI+M) P-value

1.0 1.0 1.0 1.0 1.0 –

0.85 0.85 0.85 0.86 0.85 1.000

14.30 14.30 14.30 14.00 14.20 –

0.84 0.84 0.84 0.84 0.84 1.000

15.40 15.30 15.50 15.20 15.40 –

0.83 0.83 0.83 0.83 0.83 1.000

16.80 16.50 16.30 16.10 16.50 –

0.81 0.81 0.81 0.81 0.81 1.000

18.80 18.60 18.50 18.30 18.60 –

0.099a 0.073a 0.068a 0.076a 0.069a

0.070a 0.094a 0.086a 0.066a 0.055a

0.045a 0.067a 0.083a 0.071a 0.097a

0.051a 0.092a 0.073a 0.085a 0.095a

Mean SD (n = 3); Mean values within the same row sharing the same superscript are not significantly different (P > 0.05). BI, basal ingredients; OS, O. sanctum; ST, S. trilobatum; PA, P. amarus; M, mixture of three herbs.

..............................................................................................


0.089 0.094 0.001 0.012 0.066 0.855 0.124 0.044 0.002 0.063 0.021 0.710 0.971 0.000 0.000 4.48 0.43ab 1.30 0.19ab 76.66 4.51cd 1.15 0.21a 1.25 0.20b 1.04 0.22b 2.52 0.25ab 81.00 3.19a 43.21 2.36b 21.21 2.54b 8.200 3.42bc 64.40 3.97ab 17.83 2.42b 0.20 0.02c 0.57 0.01bc 0.35a 0.22a 3.54a 0.08a 0.10b 0.18a 0.21a 2.53a 2.03a 2.36a 1.22a 4.10c 1.56a 0.03a 0.08a 4.70 1.49 93.33 1.34 1.26 1.11 2.73 84.40 48.81 26.92 14.00 61.10 18.70 0.97 0.40 0.45a 0.18a 3.89ab 0.11a 0.12b 0.21a 0.21ab 3.14a 2.14ab 2.32ab 1.97b 4.03b 1.73a 0.05b 0.04b – – – – – 37.50 0.31 21.50 1.82 11.24 1.03 4.440 0.31 67.90 4.73 12.60 1.43 0.04 0.002 0.13 0.01 dry basis) 1

Minerals (mg g

Biochemical constituents

Survival and nutritional indices

Mean SD (n = 3). Mean values within the same row sharing the same superscript are not significantly different (P > 0.05). BI, basal ingredients; OS, O. sanctum; ST, S. trilobatum; PA, P. amarus; M, mixture of three herbs.

4.60 1.40 86.66 1.25 1.28 1.08 2.68 82.03 45.97 23.60 10.80 62.80 18.21 0.24 0.57 0.51a 0.20a 4.0bc 0.13a 0.13b 0.26a 0.23ab 2.81a 2.41b 2.76b 2.0b 3.56b 2.26b 0.07b 0.02b 4.58 1.38 83.33 1.23 1.25 1.07 2.68 81.20 44.25 21.73 10.40 63.50 17.86 0.27 0.67 3.70 0.36b 1.01 0.18b 73.53 4.67d 0.86 0.10b 1.62 0.21a 0.92 0.24b 2.26 0.1b 75.45 3.21b 37.73 2.43c 20.42 2.61b 7.200 0.92c 65.40 3.52a 17.53 2.85b 0.15 0.05c 0.35 0.05c Morphometric data

Length (cm) Weight (g) Survival (%) Weight gain (g) Feed conversion ratio Specific growth rate (% day 1) Protein efficiency ratio Protein (mg g 1 wet basis) Amino acid (mg g 1 wet basis) Carbohydrate (mg g 1 wet basis) Lipid (mg g 1 wet basis) Moisture (%) Ash (%) Sodium (Na+) Potassium (K+)

1.40 0.22 0.15 0.01

P-value Diet 4 (BI+M) Diet 3 (BI+PA) Diet 2 (BI+ST) Diet 1 (BI+OS) Control (BI) Initial Parameters

Final

Table 4 Survival, nutritional indices, muscle biochemical constituents and whole-body mineral contents of M. rosenbergii PL fed with herb-incorporated feeds

higher in P. amarus-supplemented feed-fed prawn PL followed by control, and threonine was only detected in herbs mixture-supplemented feed-fed prawn PL. The non-essential amino acids asparagine and glycine were detected only in control and P. amarus-supplemented feed-fed prawn PL, respectively. In contrast, tyrosine was noted in herb-supplemented feed-fed prawn PL alone (Table 5).

The saturated fatty acids such as behenic acid and lignoceric acid were significantly (P < 0.05) elevated in P. amarussupplemented feed-fed prawn PL when compared to control and other herb-supplemented feed-fed prawn PL. However, myristic acid and palmitic acid were significantly (P < 0.05) elevated in S. trilobatum and herbs mixture-supplemented feed-fed prawn PL, respectively, and stearic acid was found to be significantly (P < 0.05) higher in control feed-fed prawn PL when compared to herb-supplemented feed-fed prawn PL. Meanwhile, lauric acid was only observed in P. amarus-supplemented feed-fed prawn PL. Further, the unsaturated fatty acids such as oleic acid, linoleic acid and arachidonic acids were significantly higher in herb-supplemented feed-fed prawn PL when compared to control. However, linolenic acid, EPA and DHA were found to be significantly higher in control feed-fed prawn PL (Table 6).

Dietary nutrients play a crucial role in culture of fish and crustaceans (Vega et al. 2006; Khan et al. 2011). In the present study, the analysed proximate compositions (protein, carbohydrate, fibre, lipid, energy, essential amino acids and fatty acids) of formulated basal feed were found to be optimum level for rearing of freshwater prawns, which is recommended by previous reports (Swamy 1995; Mitra et al. 2005). It suggests that the prepared experimental diets provide all essential nutrients for the rearing of M. rosenbergii PL. Prawns are slow and continuous feeders, and hence, the feeds should be relatively stable in water for long durations, so that the feed can be consumed without waste. Feeds with poor water stability disintegrate rapidly, cause feed waste, water pollution and give poor feed consumption rate. The stability of pelletized feeds is influenced by different factors such as the composition of the feed, nature

..............................................................................................


Table 5 Amino acid compositions (g kg feeds Amino acids Essential

Non-essential

1

dry basis) of formulated basal feed and M. rosenbergii PL fed with different herb-incorporated

Control (BI) Lysine Arginine Cystine Isoleucine Threonine Phenylalanine Asparagine Alanine Glutamic acid Tyrosine Glycine

1.64 3.28 2.32 0.68 ND 1.06 0.92 1.12 1.12 ND ND

0.20 0.27b 0.39a 0.05c

0.21c 0.03 0.03b 0.18b

Diet 1 (BI+OS)

a

1.84 3.44 3.56 2.52 ND 5.28 ND 1.72 1.72 5.28 ND

a

0.21 0.23ab 0.87a 0.21a

1.42a 0.11a 0.21a 1.20a

Diet 2 (BI+ST) 1.68 2.92 3.56 1.92 ND 4.08 ND 1.72 1.72 4.08 ND

a

0.16 0.16c 0.83a 0.4 5b

1.33ab 0.14a 0.24a 1.10ab

Diet 3 (BI+PA) 1.86 3.68 2.88 1.92 ND 2.76 ND 1.40 1.40 2.76 0.52

a

0.13 0.18a 0.31a 0.42b

0.23b

0.23ab 0.12ab 0.56b 0.01

Diet 4 (BI+M) 1.52 2.12 3.36 0.84 0.72 4.72 ND 1.64 1.64 4.72 ND

P-value

a

0.21 0.12d 0.56a 0.02c 0.03 1.21ab

0.208 0.000 0.151 0.000 0.000 0.004 0.000 0.008 0.009 0.000 0.000

0.27a 0.11a 1.11a

Mean SD (n = 3). Mean values within the same row sharing the same superscript are not significantly different (P > 0.05); ND, not detected. BI, basal ingredients; OS, O. sanctum; ST, S. trilobatum; PA, P. amarus; M, mixture of three herbs.

Table 6 Fatty acid profiles (% per 2 lL methylated fatty acid) of formulated basal feed and M. rosenbergii PL fed with different herbincorporated feeds

Fatty acids Saturated

Unsaturated

Lauric acid (12 : 0) Myristic acid (14 : 0) Palmitic acid (16 : 0) Stearic acid (18 : 0) Behenic acid (22 : 0) Lignoceric acid (24 : 0) Oleic acid (18:1n-9) Linoleic acid (18:2n-6) Linolenic acid (18:3n-3) Arachidonic acid (20:4n-6) EPA (20:5n-3) DHA (22:6n-3)

Control BI

Diet 1 (BI+OS)

Diet 2 (BI+ST)

ND

ND

ND

1.048 0.053c 18.71 2.340ab 10.03 1.210b

0.174 0.023d 15.00 1.150c

1.664 0.01 5a 16.19 1.340ab

Diet 3 (BI+PA 5%) 0.041 0.009 0.157 0.034d 15.69 1.320b

Diet 4 (BI+M 5%)

Pvalue

ND

–

1.302 0.026b 19.71 2.430a

0.000 0.038

6.885 1.530a

6.227 1.530a

5.401 1.320a

9.617 3.320b

0.053

0.762 0.023e

1.918 0.103b

1.750 0.10c

2.246 0.102ab

0.977 0.031d

0.000

0.676 0.028b

0.403 0.071bc

0.779 0.053b

1.088 0.032a

0.658 0.081

0.000

29.54 2.460ab

33.931 2.180ab

33.09 2.090ab

35.51 2.000a

32.50 2.560ab

0.084

15.28 1.460b

24.006 2.040a

22.77 2.130a

26.15 2.011a

17.83 2.090b

0.000

ND

0.000

cd

1.197 0.045a

1.083 0.081b

0.977 0.029c

0.300 0.009d

0.137 0.00d

1.517 0.018b

1.190 0.010c

1.099 0.091c

2.476 0.096a

0.000

7.626 1.023a 5.140 0.190a

5.969 1.08b 3.140 0.670b

6.762 1.01ab 3.040 0.890b

6.151 0.920b 3.417 0.390b

1.568 0.092c 2.511 0.401c

0.000 0.002

Mean SD (n = 3). Mean values within the same row sharing the same superscript are not significantly different (P > 0.05); ND, not detected. BI, basal ingredients; OS, O. sanctum; ST, S. trilobatum; PA, P. amarus; M, mixture of three herbs.

of ingredients, type of the feed processing and moisture content (Hastings 1976). The high water stability of pellet is considered one of the major advantages of artificial feed over natural feed. In this study, lowest leaching percentage of prepared diets proves that the selected basal ingredients and the binding agents (tapioca flour, egg albumin and cod liver oil) have contributed to attain appreciable water stability that prevents water pollution and nutrient leaching. Previously, Immanuel et al. (1997) and Radhakrishnan

..............................................................................................


et al. (2013) reported that the diets composed with tapioca flour, egg albumin and fish oil showed poor leaching and high water stability.

The herb-based diets can increase the survival rate and growth rate of fish and prawns due to the presence of nat-

ural active compounds (Citarasu 2010). In this study, significant (P < 0.05) elevations in survival, growth rate, weight gain, specific growth rate and protein efficiency ratio of prawns fed with herb-incorporated feeds indicated that the utilization of herbs in feed has an effect on better survival and growth of M. rosenbergii PL. However, the lower performance in feed conversion ratio in herbincorporated feed-fed prawn PL indicates that the food consumed was effectively utilized for growth. Previously, Shanthi et al. (2012), Bhavan et al. (2011, 2012), Poongodi et al. (2012) and Radhakrishnan et al. (2013) reported that the herbs such as Alteranthera sessius, Andrographis paniculata, Cissus quadrangularis, Eclipta alba, Withania somnifera, O. sanctum, Allium sativum, Zingiber officinale, Curcuma longa, Trigonella foenum-graecum have significantly promoted the survival, weight gain, protein efficiency ratio and specific growth rate of M. rosenbergii PL. Hence, the results of the current study have revealed that the survival and growth performance of prawn PL were influenced by S. trilobatum, P. amarus and O. sanctum supplementation and it might be due to the presence of its active principles in nature.

Firmness of muscle is considered as an important flesh quality trait in the aquatic animals for marketing (Brinker & Reiter 2011). The herbal growth promoters (active principles) help to induce the transcription rate, and this process leads to the increase in RNA, total amino acids, followed by an increase in the synthesis and storage of proteins in the cells, which produces better growth and muscle quality of shrimps and prawns (Citarasu 2010; Poongodi et al. 2012). In the present study, proximate compositions such as protein, amino acid, carbohydrate, lipid and ash were similar in all experimental diets, whereas these biochemical constituents were significantly (P < 0.05) elevated in herb-incorporated diet-fed M. rosenbergii PL when compared to control. It indicates that the supplemented herb additives have the ability to promote the absorption and storage of nutrients in the muscles of experimental prawn, which led to the better growth and muscle biochemical constituents. Similarly, previous studies indicate that the supplementation of herbs, such as Boerhavia diffusa, Solanum nigrum, T. arjuna, A. paniculata, C. quadrangularis, E. alba, W. somnifera, O. sanctum, A. sessius, A. sativum, Z. officinale, C. longa, T. foenum-graecum, Piper longum, Piper nigram, Z. officinale, Myristica fragrans, Glycyrrhiza glabra and Quercus infectoria, has successfully promoted the muscle biochemi-

cal constituents (protein, amino acid, carbohydrate and lipid), which seems to produce better growth and muscle quality in M. rosenbergii PL (Bhavan et al. 2011, 2012, 2013a,b; Poongodi et al. 2012; Shanthi et al. 2012; Radhakrishnan et al. 2013) and Penaeus indicus (Sambu & Jayaprakas 2001). In the present study, supplemented herbs have a pivotal role in improving the muscle biochemical constituents of M. rosenbergii PL.

In the present study, the significant elevations in sodium (Na+) and potassium (K+) levels in herb-supplemented diet-fed prawns suggest that the dietary herbs can promote the minerals utilization, which led to produce better health improvement of M. rosenbergii PL. Similarly, dietary supplementation of herbs such as A. sessius, C. quadrangularis, E. alba, P. longum, P. nigram, Z. officinale, M. fragrans, G. glabra and Q. infectoria has produced a significant improvement in Na+ and K+ contents in M. rosenbergii PL (Bhavan et al. 2013a,b; Radhakrishnan et al. 2013). Sodium (Na+) is the main monovalent ion of extracellular fluids, and it constitutes 93% of the ions (bases) found in the blood stream. The principal role of Na+ in animals is related to the regulation of osmotic pressure and the maintenance of acid–base balance. The Na+ also has an effect on muscle irritability and plays a specific role in the absorption of carbohydrate. Potassium (K+) has major action in intracellular fluid and regulates intracellular osmotic pressure and acid–base balance. Like sodium, potassium also has a stimulating effect on muscle irritability. Potassium is required for glycogen and protein synthesis and the metabolic breakdown of glucose (FAO, 1987). It has been reported that the dietary Na+ and K+ improve muscle meat quality in aquatic species such as Cyprinus carpio, Cyprinus mrigala, M. rosenbergii and Litopenaeus vannamei (Shiau & Hsieh 2001; Keshavanath et al. 2003; Roy et al. 2007; Saoud et al. 2007).

Amino acids are the building blocks of proteins and serve as body builders. They are utilized to form various cell structures, of which they are key components and serve as a source of energy. In this study, significant elevations in essential amino acids (lysine, cystine, isoleucine and phenylalanine) and non-essential amino acids (alanine and glutamic acid) of herb-supplemented feed-fed prawn PL indicate that these

..............................................................................................


herbs can influence the synthesis of these amino acids. Significant elevation of arginine in P. amarus-supplemented feed-fed prawn PL suggests that this herb has strong influence on this particular amino acid. Presence of the nonessential amino acid glycine only in P. amarus-supplemented feed-fed prawn PL shows potency of this herb on particular amino acid. In contrast, the absence of asparagine and the presence of tyrosine in all herb-supplemented feed-fed prawn PL suggest that supplemented herbs might negatively and positively have an influence on these amino acids, respectively. Similarly, Bhavan et al. (2011) reported that the herbs such as W. somnifera and O. sanctum influenced better amino acid profile and it led to better growth performance in M. rosenbergii PL. It has been reported that the dietary supplementation of medicinal herbs A. paniculata, C. quadrangularis, E. alba and A. sessius significantly influences the muscle amino acid contents in M. rosenbergii and Macrobrachium malcolmsonii (Shanthi et al. 2012; Radhakrishnan et al. 2013, 2015). Hence, this study indicates that the supplementation of herbs has an influence on the utilization and synthesis of amino acids, which led to increased protein synthesis and produced better muscle composition of M. rosenbergii PL.

Fatty acid composition of dietary lipids plays an essential role in the maintenance of proper metabolic and many physiological processes which lead to better survival and growth (Colvin 1976). In the present study, the significant elevation of saturated fatty acids, such as lauric acid, behenic acid and lignoceric acid in P. amarus, myristic acid in S. trilobatum and palmitic acid in herbs mixture-supplemented feed-fed prawn PL, suggests that supplementation of these herbs has the ability to influence on these fatty acids. However, a significant improvement of stearic acid in control feed-fed prawn PL indicates that the herbs might be suppressed progressively. Meanwhile, lauric acid was detected in P. amarus-supplemented feed-fed prawn PL alone, and it suggests that this herb has specific influence on lauric acid secretion. The unsaturated fatty acids such as oleic acid, linoleic acid and arachidonic acid were found to be significantly elevated in herb-supplemented feed-fed prawn PL when compared to control. It indicates that the experimental herbs can promote these fatty acids synthesis in M. rosebneergii PL. Significant decrease in linolenic acid, EPA and DHA in herb-supplemented feedfed prawn PL suggests that these herbs might be poorly performed on these fatty acids (Table 6). Previously,

..............................................................................................


Shanthi (2012) and Radhakrishnan et al. (2013, 2015) reported that the herbs such as A. sessius-, A. paniculata-, C. quadrangularis- and E. alba-incorporated feed-fed prawns, M. rosenbergii and M. molcolmsonii, showed better fatty acid profile. The present study also suggests that the herb-based diets can promote the muscle fatty acid contents, which paves the way for producing better meat quality of M. rosenbergii PL.

In this study, the influence of medicinal herbs (O. sanctum, P. amarus and S. trilobatum) on growth, biochemical constituents, mineral contents, profile of amino acids and fatty acids of the freshwater prawn M. rosenbergii was clearly demonstrated. Among the three herbs supplementation, P. amarus produced significantly better performance followed by S. trilobatum and O. sanctum. However, herbs mixture produced significantly poor performance in all parameters studied, and it might be due to the herbs active principles suppressed one by one. Hence, this study suggests that the individual supplementation of these herbs can be used as a feed additive in aqua feed formulations for promoting sustainable freshwater prawn culture.

Bharathiar University, Coimbatore, Tamil Nadu, India, is gratefully acknowledged for the financial support provided in the form of University Research Fellowship to the first author.

AOAC (1995) Official Methods of Analysis, 16th edn. AOAC international publishers, Arlington VA, USA. APHA (2005) Standard Methods for the Examination of Water and Wastewater, 19th edn. American Public Health Association, New York, NY, USA. Barnes, H. & Black-Stock, J. (1973) Estimation of lipids in marine animals and tissues. Detailed investigation of the Sulphophosphovanillin method for total lipids. J. Exp. Mar. Biol. Ecol., 12, 103–118. Bhavan, P.S., Jayanthi, S. & Rabecca, A.A. (2011) Growth performance of the freshwater prawn Macrobrachium rosenbergii post larvae fed with Ocimun sanctum (Tulsi) and Withania somnifera (Ashwagandha) incorporated feeds. Intl. J. Biol. Res. Develop., 1, 34–53. Bhavan, P.S., Manickam, N. & Radhakrishnan, S. (2012) Influence of herbal greens, Murraya koenigii, Coriandrum sativum and Menthe arvensis on growth performance of the freshwater prawn Macrobrachium rosenbergii post larvae. Res. J. Biotechnol., 7, 149–157.

Bhavan, P.S., Devi, N.N., Muralisankar, T., Manickam, N., Radhakrishnan, S. & Srinivasan, V. (2013a) Effects of Myristica fragrans, Glycyrrhiza glabra and Quercus infectoria on growth promotion in the prawn Macrobrachium rosenbergii. Int. J. Life Sci. Biotech. Pharm. Res., 2, 169–182. Bhavan, P.S., Saranya, C., Manickam, N., Muralisankar, T., Radhakrishnan, S. & Srinivasan, V. (2013b) Effects of Piper longum, Piper nigram and Zingiber officinale on survival, growth, activities of digestive enzymes and contents of total protein, vitamins and minerals in the freshwater prawn Macrobrachium rosenbergii. Elixir Biotechonol., 58, 14824–14828. Brinker, A. & Reiter, R. (2011) Fish meal replacement by plant protein substitution and guar gum addition in trout feed, Part I: effects on feed utilization and fish quality. Aquaculture, 310, 350–360. Citarasu, T. (2010) Herbal biomedicines a new opportunity for aquaculture industry. Aquacult. Int., 18, 403–414. Colvin, P.M. (1976) The effect of selected seed oils on the fatty acid composition and growth of Penaeus indicus. Aquaculture, 8, 81–89. Devi, P.U., Ganasoundari, A., Vrinda, B., Srinivasan, K.K. & Krishnan, M.K.U. (2000) Radiation protection by the Ocimum flavanoid sorientin and vicenin- Mechanism of Action. Radiation Res., 154, 455–460. FAO (1987) The nutrition and feeding of farmed fish and shrimp; A training manual 1; The essential nutrients. Available at: http://www.fao.org/docrep/field/003/AB470E/ AB70E00.htm. FAO (2014) National aquaculture sector overview, India National Aquaculture Sector Overview. Yemen. National Aquaculture Sector Overview Fact Sheets. Available at: http://www.fao. org/fishery/countrysector/naso_india/en. FAO (2015) FAO Global Aquaculture Production database updated to 2013 – Summary information. Available at: http:// www.fao.org/3/a-i4899e.pdf. Folch, J., Lees, M. & Bloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 266, 497–509. Hastings, W.H. (1976) Fish Nutrition and Feed Manufacture. FAO Technical Conference on Aquaculture, Kyoto, Japan, 26, May 1976. Fishery Resources and Environment Division, FAO, Rome, Italy. Hess, B. & Sherma, J. (2004) Quantification of arginine in dietary supplement tablets and capsules by silica gel high-performance thin-layer chromatography with visible mode densitometry. Acta Chromatographica, 14, 60–69. Immanuel, G., Palavesam, A. & Petermarian, M. (1997) Formulation and analysis of artificial feed from fishery wastes. J. Freshwater Biol., 9, 86–90. Jeffery, G.H., Bassett, J., Mendham, J. & Denny, R.C. (1989) Vogel’s Textbook of Quantitative Chemical Analysis, 5th ed. Addison Wesley London Ltd. England, p. 801. Keshavanath, P., Gangadhara, B. & Khadri, S. (2003) Growth enhancement of carp and prawn through dietary sodium chloride supplementation. Aquacult. Asia, 4, 4–8. Khan, N., Qureshi, N.A., Nasir, M., Rasool, F. & Iqbal, K.J. (2011) Effect of artificial diet and culture systems on sensory quality of fried fish flesh of Indian major carps. Pakistan J. Zool., 43, 1177–1182. Lowry, O.H., Rosenbrough, W.J., Fair, A.L. & Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275. Mitra, G., Chattopadhyay, D.N. & Mukhopadhyay, P.K. (2005) Nutrition and feeding in freshwater prawn (Macrobrachium

rosenbergii) farming. Aqua. Feeds Formulat. Beyond., 2, 17–19. Mohan, P.V. & Devi, S. (1996) Cytotoxic potential of the preparations from Solanum trilobatum and the effect of Sobatum on tumor reduction in mice. Cancer Lett., 110, 71–76. Mohan, P.V., Rao, J., Sumathykutty, M.A. & Devi, K.S. (1998) Cytotoxicity of extracts of Solanum trilobatum and anticarcinogenic activity of S. trilobatum. Biomedicine, 18, 106–111. Moore, S. & Stein, W.H. (1948) Photometric ninhydrin method for use in the chromatography of amino acid. J. Biol. Chem., 176, 367–388. Nichols, D.S., Nichols, P.D. & Mc Meekin, T.A. (1993) Polyunsaturated fatty acids in Antarctic bacteria. Antarct. Sci., 5, 149– 160. Oluwafemi, F. & Debiri, F. (2008) Antimicrobial effect of Phyllanthus amarus and Parquentina nigrescens on Salmonella typhi. African J. Biomedical Res., 11, 215–219. Poongodi, R., Bhavan, P.S., Muralisankar, T. & Radhakrishnan, S. (2012) Growth promoting potential of garlic, ginger, turmeric and fenugreek on the freshwater prawn Macrobrachium rosenbergii. Int. J. Pharma. Bio. Sci., 3B, 914–926. Radhakrishnan, S., Bhavan, P.S., Seenivasan, C., Shanthi, R. & Poongodi, R. (2013) Influence of medicinal herbs (Alteranthera sessius, Eclipta alba and Cissus qudrangularis) on growth and biochemical parameters of the freshwater prawn Macrobrachium rosenbergii. Aquacult. Int., 22, 551–572. Radhakrishnan, S., Bhavan, P.S., Seenivasan, C., Muralisankar, T. & Shanthi, R. (2015) Effect of native medicinal herbs (Alteranthera sessius, Eclipta alba and Cissus qudrangularis) on growth performance, digestive enzymes and biochemical constituents of the monsoon river prawn Macrobrachium malcolmsonii. Aquacult. Nutr., 21, 496–506. Roe, J.H. (1955) The determination of sugar and blood and spinal fluid with anthrone reagent. J. Biol. Chem., 212, 335–343. Roy, L.A., Davis, D.A., Saoud, I.P. & Henry, R.P. (2007) Supplementation of potassium, magnesium and sodium chloride in practical diets for the Pacific white shrimp, Litopenaeus vannamei, reared in low salinity waters. Aquacult. Nutr., 13, 104– 113. Sambu, C. & Jayaprakas, V. (2001) Livol (IHF-1000), a new herbal growth promoter in white prawn Penaeus indicus (Crustacea). Indian J. Mar. Sci., 30, 38–43. Saoud, I.P., Roy, L.A. & Davis, D.A. (2007) Supplementation of chelated potassium and arginine to diets of Litopenaeus vannamei reared in low salinity waters of west Alabama. N. Am. J. Aquacult., 69, 265–270. Shanthi, R. (2012) Influence of medicinal herbs (Andrographis paniculata, Cissus quadrangularis and Eclipta alba) in growth promotion of the freshwater prawn, Macrobrachium rosenbergii. Ph.D., thesis submitted to Bharathiar University, Coimbatore, Tamil Nadu, India. Shanthi, R., Bhavan, P.S. & Radhakrishnan, S. (2012) Influence of medicinal herbs, (Andrographis paniculata, Cissus quadrangularis and Eclipta alba) on growth, digestive enzymes, biochemical constituents and protein profile of the freshwater prawn Macrobrachium rosenbergii. Elixir Bio-Tech., 42, 6478– 6484. Shiau, S.Y. & Hsieh, J.F. (2001) Dietary potassium requirement of juvenile grass shrimp Penaeus monodon. Fish. Sci., 67, 592–595. Swamy, D.N. (1995) CIBA, Madras “Training Manual, ShortTerm Course in Biotechnological Approaches in Prawns and Fish Nutrition and Feed Technology”, 15th February To 7th March, pp. 82–88.

..............................................................................................


Tekinay, A.A. & Davies, S.J. (2001) Dietary carbohydrate level influencing feed intake, nutrient utilisation and plasma glucose concentration in the rainbow trout, Oncorhynchus mykiss. Turk. J. Vet. Anim. Sci., 25, 657–666. Vega, M.E.R., Bores, E.G., Brauer, J.M.E., Garcia, M.G.S., Suarez, L.E.C., Nolasco, H. & Cerecedo, R.C. (2006) Nutritional value of cowpea (Vigna unguiculata L. Walp) meals as

..............................................................................................


ingredients in diets for Pacific white shrimp (Litopenaeus vannamei Boone). Food Chem., 97, 41–49. Zhou, X.Q., Zhao, C.R. & Lin, Y. (2007) Compare the effect of diet supplementation with uncoated or coated lysine on juvenile Jian carp (Cyprinus carpio Var Jian). Aquacult. Nutr., 13, 457– 461.