Special Article - Diet
Int J Nutr Sci. 2019; 4(2): 1036.
Evaluation of Marine Algal Fatty Acids Supplementation Broodstock Diets on Macrobrachium rosenbergii (De Man)
Harikrishnan R1*, Balasundaram C2, Devi G3 and Balamurugan P4
1Department of Zoology, Pachaiyappa’s College for Men, India
2Department of Herbal and Environmental Science, Tamil University, India
3Department of Zoology, Nehru Memorial College, India
4Department of Biotechnology, St. Michael College of Engineering and Technology, India
*Corresponding author: Harikrishnan R, Department of Zoology, Pachaiyappa’s College for Men, Kanchipuram - 631 501, Tamil Nadu, India
Received: November 26, 2019; Accepted: December 28, 2019; Published: December 31, 2019
Abstract
The present study was evaluating the marine algal fatty acids supplementation broodstock diets on Macrobrachium rosenbergii. Four broodstock diets were formulated with 3% (diet I), 5% (diet II), 7% (diet III), and 9% (diet IV) inclusion of algal fatty acids to quantify the fatty acid requirement of the brooders for three consecutive spawning cycles. The reproductive parameters such as fecundity, Midgut gland Somatic Index (MSI), and Gonado Somatic Index (GSI) were measured. The fatty acid level in the midgut gland, ovary, and eggs were also measured. The increasing concentration of algal fatty acids in the four broodstock diets have improved the Midgut gland Somatic Index (MSI), Gonado Somatic Index (GSI), and fecundity of the brooders among three spawning cycle. The n-6 and n-3 series of fatty acids level in the midgut gland, ovary, and eggs varied among three spawning cycles. Among the four broodstock diets, diet III performance was higher than diets I, II, and IV. Maximum fatty acids in diet IV showed comparatively less performance than diet III The present study suggested that algal fatty acids supplemented broodstock diets have improved the broodstock performance in fecundity of farm reared M. rosenbergii brooders.
Keywords: Macrobrachium rosenbergii; Broodstock diet; Algal fatty acids; N-6 and n-3 fatty acids
Introduction
Research on the formulation of nutritionally complete maturation diets commercially important for crustacean is limited [1,2]. A thorough knowledge on the nutritional requirement of crustacean species is important to formulate broodstock diet to augment seed production. Moreover, research on broodstock diet for most commercially cultured shrimps such as M. rosenbergii is lacking [3]. However, few studies have been carried out the formulated broodstock diet on M. rosenbergii in various aspects of amino acid profile of eggs [4], different levels of phospholipids [1], and polyunsaturated fatty acids [1,5,6]. Although these studies have not considered the blend of essential fatty acids required to optimize the quality of the brood. Further in these studies, the source of n-6PUFA and n-3HUFA is exclusively derived from fish oil [7].
For the past two decades the formulation of aquaculture feeds using fishmeal and fish oil are main ingredients [8-11]. Aquaculture 2010 demonstrated that fish oil to the tune of 0.96 million t or around 75% of the potential supply [12]. However the escalating demand and diminishing availability of fishery byproducts have shown concern over its quality and sustainability to investigate alternative nutrient source [13]. The substitution of fish oil an alternative oil sources, is therefore imperative for the successful expansion of the industry. In the recent years, fish oil replacement has gained considerable attention [14-17].
Various alternative sources have been identified and investigated to reduce the dependency on fish oil [18-21]. The unicellular organisms such as yeast, molds, bacteria, micro algae, and fungi have been used as additives to aquaculture feed formulation. Few studies have been demonstrated that partial replacement with yeast and bacteria. However, there feeds did not yield better survival, growth nor increased the resistance to disease [22,23].
In this regard micro algae constitute a better choice in replacement of fish oil in aquaculture feed industry. The advantages of unicellular organisms in feed is the availability of perfected technical know how for mass production under controlled and environmentally safe conditions. Moreover, the composition of many microorganisms can be manipulated to ensure higher levels of protein and lipid, by enriching with specific essential amino acids or fatty acids [24- 27]. In fact, oil extracts from unicellular algae containing Long- Chain Polyunsaturated Fatty Acids (LCPUFA) are already used as nutritional supplements in human infant feeds [28]. Therefore in the present study aims to find out the marine algal fatty acids as a source of n-6 and n-3 series of fatty acids to replacement of fish oil in broodstock diets formulation to M. rosenbergii brooders.
Material and Methods
Brooder collection
Pond reared M. rosenbergii females were collected from Srikandapuram (10° 55’ N, 78° 34’ E) Nagapattinum District, Tamilnadu, India and were transported into laboratory in polyethylene bag filled with oxygenated freshwater. The total length (from tip of the rostrum to the end of the telson) and total weight of the females (after blotting them dry) and male were 15.9±0.66cm and 40.21±1.83g and 17.0±1.41cm and 60.9±0.49g. M. rosenbergii mature precociously in farms between size of 6-10g [29] whereas the wild first maturity between 20-40g [30]. Therefore in the present study was used females weighing around 40g to ensure a good quality on par with size of the wild brooders at first maturity.
Experimental design
Each brooder was tagged individually with different color threads on the telson and transferred into four experimental cement tanks (capacity 3500 L) (Table 1). Each tank was supplied aeration. Prawns were fed with commercial grow out diet during the acclimation period. Country made tiles (size 18cm) were provided as shelter to minimize the interaction of the animals [31,32]. There is no significant difference (P‹0.05) exist among the stocked prawns in terms of morphometric parameters such as total length, carapace length, postorbital length, and total weight among females at the beginning of the experiment. The experimental tanks were maintained under the ambient environmental conditions of the temperature 28±2 °C, photo period 12L: 12D cycle, ammonia 0.028mg L-1, and nitrate 1.61mg L-1, respectively recommended in a previous study [33]. Experiments were conducted in four groups of 8 females and 2 males in each at the ratio of 1 male: 4 female based on previous study [34]. These were used to replace the experimental animals periodically to quantify the fecundity, gonado-somatic, and midgut gland somatic index measurement.
Experimental diets
Total length (cm)
Carapace length (cm)
Post orbital Length (cm)
Total weight (g)
I
17.0±1.41*
8.45±0.78*
4.7±0.14*
60.9±0.49*
15.9±0.66*
7.9±0.50*
3.9±0.20*
39.61±0.98*
II
17.0±0*
8.95±0.64*
4.65±0.07*
60.3±0.28*
15.97±0.51*
8.14±0.43*
4.03±0.18*
40.21±1.83*
III
17.1±0.14*
8.75±0.21*
4.5±0.14*
60.9±0.49*
15.93±0.69*
8.03±0.33*
3.97±0.15*
40.43±0.63*
IV
16.9±0.35*
8.65±0.07*
4.5±0.14*
61.2±1.55*
15.95±0.53*
8.06±0.47*
3.91±0.12*
40.3±0.49*
Within row the asterisks represent significant differences (P<0.05).
Table 1: Morphometric parameters of Macrobrachium rosenbergii (n = 10; 8 blue claw females: 2 males) fed with broodstock diets (mean ± SD).
Experimental diets, feeding and analytical procedures
Four isolipidic (9%) and isonitrogenous (45%) broodstock experimental diets were formulated (Table 2). The n-6 and n-3 polyunsaturated fatty acids were derived from heterotrophically grown spray dried cells of Scizochytrium and Crypthecodinium sp. of algal mix (Algamac 2000, Algamac 3050, Aquagrow DHA, and Aquagrow ARA). The four broodstock diets were formulated the increasing percentage of 3% (diet I), 5% (diet II), 7% (diet III), and 9% (diet IV) inclusion of algal mix containing increasing concentration of Linoleic (18:2n-6), Linolenic (18:3n-3), Arachidonic (20:4n-6), Eicosapentaenoic (20:5n-3), and Docosahexaenoic acids (22:6n-3) with other dietary ingredients. The brooders were fed to satiation at 3% body weight twice a day at 09:30 and 18:00 h for a period of 90 days. During the experiment 20% of water was exchanged after siphoning out the feces and uneaten feed. Moulting, gonad development, spawning, and mortality were recorded daily. The proximate composition of crude protein, fat, fiber, total ash, and moisture content of the formulated broodstock diets were determined [35]. The total lipid [36] and fatty acid [37] composition of the tissue samples were analyzed through Gas Chromatography. FAME was analyzed using a Hewlett-Packard 5890 gas liquid chromatograph equipped with DEGS (Diethylene Glycol Succinate) column and Flame Ionization Detector (FID) using nitrogen as the carrier gas. The column temperature was 180 °C and injection temperature was 200 °C. The detector response was recorded. The FAME were identified and quantified by comparison of peak area and retention time of standard fatty acids.
Ingredients
Diets
I
II
III
IV
Fish meal I (Stolephorus commersonii)
16.0
16.0
16.0
16.0
Fish meal II (Sardinella fimbriata)
16.0
16.0
16.0
16.0
Prawn meal
15.0
15.0
15.0
15.0
Casein
10.0
10.0
10.0
10.0
Soybean meal
10.0
10.0
10.0
10.0
Wheat flour
10.0
10.0
10.0
10.0
Starch
10.0
8.0
6.0
4.0
Algal mixa
3.0
5.0
7.0
9.0
Cholesterol
1.0
1.0
1.0
1.0
Astaxanthin
1.0
1.0
1.0
1.0
Vitamin C
0.5
0.5
0.5
0.5
Vitamin E
0.5
0.5
0.5
0.5
Vitamin mixb
2.0
2.0
2.0
2.0
Mineral mixc
3.0
3.0
3.0
3.0
Choline chloride
1.0
1.0
1.0
1.0
BHTd
1.0
1.0
1.0
1.0
Analyzed proximate composition
Crude protein
45.4±0.12*
45.4±0.20*
45.5±0.30*
45.8±0.25*
Crude fibre
0.5± 0.1*
0.6±0.1*
0.6±0.1*
0.6±0.1*
Total Ash
11.7±0.1*
11.8±0.1*
12.1±0.1*
12.3±0.1*
Lipid
8.76±0.15*
8.86±0.05*
8.93±0.23*
9.0±0.26*
Moisture
12.5±0.1*
13.2±0.1*
12.9±0.1*
13.4±0.1*
Within row the asterisks represent significant differences (P < 0.05).
aAlgal mix comprises of Algamac 2000 and Algamac 3050 derivatives of spray dried cells of Schizochytrium; Aquagrow DHA and Aquagrow ARA a derivative of spray dried Crypthecodinium.
bVitamin mix (Nicholas Piramal India Ltd) mix formulated to provide the following per kg of feed: Vit A, 1,00,000 IU; Vit D3 10,000 IU; Thiamin, 100mg; Riboflavin, 100 mg; Pyridoxine hydrochloride, 30 mg; Cyanocobalamin, 150 mg; Nicotinamide, 1000 mg; Calcium pantothenate, 160.3 mg; Ascorbic acid, 1500 mg; a-Tocopheryl acetate, 250 mg; Biotin, 2.50 mg.
cMineral mix (Nicholas Piramal India Ltd) formulated to provide the following per kg of feed: Calcium phosphate 1290 mg; Magnesium oxide, 600 mg; Dried ferrous sulphate, 320.04 mg; Manganese sulphate, 20.3 mg; Total phosphorus in the preparation, 250.80 mg; Copper sulphate 33.9 mg; Zinc Sulphate, 22.0 mg; Sodium molybdate, 2.5 mg and Sodium borate 8.8 mg.
dBHT - Butylated hydroxy toluene
Table 2: Composition (% dry weight) of broodstock experimental diet (mean ± SD; n = 3).
Measurement of fecundity and egg quality
The maturation stages of the brooders were classified according to their ovary size and color observed through the carapace [38]. On third day of spawning, the eggs were manually removed from the abdomen of each female; clutch and female weight were determined separately. Three independent clutch weight samples at 50 mg and the number of eggs in each sample were counted. Fecundity was estimated as the number of eggs per clutch (eggs/g female). The changes during three consecutive reproductive cycles of three females from each broodstock diet experiment diets were measured to record the total weight and then sacrificed to find out Midgut Gland Somatic Index (MSI) and Gonado Somatic Index (GSI) at reproductive stage V. The reproductive performance of females fed with four broodstock diets were quantified by studying the changes in fecundity, clutch weight, gonado somatic index, and midgut gland somatic index for three consecutive spawning cycles. The fatty acid profiles of midgut gland, ovary, and eggs were also quantified. The fatty acid analyses revealed that appeared many fatty acids in trace level. These fatty acids in trace amounts, however, are not known to have any significant role neither in terms of nutrition nor embryonic development [39]; hence they have not been considered further.
Statistical analysis
Data on intermoult cycle, fecundity, female body weight, gonadosomatic index, midgut gland somatic index, and fatty acid profiles were analyzed by one way Analysis Of Variance (ANOVA) and significant difference compared at (P ‹ 0.05); the Least Significance Differences (LSD) test was applied.
Results
Proximate composition in diets
The analyzed proximate composition of lipid and protein composition of the broodstock diets were designed to comprise 9.0% and 45% of the dry weight (Table 2). The n-6 and n-3 polyunsaturated fatty acids levels were increased with increasing percentage inclusion of algal mix indicative of the high level of docosahexaenoic acid (DHA 4.8–14.0mg g-1 DW) and Eicosapentaenoic acid (EPA 4.7–13.6mg g-1 DW) in the diets (Table 3). Relatively high levels of saturated and n-6 polyunsaturated fatty acids were present in the diets. The n-6/n-3 ratio of all the diets was approximately 1. The fatty acid profile of midgut gland, ovary, and eggs of brooders fed with four broodstock diets for three consecutive spawning has been presented in Table 4,5 and 6.
Fatty acids
Diet I
Diet II
Diet III
Diet IV
-3%
-5%
-7%
-9%
14:00
7.19
11.504
16.54
20.851
16:00
4.98
7.968
11.45
14.442
18:00
6.68
10.688
15.36
19.372
18:1n-9
5.74
9.184
13.2
16.646
18:2n-6
13.09
20.944
30.11
37.961
18:3n-3
4.41
7.056
10.14
12.789
20:4n-6
1.57
2.512
3.611
4.553
20:5n-3
4.71
7.536
10.83
13.659
22:6n-3
4.83
7.728
11.11
14.007
S Saturates
18.85
30.16
43.355
54.665
S Mono-unsaturates
5.74
9.184
13.202
16.646
S n-6 PUFA
14.66
23.46
33.718
42.514
S n-3 HUFA
13.95
22.32
32.085
40.455
n-6/n-3 ratio
1.05
1.05
1.05
1.05
Table 3: Fatty acid composition (mg g-1 dry weight) of the experimental diets incorporated with algal.
Diet I
Diet II
Diet III
Diet IV
Midgut gland
Total lipids
26.7±1.31a
30.6±1.09b
32.8±1.01c
34.7±1.09d
14:00
17.99±0.07a
29.58±0.63b
41.34±0.08c
50.08±0.05d
16:00
12.47±0.06a
19.13±0.05b
29.79±0.06c
36.13±0.05d
18:00
16.03±0.06a
23.49±0.06b
36.88±0.07c
44.56±0.06d
18:1 n-9
13.20±0.06a
21.13±0.05b
29.05±0.07c
36.63±0.06d
18:2 n-6
30.77±0.06a
49.44±0.07b
69.26±0.06c
87.33±0.09d
18:3 n-3
10.23±0.06a
16.52±0.05b
23.34±0.07c
29.55±0.07d
20:4 n-6
3.65±0.05a
5.90±0.06b
8.60±0.04c
10.52±0.06d
20:5 n-3
6.12±0.05a
9.80±0.07b
14.09±0.08c
16.40±0.07d
22:6 n-3
5.79±0.08a
9.74±0.06b
13.35±0.03c
16.82±0.05d
S Saturates
46.46±0.06a
72.53±0.05b
108.03±0.04c
130.73±0.04d
S Mono-unsaturated
13.21±0.05a
21.13±0.05b
29.05±0.05c
36.63±0.05d
S n-6 PUFA
34.42±0.05a
55.34±0.04b
77.85±0.06c
97.84±0.04d
S n-3 HUFA
22.14±0.05a
36.03±0.04b
50.74±0.05c
62.74±0.05d
Ovary
Total lipids
14:00
30.7±1.25a
33.6±0.964b
35.7±0.984c
37.5±1.178d
16:00
19.42±0.05a
30.50±0.07b
42.99±0.06c
54.23±0.06d
18:00
12.96±0.06a
20.73±0.05b
27.50±0.08c
37.56±0.06d
18:1 n-9
16.9±0.8a
27.80±0.07b
38.42±0.06c
46.50±0.04d
18:2 n-6
13.78±0.05a
22.96±0.03b
31.70±0.08c
38.60±0.07d
18:3 n-3
32.11±0.04a
51.74±0.120b
74.98±0.09c
95.66±0.04d
20:4 n-6
10.81±0.07a
17.29±0.05b
24.36±0.02c
30.06±0.06d
20:5 n-3
4.25±0.07a
7.05±0.06b
10.49±0.02c
12.76±0.07d
22:6 n-3
8.96±0.08a
14.32±0.04b
20.01±0.05c
23.23±0.05d
S Saturates
9.39±0.07a
13.93±0.05b
21.10±0.06c
25.21±0.07d
S Mono-unsaturated
49.07±0.06a
79.01±0.05b
108.89±0.06c
138.25±0.06d
S n-6 PUFA
13.78±0.04a
22.96±0.05b
31.68±0.06c
38.60±0.07d
S n-3 HUFA
36.38±0.09a
58.81±0.08b
85.49±0.09c
108.42±0.05d
Eggs
29.12±0.05a
45.51±0.03b
65.43±0.06c
78.49±0.06d
Total lipids
14:00
31.73±1.150a
34.5±1.01b
36.4±0.954c
39.6±1.044d
16:00
20.15±0.08a
31.65±0.06b
43.83±0.06c
55.28±0.09d
18:00
13.45±0.05a
22.32±0.05b
30.95±0.08c
38.27±0.06d
18:1 n-9
17.37±0.06a
28.35±0.09b
39.96±0.06c
48.45±0.07d
18:2 n-6
14.36±0.06a
23.90±0.07b
33.02±0.05c
39.97±0.09d
18:3 n-3
34.05±0.09a
55.51±0.07b
80.40±0.06c
102.51±0.06d
20:4 n-6
11.92±0.06a
18.36±0.07b
25.37±0.08c
34.56±0.10d
20:5 n-3
5.83±0.06a
9.07±0.09b
12.65±0.06c
15.50±0.07d
22:6 n-3
11.79±0.06a
18.86±0.08b
28.19±0.06c
34.16±0.07d
S Saturates
12.09±0.06a
20.11±0.07b
30.01±0.08c
36.44±0.07d
S Mono-unsaturated
50.96±0.05a
82.28±0.07b
114.70±0.04c
141.96±0.03d
S n-6 PUFA
14.35±0.05a
23.90±0.06b
33.01±0.05c
39.96±0.06d
S n-3 HUFA
39.89±0.07a
64.59±0.06b
93.04±0.04c
118.02±0.05d
35.78±0.06a
57.29±0.06b
83.55±0.08c
105.11±0.05d
Within row the asterisks represent significant differences (P < 0.05).
Table 4: Total lipid (% dry weight) and principal fatty acid content (mg g-1 dry weight) of midgut gland, ovary, and eggs of females M. rosenbergii (mean ± S.D; n = 3) fed with four broodstock diet during the first spawning.
Diet I
Diet II
Diet III
Diet IV
Midgut gland
Total lipids
28.7±0.802a
31.56±1.305b
33.93±1.250c
35.8±1.054d
14:00
18.34±0.07a
30.50±0.06b
42.19±0.08c
51.11±0.07d
16:00
12.71±0.07a
19.54±0.07b
30.36±0.08c
36.86±0.09d
18:00
16.19±0.08a
24.06±0.07b
37.66±0.08c
45.54±0.07d
18:1 n-9
13.33±0.06a
21.58±0.05b
29.71±0.06c
37.47±0.08d
18:2 n-6
31.83±0.07a
51.13±0.09b
73.77±0.06c
91.12±0.07d
18:3 n-3
11.14±0.10a
17.87±0.08b
25.58±0.09c
31.97±0.04d
20:4 n-6
3.73±0.06a
5.99±0.09b
8.76±0.08c
10.94±0.05d
20:5 n-3
6.23±0.07a
10.19±0.09b
14.63±0.06c
17.08±0.07d
22:6 n-3
6.34±0.05a
9.68±0.08b
13.91±0.07c
18.22±0.05d
S Saturates
47.19±0.04a
74.07±0.05b
110.18±0.07c
133.44±0.05d
S Mono-unsaturated
13.34±0.05a
21.60±0.07b
29.71±0.04c
37.46±0.07d
S n-6 PUFA
35.53±0.04a
57.08±0.05b
82.50±0.04c
102.05±0.05d
S n-3 HUFA
23.72±0.04a
37.69±0.06b
54.09±0.07c
67.26±0.06d
Ovary
Total lipids
14:00
32.1±1.258a
34.7±1.058b
36.8±1.10c
39.7±0.850d
16:00
19.77±0.05a
30.51±0.06b
43.84±0.07c
55.26±0.05d
18:00
13.20±0.06a
20.34±0.07b
28.06±0.05c
38.29±0.06d
18:1 n-9
17.04±0.05a
28.34±0.06b
39.19±0.04c
47.48±0.08d
18:2 n-6
14.07±0.05a
23.44±0.08b
32.35±0.06c
39.13±0.07d
18:3 n-3
34.04±0.06a
54.89±0.07b
79.79±0.06c
104.40±0.06d
20:4 n-6
11.29±0.04a
18.00±0.07b
24.86±0.05c
33.28±0.09d
20:5 n-3
3.79±0.06a
5.54±0.05b
8.77±0.09c
11.08±0.08d
22:6 n-3
9.18±0.05a
14.72±0.07b
20.05±0.05c
23.92±0.06d
S Saturates
9.66±0.05a
15.17±0.06b
21.69±0.09c
25.92±0.05d
S Mono-unsaturated
49.99±0.05 a
79.15±0.06 b
111.07±0.05c
140.99±0.04d
S n-6 PUFA
14.08±0.06 a
23.43±0.05 b
32.34±0.05c
39.13±0.04d
S n-3 HUFA
37.80±0.05a
60.41±0.05b
88.52±0.04c
115.46±±0.06d
Eggs
30.14±0.05a
47.83±0.04b
66.56±0.06c
83.06±0.04d
Total lipids
14:00
32.83±1.01a
35.8±1.054b
38.7±0.721c
40.6±0.700d
16:00
20.49±0.06a
31.66±0.08b
45.50±0.07c
53.17±0.05d
18:00
13.69±0.06a
22.73±0.07b
31.50±0.05c
39.01±0.08d
18:1 n-9
17.72±0.05a
28.87±0.04b
42.74±3.44c
49.39±0.06d
18:2 n-6
14.65±0.06a
24.81±0.07b
33.66±0.05c
40.80±0.08d
18:3 n-3
36.00±0.06a
58.23±0.07b
83.99±0.05c
112.00±0.07d
20:4 n-6
11.47±0.05a
18.47±0.06b
25.86±0.05c
33.66±0.06d
20:5 n-3
4.34±0.07a
6.66±0.05b
12.83±0.04c
11.17±0.06d
22:6 n-3
12.03±0.07a
19.23±0.06b
28.74±0.08c
34.84±0.05d
S Saturates
12.33±0.06a
20.50±0.08b
30.56±0.05c
37.15±0.09d
S Mono-unsaturated
51.88±0.04a
83.22±0.05b
117.69±0.05c
141.56±0.06d
S n-6 PUFA
14.65±0.05a
24.80±0.04b
33.67±0.05c
40.79±0.06d
S n-3 HUFA
40.32±0.05a
64.89±0.06b
96.83±0.05c
123.14±0.04d
35.81±0.05a
58.13±0.04b
85.14±0.05c
105.61±0.06d
Within row the asterisks represent significant differences (P < 0.05).
Table 5: Total lipid (% dry weight) and principal fatty acid content (mg g-1 dry weight) of midgut gland, ovary, and eggs of females M. rosenbergii (mean ± S.D; n = 3) fed with four broodstock diet during the second spawning.
Diet I
Diet II
Diet III
Diet IV
Midgut gland
Total lipids
30.53±0.874*
33.43±0.757*
35.5±0.793c
36.8±0.954d
14:00
18.70±0.06*
31.09±0.08*
43.85±0.09c
53.17±0.05d
16:00
12.96±0.06*
19.96±0.09*
30.94±0.05c
37.57±0.08d
18:00
16.72±0.06*
25.14±0.07*
38.44±0.08c
47.48±0.10d
18:1 n-9
14.08±0.07*
22.50±0.06*
31.04±0.07c
39.13±0.06d
18:2 n-6
33.39±0.06*
53.64±0.08*
78.30±0.07c
96.81±0.05d
18:3 n-3
11.69±0.07*
18.69±0.06*
26.39±0.09c
33.90±0.07d
20:4 n-6
3.79±0.07*
6.09±0.06*
9.95±0.07c
11.63±0.06d
20:5 n-3
6.84±0.05*
10.95±0.07*
15.73±0.06c
18.45±0.05d
22:6 n-3
6.63±0.06*
10.45±0.07*
15.00±0.08c
18.93±0.06d
S Saturates
48.35±0.05*
76.11±0.06*
113.18±0.07c
138.20±0.06d
S Mono-unsaturated
14.07±0.05*
22.52±0.06*
31.04±0.06c
39.15±0.04d
S n-6 PUFA
37.18±0.07*
59.72±0.06*
88.24±0.06c
108.43±0.05d
S n-3 HUFA
25.15±0.05*
40.06±0.06*
57.08±0.05c
71.26±0.07d
Ovary
Total lipids
14:00
33.7±1.201*
35.6±1.250*
37.6±1.30c
40.46±0.611d
16:00
20.14±0.05*
32.24±0.08*
43.00±0.07c
56.17±0.05d
18:00
13.47±0.08*
20.74±0.06*
29.23±0.05c
39.00±0.07d
18:1 n-9
17.37±0.06*
28.89±0.08*
40.72±0.05c
48.44±0.05d
18:2 n-6
14.65±0.06*
23.90±0.07*
33.68±0.06c
39.97±0.07d
18:3 n-3
36.65±0.05*
58.66±0.07*
84.30±0.06c
106.30±0.07d
20:4 n-6
11.44±0.08*
18.36±0.07*
25.41±0.07c
33.28±0.08d
20:5 n-3
3.94±0.05*
6.29±0.07*
9.24±0.08c
11.86±0.07d
22:6 n-3
9.43±0.06*
14.35±0.08*
21.14±0.07c
25.29±0.06d
S Saturates*
11.13±0.06*
15.46±0.05*
22.25±0.07c
27.34±0.08d
S Mono-unsaturates**
50.95±0.06*
81.80±0.06*
112.39±0.07c
143.73±0.05d
S n-6 PUFA
14.65±0.06*
23.88±0.04*
33.66±0.03c
39.94±0.04d
S n-3 HUFA
40.60±0.06*
64.94±0.05*
93.51±0.05c
118.15±0.07d
Eggs
32.01±0.06*
48.14±0.05*
68.72±0.07c
85.83±0.05d
Total lipids
14:00
34.5±0.945*
36.5±0.800b
38.4±1.054c
42.6±0.700d
16:00
20.85±0.05*
32.80±0.07b
45.49±0.06c
55.29±0.08d
18:00
14.20±0.06*
23.54±0.07b
32.65±0.05c
39.00±0.06d
18:1 n-9
18.38±0.06*
29.40±0.07b
40.72±0.06c
51.36±0.09d
18:2 n-6
15.22±0.06*
24.35±0.07b
35.00±0.07c
42.44±0.05d
18:3 n-3
39.29±0.07*
62.84±0.06b
90.34±0.07c
113.89±0.05d
20:4 n-6
12.36±0.06*
19.42±0.08b
26.90±0.07c
35.19±0.06d
20:5 n-3
4.72±0.05*
7.68±0.06b
10.85±0.07c
13.69±0.09d
22:6 n-3
12.50±0.07*
19.97±0.04b
29.80±0.05c
36.20±0.08d
S Saturates
12.80±0.08*
21.26±0.06b
31.68±0.05c
38.51±0.07d
S Mono-unsaturated
53.42±0.04*
85.70±0.07b
118.84±0.05c
145.60±0.06d
S n-6 PUFA
15.23±0.05*
24.35±0.06b
35.00±0.04c
42.45±0.05d
S n-3 HUFA
43.99±0.05*
70.50±0.05b
101.16±0.03c
127.55±0.06d
37.64±0.06*
60.64±0.0b
88.35±0.06c
109.88±0.05d
Within row the asterisks represent significant differences (P < 0.05).
Table 6: Total lipid (% dry weight) and principal fatty acid content (mg g-1 dry weight) of midgut gland, ovary, and eggs of females M. rosenbergii (mean ± S.D; n = 3) fed with four broodstock diet during third spawning.
First spawning
Total lipid content of the midgut gland, ovary, and eggs of females fed with broodstock diets were found to be accumulating among diets with increasing concentration of algal mix. Such an increase in the accumulation is statistically significant (P‹0.05) (Table 4). The increasing accumulations of total lipids elucidate the maximum utilization of the broodstock diet. The fatty acid profiles of the organs indicate that saturated fatty acids are predominately found over than the n-6PUFA and n-3HUFA. There is a significant difference observed in the accumulation of the principal fatty acids on the basis of the diets (P‹0.05). Over accumulation of saturated fatty acids such as myristic (14:0), palmitic (16:0), and stearic (18:0) acids emphasize their nutritive role in embryonic stage. The percentage accumulation of various types of fatty acids varies among the organs, which differed from one another. For example in midgut gland the range for saturated 2.2 to 2.6, mono-unsaturated 2.2 to 2.3, n-6 polyunsaturated 2.30 to 2.38, and n-3 highly unsaturated fatty acids 1.19 to 2.34, respectively. Similarly ovarian fatty acid profile revealed that more percentage accumulation of fatty acids, such as saturated 2.4 to 2.7, monounsaturated 2.3 to 2.5, n-6 polyunsaturated 2.45 to 2.9, and n-3 highly unsaturated fatty acids 1.7 to 2.45, which is differed significantly (P‹0.05). It is interesting to note that when compare with midgut gland and ovary, the accumulation of lipids is higher in eggs. The accumulation of various types of fatty acids, such as saturated 2.5 to 2.8, monounsaturated 2.4 to 2.6, n-6 polyunsaturated 2.6 to 3.7, and n-3 highly unsaturated fatty acids 2.5 to 2.7 in the eggs of prawns fed with four diets are also differed statistically (P ‹ 0.05). Increased accumulation of total n-6 polyunsaturated and n-3 highly unsaturated fatty acids in eggs is due to satisfy the nutrient requirement of the growing embryos.
Second spawning
The fatty acid profile of prawns fed with four broodstock diets at second spawning revealed that there is a significant difference in accumulation (P‹0.05). The accumulation range of fatty acids aree saturated (2.25 to 2.65), monounsaturated (2.25 to 2.35), n-6 polyunsaturated (2.36 to 2.45), and n-3 highly unsaturated fatty acids (1.25 to 2.53) in the midgut gland among four diets were differed significantly (P‹0.05). Regarding the fatty acid profile of ovary, there Within row the asterisks represent significant differences (P ‹ 0.05). was significant difference (P ‹ 0.05) found in saturated (2.45 to 2.75), monounsaturated (2.35 to 2.55), n-6 polyunsaturated (2.40 to 2.65), and n-3 highly unsaturated (1.75 to 2.60) when compared to first spawning. However, eggs are found to have a maximum accumulation of fatty acids when compared to midgut gland and ovary, which is statistically significant (P‹0.05) (Table 5).
Third spawning
The individual fatty acids studied in the third spawning indicates that there was significant difference in the percentage of accumulation in all organs with reference to diets. Increase in the level of fatty acids with regard to spawning cycles is statistically significant at 5% level. In midgut gland similar observation was found in the case of n-6 PUFA and n-3 HUFA. Similar trend was observed in ovary and eggs of three consecutive spawning cycles, the accumulation of fatty acids are in the following order: eggs ‹ ovaries ‹ midgut gland. The four broodstock diet has no influence (P›0.05) on the duration of the intermoult period and breeding frequency but had an influence (P‹0.05) on GSI and MSI (Table 7). The diets had significant influence (P‹0.05) on the fecundity and clutch weight. The number of eggs produced per spawn varied considerably among diets. But there was direct relationship exist between the number of eggs produced or spawn and the diets. The relationship between the number of eggs per spawning event (NES) and female size (W; in g) in each diet is: Diet I = 955+1147 W (r2 = 0.84); Diet II = 909+2088 W (r2 = 0.962); Diet III = 1000+1210 W (r2 = 0.809), and Diet IV = 811+6931 W (r2 = 0.731) among three consecutive spawning cycles. The egg production efficiency of brooders fed with four broodstock diets were ranged from 914 eggs g-1 body weight in diet I and 982 eggs g-1 body weight in diet IV in the first spawning cycle, which is significantly increased in the second spawning (P‹0.05) but no such difference observed (P>0.05) in the third spawning. The regression between female size and egg production efficiency (number of eggs per female wt; eggs g-1) resulted in low relationship coefficient among diets; Diet I (r2 = 0.044); Diet II (r2 = -.226); Diet III (r2 = -.164), and Diet IV (r2 = -.378) among three spawning cycles.
Diets
I
II
III
IV
Intermoult period First spawning
26.9±0.83*
25.9±1.35*
26.0±1.60a
26.5±1.07*
(days) Second spawning
27.3±1.04*
26.9±1.34*
28.0±1.07a
27.0±1.07*
Third spawning
28.0±1.07*
28.1±0.83*
28.4±0.52a
28.0±0.76*
GSI (%) First Spawning
9.83±0.026*
9.99±0.035*
10.26±0.055bc
10.45±0.050*
Second Spawning
10.26±0.038*
10.27±0.045*b
10.31±0.066bc
10.19±0.036*
Third Spawning
10.23±0.05*
10.21±0.051*
10.19±0.04bc
10.26±0.05*
MSI (%) First Spawning
4.91±0.040*
5.156±0.297*
5.13±0.05ab
5.24±0.045*
Second Spawning
5.03±0.025*
5.133±0.035*
5.16±0.04ab
5.13±0.045*
Third Spawning
5.15±0.050*
5.096±0.050*
5.12±0.05ab
5.18±0.050*
Within row the asterisks represent significant differences (P < 0.05).
Table 7: Reproductive performance, GSI and MSI of females M. rosenbergii (mean ± S.D; n = 3) fed four experimental diets during consecutive three spawning.
Discussion
Nutrition is an important factor which influences the maturation of wild as well as captive crustacean broodstock [2,40]. The growth and fecundity of an organism depends upon a number of environmental and physical factors, including the impacts of food quantity, composition, and seasonal variability [41]. Recent researches on nutrition were mainly focused on the use of highly unsaturated fatty acids on the reproductive performance. Marine fish oil is the main source of highly unsaturated fatty acids. In view of the inconsistent availability of fish oil to satisfy the n-3 highly unsaturated fatty acids and other sources are being tested. Marine algae are an alternate to replace conventional source and satisfy the current requirement of highly unsaturated fatty acids. In this regard, many research being carried out with algae as a nutrient source for fishes [42] and larvae of P. vannamei [43].
In the present study incorporation of heterotrophically grown Scizochytrium and Crypthecodinium sp. in the diet augmented the reproductive performance of M. rosenbergii brooders; this resulted in a significantly increased fecundity and three consecutive berried moults as evident from earlier studies. In corporation of fatty acids in midgut gland, ovary, and eggs of females in the present study is in agreement previous study in the same species [1,3]. The egg production efficiency of M. rosenbergii studied in the farms before conducting the experiment over a 218 M. rosenbergii females ranged from 767.1 eggs g-1 to 874.4 eggs g-1 body weight. When fed with formulated broodstock experimental diets significantly increased (P‹0.05) from 914 eggs g1 (diet I) to 982 eggs g-1 (diet IV) in the first spawning. This is the first study such comparative analyzed in M. rosenbergii to improve the brooder quality. At the same time compare to the wild brooder with the present result revealed that the average efficiency of egg production was 844.41 eggs g1 body weight of wild M. rosenbergii [34].
In the present study significantly increase the fecundity of experimental brooders when compare to the wild brooders. The efficiency of egg production also increases with female size [44] as reported in M. rosenbergii [1,45,46]. Previous studies carried out in M. rosenbergii for a period of 180 days on maturation diet fortified with fish oil based different levels of n-6 and n-3 HUFA showed an improved fecundity and larval viability [1,3]. Addition of higher amounts of linoleic acid (18:2n-6) from 3 to 13mg g-1 improved the efficiency of egg production from 1282.5±254.3 to 1570±203.1. Moreover, high levels of 18:2n-6 and n-3 HUFA (13 and 15mg g-1 DW respectively) improved the fecundity, egg hatchability, and overall quality of the larvae.
The dietary n-6/n-3 ratios of these diets are 0.94, 2.64, and 0.70; of these 0.94 produced a higher fecundity and larval quality. In this present study with algal fatty acids indicates that the increased amount of linoleic acid (13.09 to 37.96 mg g-1 DW) and n-3 HUFA (13.95 to 40.45mg g-1 DW) in the experimental diets significantly improved the fecundity and enhanced egg production efficiency. The spurt in egg production efficiency in first spawning followed by the stagnation in the subsequent spawning may be due to the maximum level attained that was maintained. The maturation performance, offspring quality, and lipid composition of M. rosenbergii females fed with chosen levels of phospholipids do not have any specific role on reproduction, however, linoleic acid significantly enhanced the fecundity, but higher level of n-3 HUFA had no such effect [1].
The egg production is not only determined by the linoleic acid content alone, these variations in fecundity may be due to different conditions of female maintenance in the laboratory, physiological conditions of the experimental animals, and season (personal communication, Sorgeloos). Normally female M. rosenbergii mature when they reach a size of 15-20g body weight while, broodstock ponds they mature earlier (minimum size of 6.5-10g) [29]. Use of such precociously mature females in hatcheries results in poor egg and larval quality; subsequently offspring of these females mature even more precociously still worsening the quality of offspring. In the wild the size at first maturity ranges from 20-40g [30]. Quality of eggs spawned by a captive broodstock remains a major constraint for the production of viable post larvae in commercial hatcheries [47], which is to a great extent influenced by the quality of diet [48]. Therefore in the present study suggested that algal fatty acids supplemented broodstock diets have improved the broodstock performance in fecundity of farm reared brooders and its for sustainable aquaculture practice. This study indicates that an algal fatty acids supplement instead of fish oil on the nutritional requirement of broodstock of grow-out crustaceans and even widely cultured organisms. Also further investigation on the broodstock performance at fecundity level before recommended the alage fatty acids supplementation instead of fish meal in aquaculture is warranted.
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