Estimation of growth parameters in fingerlings of Labeo rohita ( Hamilton , 1822 ) fed with exogenous nutrizyme in Tarai region of Uttarakhand , India

Biotechnological research and development are moving at a very fast pace. The application of biotechnology in the fisheries sector is a neoteric practice. Nevertheless, it is a promising area to enhance fish production. Efficacy of exogenous nutrizymes in fish feed is being reinvestigated in the present time. A feeding experiment of 90 days was conducted to assess the efficacy of exogenous nutrizyme supplement diet on weight gain and growth performance of Labeo rohita fingerlings. Three supplementary diets (D1, D2 and D3) were prepared containing 30±0.6% crude protein, 4.8±2% crude fat and 11.6±0.2% crude fibre. Experimental diets D1, D2 and D3 were supplemented with papain at the level of 5, 10 and 12 g per kg of feed respectively. Feeding was done at the rate of 5%, twice a day. Rearing of fingerlings on diet D2 resulted in better growth of L. rohita fingerlings compared to those with the experimental diet, D3 and D1 followed by control diet D0 which was without any supplementation. Comparison of growth performance was done on basis of growth rate, nutrient digestibility, gross protein retention (GPR), feed conversion ratio (FCR), protein efficiency ratio (PER) apparent net protein utilization (ANPU) and survival percent.


INTRODUCTION
Over the last decade, the world has witnessed spectacular growth in the aquaculture industry of many developing countries.As a result, aquaculture has been contributing significantly to food security and poverty elevation.It is further anticipated that world aquaculture production will continue to increase and since nutrition and feeding play a pivotal role in sustainable aquaculture, use of nutritionally balanced and complete formulated feeds will, continue to play a dominant role in finfish and shellfish production.
Hence, alternative and biotechnologically improved feed ingredients should be sought along with improvements in pond management and manipulation of pond productivity.
Plants are a good source of enzymes, including bromelain from pineapples, papain from papaya, nanokinase from soy fermentation and others.They are protein-digesting enzymes common in commercial and industrial use.Plant ingredients are also found to be rich in protein content and also other nutrients.A number of published reports are available regarding the efficacy of plant feedstuffs as alternative protein sources in fish feeds (Singh et al., 2008).The quality of aqua feed however, not only depends on the nutrient composition and nutrient balance, but also on the effective utilization of nutrients by the animal.
Fish/shellfish fed plant-based diets show lower nutrient digestibility, due to the high level of carbohydrate and various antinutritional factors (ANFs) (Tacon, 1990).Moreover, the metabolic activity of fish/shellfish during the juvenile stage is quite high, and consequently requires a nutrient dense (mainly high protein) diet in order to optimize survival and growth.However, the digestive system is not well developed enough to digest the nutrient-dense diet.Improper digestion and malabsorption of nutrients often have far-reaching effects that include reduced growth, impaired immunity, allergic reaction and poor wound healing.Nutrizymes are viable solutions for these problems.
Nutrizymes are nutraceuticals from exogenous enzymes that increase the digestibility of feed ingredients, thereby reducing nutrient excretion into the environment and ultimately enhancing growth.They facilitate a considerable saving in production costs and infrastructure, and offer nutritional consistency and offthe-shelf convenience.Nutrizymes provide additional commanding tools that can inactivate ANFs and enhance the nutritional value of a plant-based diet.They also provide a natural way to transform complex feed components into absorbable nutrients.One such nutrizyme is protease (Sinha et al., 2007).Papaya, Carica papaya is one of the herbal sources of proteolytic nutrizyme.
Papain is derived from papaya (C.papaya) leaf, unripe fruit and papaya latex, a milky fluid that oozes out of green papaya.It is from the cysteine proteinase family.Papaya leaves contain around 9% protein and 5.3% papain and also contain vitamin C (286 mg/100 g) and vitamin E (30 mg/100 mg) (Singh et al., 2011;Boshra and Tajul, 2013).Papain can hydrolyze lipids and carbohydrates as well as proteins, and it has been shown to have activity in a wide range of pH and temperature (Miyamoto et al., 2004).Thus the objective of present study was to evaluate the effect of exogenous papain supplementation on growth performance and survival of Labeo rohita fingerlings.

Fish and husbandry
L. rohita, the most favoured fish among Indian major carp, was selected for the study.Fingerlings of L. rohita, average weight 4.0±1 g, were obtained from instructional fish farm, College of Fisheries, Pantnagar and were used in the experimental studies.Fish were acclimatized for 2 weeks before experimentation.

Experimental site
The experimental site is located at the College of Fisheries, Pantnagar (29°N lat., 79.3°E long at an altitude of 243.8 m above mean sea level in tarai belt of shivalik range of himalyan foot hills).The study was carried out in the four outdoor cemented rectangular raceways of the size 8.73 m x 1.63 m with the average water depth of 0.84 m, which was maintained throughout the study.Tube well oxygenated water was used for fish culture in the raceways.

Raceway management
Prior to the experiment, the raceways were cleaned thoroughly with the lime water and filled with tube well water.The flow rate of 25 L/ min was maintained in all the raceways.Periodically cleaning of the raceways was done every 15 days interval for removal of excess algal biomass.

Experimental plan
Three levels of papain (R1=5 g, R2=10 g and R3=15 g) were used according to the body weight during the study and fed to three experimental groups each having two replicates.One control group was maintained on pelleted feed without papain supplementation.
Feeding was done at the rate of 5% body weight daily in each experimental feeding group after ascertaining the total group weight on weekly basis.The experiment was conducted for a period of 90 days.

Diet preparation
Three identical diets (approximately 30% crude protein) were prepared with similar ingredient composition (Fish Meal, 10%; Rice Bran, 45%, Mustard oil cake 25% and Soyabean oil cake, 25%).and subjected to proximate analysis following standard procedure (AOAC, 1998) (Table 1 and Figure 1).All the ingredients were properly weighed as per their inclusion rates in the four experimental diets and were finely powdered with the help of grinder machine.The feed was soaked with water for about 8 to 10 h.The mixture was then passed through a pelletizer (2 mm dia) and pellets were collected on aluminium tray.The pellets were then kept for drying in the hot air oven at 60°C for 24 h, till the moisture content was reduced to less than 10%.The dried pellets were then broken into small pieces and packed in air tight plastic containers, labeled and stored in cool and dry place (Papain and chromic oxide (1%) as an indicator for determining nutrient digestibility was added after soaking at required levels).One control feed was formulated without any papain supplementation.The dried pelleted diets were packed in air tight polythene bags and stored at -20°C.The feed was offered in the feeding trays, which were kept at the bottom of raceway.Regular monitoring for the consumption of feed was done, and any uneaten feed was removed daily.

Physical evaluation of formulated diets
Water stability and sinking rate was calculated for all the diets.The water stability of different feeds was determined by the wet durability method (Jayram and Shetty, 1981) at varying periods of 1 to 4 h durability test.The sinking rate of the experimental diets was determined after their preparation.Glass aquarium tank (0.35 m x 0.35 m × 0.60 m) with 28 cm depth of water was chosen for the above study.Equal size experimental pellets of diets were gently released at the surface of water in aquarium and time taken by the pellet to reach the bottom was recorded by using stopwatch.The average time taken by each type of diet to reach the bottom of the aquarium was recorded separately and the mean value of sinking rate was calculated and expressed as cm/sec.

Sampling schedule
To record the growth parameters sampling was done fortnightly.
The physicochemical parameters of the water were monitored weekly.

Growth performance
Growth parameters were assessed in terms of weight gain (WG), specific growth rate (SGR), condition factor (CF), food conversion ratio (FCR), apparent net protein utilization (ANPU), gross protein retention, protein efficiency ratio (PER), apparent protein digestibility % and survival.Final sampling was done after 90 days by weighing all the surviving fish from raceways.

Condition factor
Weight of fish (g) K =  100 Length of fish 3 (cm)

Feed conversion ratio
Dry weight of food consumed by fish (g) FCR = Live weight gain by fish (g)

Protein efficiency ratio
PER= Increase in mass of animal / Mass of protein in feed.

Gross protein retention
Protein content of the feed was measured using Kjeldahl method (AOAC, 1998) and was calculated using 6.25 as a conversion factor.Protein content of fish muscle was calculated using Lowry et al. (1951) method.

Survival rate (%)
Survival rate = (Final number of fish/Initial number of fish) × 100

Feed consumption
Percent of feed consumption for all diets were calculated by the difference of the amount of feed given and amount of uneaten feed/feed given ×100.

Physicochemical parameters
The water samples were collected in sampling bottles from each raceway between 10-11 AM for the estimation of pH, dissolved oxygen, free carbon dioxide and alkalinity.Dissolved oxygen was measured at raceway itself while rest of the parameters was analyzed in the laboratory methods outlined by APHA (2005).

Statistical analysis
Experimental data was subjected to the statistical analysis following the completely randomized design (CRD) and the variation among the treatment means was tested for the significance by analysis of variance techniques as described by Gomez and Gomez (1984).Level of significance used for F and t-test were P= 0.05 from the table given by Fisher.

Physical characteristics of formulated diets
The measurement of physical characteristics of formulated diets like texture of pellets, sinking rate, water stability are some of the essential features in view of their reaction to water at the time subjection for feeding and ultimately their consumption by fish.The feed should be rigid enough not to disintegrate and brittle but to sustain firmness when put in water for feeding.If the feed pellets disintegrate it would not be consumed by fish and would settle at bottom as waste to invite danger of deterioration in water quality parameters.Many intrinsic factors also enter into the choice of feed ingredients, which have vital significance on the economic viability of supplementary feeds such as their availability, cost on their procurement and factors of applicability of formulated feeds in the aquatic environment such as maintenance of texture, mechanical strength, size, density, fragility etc. (Glencross et al., 2007).
The formulated diets when seen under the subjection of reaction to water suggested varying results.The water stability of the pellets was determined over a period of 1 to 4 h.The percentage of dry matter obtained after immersing the feeds indicated decreasing trend with the passage of each hour for all the diets.This decrease between 1 to 4 h was from 86.45 to 75.68% for diet D0, 85.14 to 73.28% for diet D1, from 85.62 to 74.61% for diet D2, from 86.83 to 76.71% for diet D3.It was also seen that the reduction in dry matter was maximum during the 3 rd and the 4 th hour of immersion.This clearly suggests that with the increase in time of immersion all the diets acquired water through absorption and are liable for disintegration.It also means that the diets absorbed less moisture between the 1 st and 2 nd hour of immersion and retained proper texture and shape which makes them available for fishes up to two hour after immersion.However, there was no significant difference in water stability among all experimental diets (D0, D1, D2 and D3).The average values of sinking rate of pellets (formulated diet) suggested that the average sinking rate was almost similar (non significant) for all diets D0, D1, D2 and D3, with an average value of 6.04 m/s in D0, 6.05 cm/s in D1, 6.04 cm/s in D2 and 6.06 cm/s for diet D3.

Growth parameters
The cost of feed ingredients and other inputs are increasing, while market costs for the major cultivable finfish and shellfish species have remained static or are decreasing.It is, therefore, likely that increased aquaculture production will be from herbivorous/omnivorous fishes in developing countries of Asia and other parts of the world.Aquaculturist can reduce the current dependence on natural marine resource to farm carnivorous finfish and shellfish through the use of the low cost, locally available, alternative feed ingredients (Hasan, 2001).The idea of introducing exogenous enzymes into fish feed is not new but their efficacy is being reinvestigated.The use of biotechnologically improved products and appropriate use of locally available feed ingredients in semi intensive aquaculture is still needed.Although fish larvae have sufficient levels of digestive enzymes to digest live food organisms at first feeding, there may not be enough for the digestion of microparticulate diet common in modern fish culture practice.
The microparticulate diets are rich in nutrients (mainly protein) as well as other antinutritional factors that larvae find hard to digest.Binders used for microbound diets as well as proteins and synthetic polymers used for crossbinding with encapsulation methods etc. have been found to be difficult to digest by fish larvae.Moreover, microparticulate diet contains 60 percent to 90 percent dry matter, compared with only 10 percent in zooplankton.This may lead to insufficient digestibility, as it is much harder to digest dry, hard particles than live organisms.
Dietary supplementation of proteolytic nutrizyme can overcome this problem.The statistical analysis of different growth parameters of Labeo rohita fingerlings at the end of experimental period indicate significant differences in the final body weight between the four groups (Table 2 and Figures 2 and 3).
Fingerlings of experimental tank T2, kept on diet D2 were the fast grower followed by fingerlings of tank T3, T1 and least in control group.The specific growth rate and condition factor take almost the same pattern of weight gain in which fingerlings fed with diet D2 had highest SGR and CF followed by tank D3, D1 and D0 fed groups.The differences in growth performance of L. rohita fingerlings between control and treated group could be attributed to the quality of diets.The reduced growth rate in control treatment could be due to the presence of anti-nutritional factors in feed which in turn have an adverse impact on growth performance and availability of various dietary nutrients (Spinelli et al., 1983;Richardson et al., 1985).Better results in papain fed groups suggest that nutrizyme supplementation in the diet may be effective in reducing either anti nutritional factors or adverse consequences of phytate from plant origin ingredients of feed, which is supported by the finding of Liu (1997).This may also be linked to the activity of papain to dephosphorylate the phytic acid and phytate phosphorus to increase the phosphorous availability (Lanari et al., 1998).
The reduction of phytate-protein complexes in the gut increases nutrient availability (Liebert and Portz, 2005).The improvement in growth rate of fish after papain supplementation is consistent with other studies where fish were fed either phytase supplemented diets (Jackson et al., 1996) or phytase pretreated ingredients (Vielma et al., 2002).Thus it is clear from the present study that papain increased the availability of proteins by its proteolytic activity and thus contributed to the growth performance of L. rohita fingerlings.
Lowest FCR value of 2.05 was obtained in D2 treatment group in which papain was supplemented at a level of 10 g/kg feed followed by D3 and D1.Exogenous application of enzyme resulted in improvement in FCR when compared to control.The reason may be due to increased metabolism in fish fed on papain supplemented feed which in turn resulted in better FCR.Papain is a protease enzyme that hydrolyzes proteins to short peptides in diet, which is the key factor to increase protein digestibility and fast absorption, and helps to increase growth factors (Wong et al., 1996).
Highest apparent protein digestibility was found in D3  group and the lowest was found on control group.The feed supplemented with papain showed higher protein digestibility values as compared to control feed where no enzyme supplementation was done.
The digestibility values recorded in the common carp fed on papain supplemented diet (2%) clearly indicated that exogenous enzymes play a considerable role in fish digestion process and added enzymes advantageously influence fish growth and food utilization (Singh et al., 2011).Improvement in protein digestibility of feed by papain supplementation may also be attributed to the activity of papain to dephosphorylate the phytic acid and phytate phosphorus to increase the phosphorus availability (Lanari et al., 1998).
Protein efficiency ratio is a measure to show as to how well the protein sources in the diet could provide essential amino acid requirement of the fish.Hence, the PER in the range of 1.12 to 2.30 (g/g) could favour fat deposition in rohu and this finding is in agreement with the work done by Desilva and Anderson (1995) and Manush et al. (2013).During the whole experiment there was an increase in protein efficiency ratio in all treatments and it was always higher than the control treatment which could be due to the exogenous application of vegetable pepsin.
Gross protein retention ranged in between 24.25 to 26.98.However, the highest Gross Protein Retention (GPR) was recorded in D2 feeding group (P < 0.05).Exogenous application of enzyme in fish feed breaks the anti-nutritional factors present in plant origin food stuffs, hence making more protein available to fish which in turn results in better protein retention by the fish.The GPR values recorded in the present study are in agreement with those of Jana et al. (2006) who recorded a GPR value in the range of 28 to 31.05% in milk fish Chanos chanos.
The data on apparent net protein (%) reveals that the highest and lowest ANPU values were shown by D2 and control treatments, respectively.Similar results were found by Siddhuraju and Becker (2000).The reason for higher ANPU in case of papain supplemented feed when compared to control treatment can be ascribed due to action of papain against anti-nutritional compounds in feed and therefore, more amino acid availability to the experimental fish.
Similar results were stated by Singh et al. ( 2011) concluding that addition of papain at the level of 2% to the feed resulted in lowest feed conversion ratio (FCR), better growth rate, high protein digestibility, higher protein efficiency ratio (PER), good gross energy retention, better apparent net protein utilization (ANPU), energy conversion efficiency ECE (%) and nitrogen retention efficiency (NRE) enhancing the overall growth and production of common carp.
The degree of survival and well being of carp larvae dictate the success and failure of fish culture.Due to non availability of proper larval diet the rate of survival remains low therefore development of fish farming at the commercial scale is limited due to difficulties in producing sufficient quality of juvenile and high mortality.100% survival rates were achieved in the present study in D2 groups followed by D3 and D1 showing remarkable acceptability of nutrizyme.Similar results of more than 95% fish survival were obtained by Tewari and Ram (2012).The results for feeding trial by Patil and Singh (2014) also indicated that the 0.1% supplementation of papain to the diet resulted in better growth, survival and feed utilization of post-larvae of M. rosenbergii.
Protease enzyme papain is principle and most active enzyme in nature, green papaya and possesses a powerful digestive action superior to pepsin and pancreatin (Trivedi et al., 2013).Papain, enzyme acts as a biological catalyst which improves nutrient availability from feed stuffs, increase digestibility of low quality feed, lower feed costs and reduces output of waste into the environment.Almost similar status of all physico chemical parameters (Temperature, pH, dissolved oxygen, free carbon dioxide and alkalinity) was found without any adverse effect of the use of papain on the water quality.
From the findings of this study, it can be concluded that adding of papain to the feed will result in efficient protein metabolism by hydrolyzing of proteins and forming short peptides in diet, leading to an increase in digestibility and fast absorption of proteins, which in turn helps to increase growth factors.Moreover, rohu showed higher feeding responsiveness to nutrizyme supplemented diets.Hence, for better effective results papain can be added at the level of 10 g per kg fish feed to enhance overall growth and production of L. rohita fingerlings.Papain is an eco friendly growth promoting agent and has no deteriorating effect on the aquatic environment.It can thus be used in aquafeed to increase nutrient digestibility in addition to improving the health status of cultured aquatic organisms.
different superscripts in the same column are significantly different (<0.05).Values given in the table are means (n = 3).

Figure 2 .
Figure 2. CF, SGR, FCR and PER of Labeo rohita fingerlings fed on papain supplemented diets at three different levels for a period of 3 months in experimental raceways.

Figure 3 .
Figure 3. Net Weight Gain, APD%, GPR% and ANPU% of L. rohita fingerlings fed on papain supplemented diets at three different levels for period of 3 months in experimental raceways.

Table 1 .
Proximate composition (%) of different type of test diets.

Table 2 .
Growth parameters of L. rohita fingerlings fed on papain supplemented diets at three different levels for a period of 3 months in experimental raceways.
d *Values bearing different superscripts in the same column are significantly different (<0.05).Values given in the table are means (n=3).