Abstract: Azolla Protein Concentrate (APC) mixed with dry Spirogyra Powder (SP) at 4:1 ratio was evaluated in the diet of Labeo rohita fry as a substitute of fish meal. One hundred and sixty fry (avg. wt. 1.3±0.2 g) were distributed in five experimental groups with each of four replicates and fed for 60 days. Five isonitrogenous and isocaloric diets were prepared by replacing fish meal with APC-SP mixture on equal protein basis viz., T1 (0%); T2 (25%); T3 (50%); T4 (75%) and T5 (100%). Crude protein content of the APC increased to 44.96% than the dry azolla containing 24.06% CP. Growth and nutrient utilization of the Labeo rohita fry were evaluated in terms of percent weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, net protein utilization, protease activity, in vitro protein and carbohydrate digestibility. Highest percent weight gain (135.57), SGR (1.45%), protease activity (18.56 units mg protein-1), protein digestibility (93.16%) carbohydrate digestibility (35.08%), FCE (0.75), PER (1.9), NPU (28.90) was recorded in control (0% APC-SP), which was similar with T2 and T3 groups but decreased significantly (p<0.05) as the APC-SP content increased in the diet. There were no histological changes except the vacuolation of hepatic cells with increased concentration of APC-SP in the diet. No mortality was registered in any of the experimental groups signifying no adverse effect of feeding APC-SP. It concludes that APC is a good source of protein and can be used to the maximum extent of 16.25% by replacing 10% fish meal in the diet of Labeo rohita fry.
Introduction
Aquaculture has been considered as answer to the shortfall from the global capture fisheries production. The intensification of aquaculture has led to a dependence on artificial feed ingredients especially on fishmeal due to its high quality protein and presence of some unidentified growth factors. Increased demand of this noble ingredient concomitant with its static production has put extra pressure on the animal or fish nutritionist to search for the alternate protein sources. Hardy (2000) predicted that the fishmeal requirement during the year 2010 would be 2.83 mmt, which is 44% of the total global fishmeal production. This is somewhat 716000 mt over estimate of fishmeal use in 2000. If fishmeal production remains same as of today, the demand in aquaculture sector may go upto 63% of the total global fishmeal production. Hence, alternate protein sources need to be explored to overcome this problem.
Lot of works have been done to include many of the unconventional protein sources to be used in animal or fish feed, but use of aquatic plant has given little emphasis. In this connection aquatic weeds or plant fodder, rich in protein may be considered as the alternative protein sources in the aqua-feed as suggested by Chiayvareesajja et al. (1989) Ray and Das (1995). Aquatic weeds have been used as complete or partial replacement of the fishmeal. These include lemna, eicchornia, azolla, najas etc. in different forms such as meal or protein concentrate.
Azolla is a heterosporous free-floating aquatic fern that lives symbiotically with the nitrogen fixing algae, Anabena azollae (Peters et al., 1979). It has attracted the attention of many livestock, poultry and fish farmers due to its high protein content (19-31%) and a favourable essential amino acid composition for animal nutrition (Buu, 1971; Antoine et al., 1987; Santiago et al., 1988). The growth rate of azolla is very rapid and it becomes double the weight in 2-3 days. It has been used as fresh feed in cage culture (Cagaun and Nerona, 1986) in integrated rice-azolla-fish (FAO, 1988).
Algae have been identified as an alternate protein source in the fish diet, particularly in the tropical developing countries, where algal production rates are high (Venkataraman et al., 1980). The most commonly mass cultured algae evaluated, as protein sources in the fish feed, are unicellular micro algae chlorella, Scendesmus and Spirulina (Terao, 1960; Stanley and Jones, 1976; Sandbank and Hepher, 1978). However, from practical point of view, these are costly to produce and harvest. Hence, it appears logical to evaluate other types of algae, which grows extensively under natural conditions and incurs little or no production cost.
The filamentous green alga, Spirogyra maxima grow extensively in freshwater ponds, shallow ditches and in inundated areas. It is frequently a problem in nursery ponds trapping the fries, impediment to boating, when in bloom and on decay it produces offensive odour together with dissolved oxygen depletion resulting in fish kills.
Though azolla has been used in various forms such as fresh (Almazan et al., 1986), sun dried (Almazan et al., 1986; Santiago et al., 1988; Joseph et al., 1994), either singly or in combination (Antoine et al., 1987; El-Sayeed, 1992; Micha et al., 1988), its use as a protein concentrate has not been reported till date.
In the present study two aquatic plants were selected with specific purposes. Though the protein content of both the aquatic plant is similar, azolla was selected due to its better protein quality and spirogyra due to its abundant availability. Azolla Protein Concentrate (APC) was made for more addition of protein from azolla so as to replace fishmeal. Hence, in the present study a mixture was prepared with APC and dry spirogyra with a ratio of 4:1 and added in the feed at a graded level as a substitute of fishmeal in the diet of Labeo rohita fry.
Materials and Methods
Experimental Design
The experiment was conducted from 21st December, 1998 to 18th March, 1999
at Digestive Physiology, Nutrition and Feed laboratory of Central Institute
of Fisheries Education, Versova, Mumbai. Labeo rohita fry were procured
from Khapoli fish seed farm, Maharasthra, India and acclimatized for 15 days
in a FRP circular tank (1000 L). One hundred and eighty fry of uniform size
(1.3±0.2 g) were distributed in 5 treatments with each of 4 replicates.
Nine fry were stocked in each tub containing 40 L water. Round the clock aeration
was provided to all the tubs to maintain the dissolved oxygen level. Experimental
tubs were cleaned manually by siphoning at least 50% of the water daily along
with the faecal matter and left over feeds. The siphoned water was replaced
by equal volume of fresh water. Feed was given twice daily at 09.00 and 16.00
h at 5% of their body weight. Feeding rate was adjusted by daily observation
of the refusal.
Diet Preparation
Preparation of Azolla Protein Concentrate (APC)
APC was prepared by modifying the method of Harendranath and Singh (1984).
Azolla caroliniana was collected from azolla culture unit of Central
Institute of Fresh water Aquacultute, Bhubaneswar, Orissa, India. It was then
pulped in a domestic mixer with little addition of water to facilitate better
maceration. The pulp was than hand pressed in a cloth of 5 μ mesh size
to collect the juice and acid precipitated by using 2% HCl with a juice to acid
ratio of 9:1. Complete precipitation of protein was done within 90 min. The
supernatant was filtered off and the precipitate was dried in a hot air oven
at 50°C until complete drying and used in the feed.
Spirogyra maxima was collected locally, washed thoroughly to remove the adhering dirt followed by drying in a hot air oven at 60°C. It was pulverized to fine powder and mixed in the feed.
Experimental Diets
Before feed formulation the proximate composition of the feed ingredients
especially APC, fishmeal, spirogyra were determined (Table 1).
Five semi-purified diets were prepared by gradual replacement of fishmeal with
a mixture of Azolla Protein Concentrate (APC): Spirogyra Powder (SP) at a ratio
of 4:1. Equal amount of protein from the replaced fishmeal was substituted with
same amount of protein from APC-SP viz., T1 (0% substitution, control),
T2 (25%), T3 (50%), T4 (75%), T5
(100%) (Table 2). The percent inclusions of different ingredients
were adjusted to make all the diets iso-proteinous (39-40%) and iso-caloric
(347-387 Kcal DE/100 g). All the ingredients except the vitamin, mineral mixture
were mixed thoroughly in a mix blender and the required amount of water and
oil were added. Mixed ingredients were kept in an airtight polyethylene packet
for one hour for proper conditioning followed by steam cooking for 20 min in
a pressure cooker. Vitamin and minerals were added to the feed mix after cooling
and passed through an extruder with a 2 mm die (Twin Screw Extruder, Basic Technology
Private Ltd., Calcutta-12) at barrel screw speed of 430 rpm, feeding rate of
90 rpm, barrel temperature of 90°C and cutter speed of 1100 rpm. Pellets
thus obtained were dried at 60°C in an oven and put in airtight polyethylene
bag until further use.
Growth Parameters
Fishes were weighed at every 15 days to assess the growth performance in
terms of percent weight gain, Specific Growth Rate (SGR), Feed Conversion Efficiency
(FCE), Protein Efficiency Ratio (PER) and protein gain. Fishes were counted
at the end of the experiment and compared with the initial stock to calculate
survival.
Water Quality Parameters
Temperature, pH, dissolved oxygen, ammonia, free carbon dioxide and total
alkalinity were measured once in every week according to standard methodology
(APHA, 1985). Water temperature of different experimental groups ranged from
22.1-26.8°C. pH value in all the experimental groups varied within a range
of 7.2-7.7. Dissolved oxygen of all the experimental tubs recorded was within
the range of 6.15-6.97. Free carbon dioxide was not detected in any of the experimental
container. Range of total alkalinity and hardness were 183-210 and 153-167 ppm,
respectively. Ammonia and nitrate concentration of different container were
within the range of 0.62-0.92 and 0.53-0.85 ppm, respectively.
| Table 1: | Proximate composition (% DM basis) of the ingredients |
| Table 2: | Composition of the ingredients for 100 g experimental diets of the different groups |
| *Composition of vitamin mineral mixture of 1 kg: Vitamin A - 50,00,000 IU; Vitamin D3 - 10,00,000 IU; Vitamin B2 - 2.0 g; Vitamin E - 750 units; Vitamin K - 1.0 g; Calcium pantothenate 2.5 g; Nicotinamide - 10.0 g; Vitamin B12 - 6.0 g; Choline Chloride - 150.0 g; Calcium - 750.0 g; Manganese - 27.5 g; Iodine - 1.0 g; Ion - 7.5 g; Zinc - 15.0 g; Copper - 2.0 g; Cobalt - 0.45 g | |
Protease Assay
The reaction mixture containing 100 μL Hammerstein casein (1 mg mL-1),
780 L buffer (50 mM Tris-HCL, pH 8.1 containing 2 mM CaCl2) and 20
μL of extract was incubated at 37°C.
Bio-chemical Analysis of Feed and Tissues
Feed and feed ingredients were analysed for the proximate contents viz.,
crude protein, ether extract, ash, crude fibre and nitrogen-free-extract content
as per standard methods of AOAC (1990). Similarly, tissues were analysed at
the end of the experiment using the similar methodology.
Statistical Analysis
Data were statistically processed for one-way analysis of variance (ANOVA)
and significance differences between two groups were compared by Duncan’s
multiple range tests at 5% level of significance. All the analysis was done
by SPSS software (version 11).
Results
Proximate Composition of Feed, Feed Ingredients and Tissues
Proximate composition of feed ingredients and feed are given in Table
1 and 2, respectively. Protein content of APC increased
to 87% than the protein content of dry azolla. Fibre and ash content of APC
was reduced to the extent of 82.33 and 77.86%, whereas, the lipid contents increased
to 71%.
Crude protein and lipid content of the experimental diets was within the range of 39.11-40.82 and 9.40 to10.15%, respectively. Crude fibre content widely varied between 4.3-15.5%. Ash content was recorded in the range of 9.82%-10.35%. Soluble carbohydrate (NFE) ranged from 26.17-34.92%.
Crude protein content of different experimental groups ranged from 81.69 to 83.56%. Lipid content varied from 6.23 to 7.17%. Moisture and total ash content varied within a range of 81.87-83.78 and 8.83-10.91%, respectively (Table 3). However, difference in mean values among different groups were not significantly different (p>0.05).
Growth Parameters
Growth parameters of different experimental groups are given in Table
4. Weight of fish at 15 days interval are given in Table 5.
Highest body weight gain% was recorded in control group, which were similar
with T2 and T3 groups and lowest weight gain% in T5
group. Weight gain % of T1, T2 and T3 groups
was significantly higher (p<0.05) than the T4 and T5
groups. Similar trend was also recorded for SGR and FCR. Protein efficiency
ratio of T4 and T5 were similar and significantly lower
(p<0.05) than the T1, T2 and T3 groups.
But NPU was lowest in T5 group and less than (p<0.05) T4
group. There was no significant variation in NPU of T1, T2
and T3 groups.
| Table 3: | Biochemical composition of the tissue of fingerlings of Labeo rohita of different experimental groups (% DM basis) |
| Table 4: | Growth and nutrient digestibility of different experimental groups1 |
| 1-Means of 3 replicates ±SEM. Means in the same row sharing same superscripts are not significantly different (p>0.05) m = 100(Final body wt (g) - Initial body wt (g)/Initial body wt (g), n = 100(Loge Final body wt - loge initial body wt/Experimental duration (60 days), p = Feed intake (g)/Weight gain (g), q = Weight gain (g)/Protein intake (g), r = protein retention/ protein fed | |
| Table 5: | Body weight (g) of different experimental groups at 15 days interval during experimental period |
Enzyme Activity and In vitro Digestibility
Specific protease activity of the different experimental groups are given
in Table 4. Higher protease activity was recorded in T1,T2
and T3 groups, while the lowest in T5 group. Protease
activity of T4 and T5 group was significantly lower (p<0.05)
than the other groups. However, no significant difference was found among T1,
T2 and T3 groups.
Carbohydrate digestibility of T4 and T5 groups were significantly lower (p<0.05) than the T1, T2 and T3 groups. However, there was no difference between T1, T2 and T3 groups. Protein digestibility of T1, T2, T3 and T4 groups were similar (p>0.05) except T5 groups. However, there was no significant difference between the protein digestibility of T4 and T5 groups.
Histology of Hepatic Tissue
There were absolutely no histological changes in the hepatic cells of the
control and T2 groups. Cells were moderately hypertrophy in the T3
and T4 groups, whereas maximum hypertrophy was seen in T5
groups.
Discussion
Proximate Composition of APC
Increase in protein content of APC as compared to sun-dried azolla is because
of extraction of protein along with removal of fibre. Acid precipitation using
HCl followed by thermal coagulation was found to be effective than the only
heat coagulation. Borhami and El-shazly (1984) had also reported HCl (pH 2)
was more efficient for coagulating of about 30% more protein than heating, while
extracting protein from water hyacinth. Kositsup et al. (1992) observed
a higher extraction of 23% from water hyacinth using acid and thermal coagulation.
Growth Parameters
There was significant decrease in weight gain % and SGR of T4
and T5 groups when the level of APC-SP mixture increased in the diet.
However, there was no significant difference (p>0.05) within T1,
T2 and T3 groups. This indicates that 16.25% APC-SP mixture
can replace 10% of fish meal in the diet of L. rohita fry. Although literature
is lacking but in a similar type experiment Ogino et al. (1978) had reported
that LPC from rye grass could replace upto 43 and 40% of the total protein in
diets for carp and rainbow trout, respectively. Similarly cowpea protein concentrate
could replace upto 30% fishmeal in tilapia (Olvera et al., 1997). Feeding
fresh or dried azolla to fish resulted in poor growth (Almazan et al.,
1986; Antoine et al., 1987; Micha et al., 1988). El-Sayeed (1992)
had reported that azolla could be used only by replacing 25% of fishmeal in
the diet of tilapia. However, APC-SP could replace upto 50% protein from the
fishmeal of control group in the present experiment. It appears that APC could
be better utilized by the Labeo rohita fry. Growth rate was favourable
for the L.rohita fry when 50% of total protein from fish meal was replaced
by APC-SP, after which it reduced significantly implying low protein utilization
at higher inclusion of APC-SP. This may be due to imbalanced amino acid make-up
as it is evident from low NPU from T4 and T5 groups. Protein
utilization was reduced when essential amino acid in required proportion and
amount are not available (De Silva, 1995). Almazan et al. (1986) had
reported that Azolla pinnata strain was limiting in tryptophan and slightly
deficient in threonine. Hence, increased concentration of azolla in the diet
increases the demand for the deficient amino acid.
High FCR was registered at high inclusion level of APC-SP, especially when protein from APC-SP increased more than 50% of fishmeal protein in T4 and T5 groups. This is due to low nutrient utilization from APC-SP at high inclusion level (>24.37%). Increased FCR due to higher feeding of azolla had also reported by many workers (Joseph et al., 1994 in Etroplus suratensis; Fasakin and Balogun, 1998 in Clarias gariepinus; Almazan et al., 1986 in Oreochromis niloticus; Appler and Jauncey, 1983).
Similar trend was also observed for PER as with FCR. The PER of T4 and T5 groups were similar and were significantly lower (p<0.05) than T1, T2 and T3 groups. Increased level of APC-SP with concomitant decrease of fishmeal might have resulted essential amino acid deficiency and thus low utilization of protein. Most of the workers were also found the same results when they used alfalfa and Azolla pinnata in the diet of tilapia (Sntiago et al., 1988; Olvera-Novoa et al., 1990). Fasakin and Balogun (1998) had reported reduced PER and NPU with increasing level of azolla in the diet of Clarias gariepinus.
Enzyme Activity and In Vitro Digestibility
Protein digestibility of T5 group containing only APC and no
fishmeal was significantly lower (p<0.05) than the other groups except T4.
Ogino et al. (1978) also reported a decreasing trend of protein digestibility
when LPC of the rye grass was used at higher concentration. Similarly Appler
and Jauncey (1983) reported at higher levels of algae in the diet, the decrease
in APD with increasing level of algal meal might be due to an actual reduction
in the availability of the protein or a decrease in gastro-intestinal passage
time. Increasing fibre contents result in more rapid passage of food through
the gut of higher animals (Bender, 1967), thus decreasing the time available
for digestion and absorption of the diet. A negative correlation was found out
between protease activity and APC content of the feed (r2 = 0.87,
Fig. 1). Kawai and Ikeda (1972) and Shcherbina et al.
(1976) had reported adaptive changes in the activity of the proteolytic enzymes
in Cyprinus carpio in relation to the type of diet. Fishmeal based diet
in T1 group may be more adaptive for proteolytic enzyme, which gradually
decease due to the presence of APC-SP. Moreover, the proteolytic activity might
have decreased probably due to the presence of trypsin inhibitors, which are
reported to occur in vegetative tissues of several aquatic plants (Gleen et
al., 1982; Yousif et al., 1994). The trypsin inhibitors impairs the
digestion and absorption of protein (Olvera-Novoa et al., 1990).
Carbohydrate digestibility was recorded in decreasing mode as NFE content increased in the diet. Fish have a genetic inheritance to low carbohydrate utilization (Palmer and Ryan, 1972). Moreover, NFE which normally represent available carbohydrate in cereals and pulses may be of limited value for weeds due to physico-chemical characteristics of gel polysaccharides and water soluble nature of fibre in the diet.
Histological Study
However, results show no mortality was registered in any of the experimental
groups, which explain no adverse effect of APC-SP for short term feeding. However,
it’s effect in long term feeding needs confirmation.
| Fig. 1: | Relationship between APC-SP content of feed and protease activity |
Conclusions
It concludes that fresh water aquatic plants can be used in the diet of L. rohita fry as protein source. Preparation of azolla protein concentrates enhanced the protein percent nearly 1.87 times than its normal protein content. Azolla protein concentrate and spyrogyra mixture (4:1) can be used maximum upto 16.25% by replacing 10% fish meal in the diet of L. rohita fry. However, detailed studies on its amino acid make-up of these ingredients may help for its higher inclusion level by supplementing the deficient amino acids. Short term feeding had no adverse effect on L. rohita but long term effect needs further research.
Acknowledgements
We gratefully acknowledge the present Director, Central Institute of Fisheries Education for providing the facilities for the conduct of this work. We are also thankful to Indian Council of Agricultural Research for financial support for this experiment.