A Study on Nutritional Profile of Liver from Captured and Cultured Rohu, Labeo rohita (Cypriniformes: Cyprinidae)

Fish production and fish processing waste have straight connection. In India, the waste produced during the processing of fish is predicted to be approximate 3.6 million metric tonnes, 48 per cent of the total body weight of Indian and exotic major carps is thrown away as waste (non-edible ). The present research, it was conducted to compare the total lipid content (TLC) and fatty acid composition from the liver of captured and cultured fish, Labeo rohita (Hamilton) having weight more than 500 gram during different months as well as to evaluate its nutritional quality. Maximum total lipid content (33.33±0.14%) was found in the liver of cultured fish in May month, while the minimum (15.26±0.24%) was in the liver of captured fish in the month of January. During the study, total lipid content was found to be considerably elevated in cultured than captured Fish, Labeo rohita (Hamilton). The amount of three major groups of fatty acids namely polyunsaturated fatty acids, monounsaturated fatty acids and saturated fatty acids was found to be maximum in captured fish during the month of March 97.19±0.96%, 61.30±0.56% and 95.39±0.31% month of April respectively. Total n-6 polyunsaturated fatty acids were observed to be highest (21.95±8.05%) in the waste of liver in cultured fish during the month of March. Hence, it is concluded that the processing waste (liver) of the captured and cultured, Labeo rohita (Hamilton) is a prosperous resource of the essential fatty acids i.e. PUFAs and total lipids. Further, it is observed that captured species are rich in fatty acid composition as compared to cultured species. Food industries can manufacture by-products from these high nutritional value contents of waste for human utilization. EPA and DHA also reduce the risk of various life threatening diseases.


INTRODUCTION
In this contemporary era, fish is the economical and primary resource of top-quality animal protein cheerfully accessible to the masses all over the world. Fish requirement is increasing day by day, in every nook and corner of the world due to its nutritional values. Approximate 167.2 million tonnes of fish production recorded by capture fisheries and aquaculture from which 146.3 million tonnes was consumed as foodstuff by human being left over 20.9 million tonnes was discarded as waste during the process of making of different fish products [1]. The tremendous increase of fish waste production can cause loss of finance as well as environmental pollution problems. Worldwide annually, almost63 million metric tons of waste produced during the processing of fish by the fisheries industries, which is rich in various biomolecules such as lipids, protein, chitin and carotenoids [2]. Frequently, to get rid of from this fish waste it is discarded in dump ground or throw out at sea rather than its utilization for various purposes, however, there are alternative uses that can increase economic value [3]. During fish processing, the waste water is also generated which is having fair amount of lipids and protein (6 mg /l and 2 mg /l, respectively) [4].
Fish processing waste is a big menace of present times, various essential biomolecules can be recovered from it such as proteins and lipids which will not alone generate an additional returns by preparing some nutritional food items but also solves the problems of its disposal.
Recently Japan has shown the way, various food processing companies have been involved in the reduction of this waste by utilizing it for production of condiments and valuable materials such as fine chemicals [5]. In different tissues of the fish for instance muscles, liver, under skin layers also, in the body cavity fat synthesis from fatty globules occurs and accumulated in all the organs which are discarded by considering it waste during the course of processing [6][7][8]. Liver oil extracted from the fish is commonly a rich source of numerous beneficial fatty acids which includes long -chain polyunsaturated fatty acids (PUFAs) such as omega-3 fatty acid and fat-soluble vitamins A, D and E [9][10][11]. Same can be extracted from nonconsumable parts which are presumed as waste like skin, viscera head and central bones [12][13][14][15] [8]. Above enlisted discarded tissues possibly could play a pivotal role towards the generation of nutritional food items.
Various disorders like high blood pressure, gout, coronary heart diseases, arthritis and plethora of additional health issues which resulted, due to intake of excessive cholesterol enriched foodstuff can fend off by addition of supplements manufactured from fish lipid in diet [16]. So, waste generated during the processing of fish is loaded resource of a So, fish processing waste is a rich source of total lipid content, polyunsaturated fatty acids, specific omega-6 and omega-3 PUFAs, enzymes and minerals that can be used for food, various food products, agricultural activities, pharmaceutical and other industrial benefits [17]. Freshwater and marine water fish oils and fats were observed to be enriched with superior quality omega-3 long chain poly-unsaturated fatty acids predominantly, EPA, 20:5n-3, DHA, 22:6n-3 as well as its originator, alpha linolenic acid [18][19][20][21][22][23]. This higher content of polyunsaturated fatty acids (PUFAs) in fish and fish processing waste are helpful for cardiovascular fitness [24], arteries and blood vessels functioning [25], reduction of blood clotting [26] [27]; [25].
Highest concentrations of omega-3 fatty acids (EPA and DHA) are generally detected in the tissues of fish parts like viscera that are discarded. Lipids rich in Polyunsaturated fatty acids fight against thrombotic, aging, cholesterelemic, inflammatory and cancer like disorders and act as inhibitory drugs which further stimulate immune system as well as used as immunosuppressant therapeutics [28]. EPA and DHA (Omega-3 fatty acids) are also helpful as of the prevention from neuropsychiatric illness [29]. Subsequently, the probability of fish waste selection as compared to muscle for lipid extraction is inexpensive source which could prospectively make remarkable profits for environment and fish processing industries. Evaluation of the oil extracted from the composite samples of liver tissue reflects the existence of the significant essential and nonessential polyunsaturated fatty acids. Table-sized fresh specimens of captured and cultured samples of Labeo rohita (Hamilton) more than 500g were obtained from the collection sites (Ludhiana district) every month from December 2015 to May 2016. Every fish sample was individually enclosed in a marked sanitary sealed zip-up plastic bag and immersed in abundant compressed ice cubes in the cold storage box. Ambient temperature was recorded at the time of collection of the samples. The samples were transferred to the fisheries research laboratory and store up in a deep refrigerator at low temperature (-20 0 C), in order to keep safe its lipids and fatty acid quality as well as quantity.

Biometric Measurements
Fish samples were brought to the laboratory and defrosted approximately for 7-8 hours in a freezer at optimum temperature 5°C and measurements such as : total length, standard length were taken in cm and weight was recorded in gram. Total length and standard length were measured using a measuring scale. The weight was taken with a Goldtech top pan electronic weighing balance model GTA 6K / Fabr. Nr.01113254.

Sample Preparation
Each fish specimen was washed with running water. Liver from captured as well as cultured fish were removed and their weights were recorded. The livers so collected from the captured and cultured species were then separately pooled jointly for the preparation of composite liver samples. The entire process was ended on crushed frost and took about 10 minutes. The prepared samples were stored at-20 0 C until analyzed.

Extraction of total lipid content (%):
The quantity of total lipid content had been estimated as per Soxhlet lipid extraction/ solvent extraction method [30].
Calculations: Total lipids (%) Where, a = Weight of the composite sample (fish flesh) taken. W1 = Weight of empty crucible. W2 = Weight of crucible with extracted lipids.
Composition of Fatty Acids: Analysis of fatty acid composition was done on Gas Liquid Chromatography [31] from Punjab Agricultural University, Ludhiana. Fish liver fatty acids were converted into vaporized form after transferred them into methyl/ethyl esters [32]. The identity and quantity of these esters were done after injecting into GLC along with compared with standard set values of esters.

RESULTS
Biometric measurements of captured and cultured fish and weight of the processing waste during different months: The biometric data (Table.1) of captured and cultured Labeo rohita (Ham.) have been obtained. Total length, Standard length, Body width as well as Body weight have been measured. Biometric data of a fish is very important and it is specific for specific weight group as well in males and females. The lengths of the body directly correspond to the weight of the fish. It is to be concluded that the processing waste generation was highest in captured species in the May month.

Total lipid content (%) of the Fish Waste:
Results obtained on the inter specific divergences in the TLC (total lipid content) and fatty acid profiles of liver tissue (processing waste) from captured and cultured fish, Labeo rohita (Ham.) more than 500gm weight have been analyzed (Table 2). It has been observed that the liver of cultured fish during all the six months had significantly higher (p<0.05) TLC than captured fish. Maximum TLC (33.33±0.14%) was reported in cultured species liver during May month, while the minimum (15.26±0.24%) was found in the liver of captured fish in the month of January. The TLC in liver of cultured fish increased in all the months when compared with captured fish (Fig..1).
Liver of captured fish in the month of March contained maximum DHA (76%) in the n-3 PUFAs followed by linolenic (13%) and DPA (11%), however, in the liver of cultured fish, n-3 PUFA has contained DPA (99%) followed by1% EPA. During the month of April, EPA was the major contributor in the liver of captured and cultured fish and contained maximum 62% and 97% to the n-3 PUFAs, respectively. Liver of captured and cultured fish, DHA contributed maximum 88% and 95%, respectively to the n-3 PUFAs in the month of May. The Linoleic acid was the major contributor to the total n-6 PUFAs in the liver of captured fish contributing 100%. In cultured fish liver, AA (81%) was the major contributor followed by linoleic acid (19%).

Monthly comparison of the major fatty acids from liver of captured and cultured fish
Labeo rohita (Hamilton) during different months: Data on major groups of FAs in liver tissue of captured and cultured fish species (Labeo rohita) during different months revealed that there were variations in the pattern of occurrence of different groups of fatty acids in their liver. The mean total n-3 fatty acids in Labeo rohita (Table  3) were maximum (78.55±1.70%) in the liver (captured) in the month of March and minimum (10.94±0.43%) in the captured fish during the month of April.  Similarly, the omega-3/omega-6 ratio (n-3/n-6) was observed highest (5.33±0.09%)) in captured fish in the month of February and minimum (0.80 ± 0.08) in the cultured fish in the month of January.
The maximum mean MUFAs (58.94±0.18%) were present in captured fish in the month of April and the minimum (1.83±0.67%) in cultured species during the month of December. Similarly, the mean total SFAs were maximum (82.72±6.62%) in captured rohu in May and minimum (2.20±0.10%)) in the captured fish in December. It has been inferred from the results that during different months, the processing waste of Labeo rohita, the liver of captured fish was best as it enclosed highest amount of omega-3 FAs, mean total PUFAs in March, highest ratio of omega-3/omega-6 in February, maximum MUFA in April, maximum SFAs contents in May when it is compared with cultured species (Table 3).

DISCUSSION
Sharma et al. [33] studied comparative FAs profile of liver, muscle and brain tissue of the cultured and captured tropical fish, Labeo rohita. Significantly higher (p<0.05) TLC (lipid contents), SFAs and MUFAs were reported in cultured fish than its wild counterpart, while in captured species highest omega-6 and omega-3 PUFAs were detected. Similarly, it has also been revealed during the present study that maximum TLC ( total lipid content) was scrutinized in cultured Labeo rohita (33.33%) liver as compared to captured species (29.06%) and higher SFAs level in all the months (except in the month of Febuary was observed in captured Labeo rohita liver as compared to cultured species during different months. Further, the presence of higher TLC (total lipid content) quantity in cultured fish liver perhaps due to supplementary feed having containing high SFAs amount fed to cultured fish. The marked difference in the lipid content of wild fish (captured) seems to be due to scarcity of food. Resultantly, the scarcity of food in natural conditions can cause slow growth of fish. Hussain et al. [34] analyzed the comparison of nutritional profile of head from captured as well as cultured Catla catla and recorded lipid contents as 7.56±0.46% and 11.90±0.25% respectively.
Nazeer et al. [35] studied the lipid profile of Threadfin Bream (Namipterus japonicas) organs and observed that liverhas stored a major amount of lipids (6.22%) in comparison to muscle (2.7%) and skin (1.0%). Further it has been observed that from fish physiological point of view significant quantity of two omega-3 polyunsaturated FAs: Eicosapentaenoic and Docosahexaenoic FAs were found more 1.6% and 0.5% in skin and 1.6% and 0.6% in liver as compared to muscle 1.4% and 0.4%. Khoddami et al. [8] studied on oil fatty acid profile squeezed from the waste of Sardinella lemuru which includes viscera (intestine and liver) and head. During their study, valuable quantity of oil has been taken out from these wastes and reported less than 6% of oil which was highest in the liver (5.80%). The palmitic, stearic, oleic and docosahexaenoic acid were 27.80-35.56 %, 5.90-9.30%, 15.47-21.79%, 11.87-15.95%, respectively reported to be predominant fatty acids in the waste of sardine. However, liver, head and intestines oil samples omega-3/omega-6 ratio represented value more than 1. Sardine waste is a good nutritional resource for human diet because having, greatest quantity of omega-3 FAs , lipids and omega-3/omega-6 ratio. The above studies were found to be similar to the present studies in their fatty acid composition: palmitic acid (C16:0; 2.19-35.82%) during the month of January, stearic acid (C18:0; 0.01-5.80%) and docosahexaenoic acid (DHA; C22:6; 4.06-39.49%) during the month of May and oleic acid (C18:1c; 4.75-25.06%) in March month. The total lipid content in the present study on cultured species liver in the month of May has been found to be (33.33±0.14%). Kaur et al. [36] reported the total lipid contents from the liver of C. carpio and it was found to be22.7%, however, Hassan et al. [37] recorded very low lipid contents i.e. 7.43% in Catla catla.
Bajwa and Kondal [38] studied deviation in total protein and total lipid content of Wallago attu (catfish), Channa striatus (Snakehead Murrel) waste in diverse months. In the waste of Channa striatus, lowest amount of TLC (total lipid content) (2.63±0.23 gram per 100 g) was monitored in May month, however highest TLC (5.38±0.15%) was recorded in the month of February. In the waste of W. attu, greatest amount 11.20+0.24 gram per 100 g TLC (total lipid content) was examined during February month and minimum TLC (7.38±0.25) recorded in May month. The TLC (lipid content) in processing waste of W. attu and C. striatus species raised extensively (p<0.05) primarily from December-February and afterward diminished considerably (p<0.05) up to May month. Conversely, from December to January (preparatory phase of spawning) total protein extracted from the waste of catfish as well as murrel was recorded least amount furthermore, attained its utmost value in March to May (prespawning phase). Highest and lowest values of protein content from snakehead murrel processing waste was found 88.20±1.05 mg per g and 52.30+1.30 mg per g in May and December month, respectively. In the waste of W. attu fish minimum total soluble protein (75.20±2.77 mg/g) was present in the month of December and maximum amount (103.0+2.63 mg/g) was recorded during the month of May. Similarly, it was observed during the present investigations on fish, Labeo rohita (Hamilton) that minimum TLC captured and cultured species during the December and January (starting period of spawning) and achieved its utmost value in March to May (pre-spawning phase).
Rani and Sehgal [39] reported maximum values of TLC in liver (17.80±0.45%) and kidney (21.90±0.80%) in 751-1000g weight group of freshwater food fish, Catla catla (Ham.) when thrice (250-500g, 501-750g and 751-1000g) weight clusters of the Catla catla were evaluated for comparison. The above mentioned values (TLC) were close to liver (15.26±0.24%) in February in March of the present studied captured fish, Labeo rohita.. Saify and [40] estimated fatty acid constitution of lipid extracted from the liver of two salt water habitat fishes, Carcharhinus bleekeri and Eusphyra blochii and reported 66.19%, 39.94% lipid value in the liver tissue of Eusphyra blochiiwas and Carcharhinus bleekeri, respectively. Similar values of lipid content was reported in cultured fish (33.33±0.14%) during the present studies in the month of May.
Fatty acid composition in liver, muscle of Perca fluviatilis (wild perch) reared in pond reported significantly highest values of arachidonic acid (7.44±0.80%), EPA (7.81±2.69%) and DHA (24.94±2.94%) in wild perch as compared to pond cultured species. DHA was found to be comparable in pond cultured and wild fish.
It is revealed from the above discussion that the fish processing waste is very nutritious having high TLC (total lipid content) and FAs (fatty acids) from the captured and cultured fishes. From the fish processing industries, fish waste and wastewater goes into the freshwater lakes, ponds and on land which causes environmental pollution. Although, mostly fisheries industries putting efforts for utilization and treatment of waste that is produced during the processing of fishes, the techniques adopted for them are not fulfilled completely chiefly because of plethora of factors such as, fish waste nature, little returns while sold for trade purpose in fish market and limited land hence it is dumped in the open. Considering the appropriate quantity of lipids, omega-3 (n-3) fatty acids and omega-3/omega-6 (n-6/ n-3) ratio, liver tissue of captured and cultured Labeo rohita may perhaps utilized as a respectable alternate resource for fish lipid extraction. The foremost benefit of fish processing waste lipid is that this by-product is much economical than lipid extracted from fish muscle. Lipid collected from fish waste is taking into account as the vastly eye catch seeking resource for industrial utilization in addition to human consumption.

CONCLUSION
Captured Labeo rohita (Hamilton) generated higher amounts of waste than cultured fish during their processing in all the six months. Minimum TLC in captured and cultured species was observed during the December and January (preliminary period of spawning stage) furthermore, attained its highest value in March to May (pre-spawning stage). It is observed that in liver tissue of captured species total omega-3 (n-3) fatty acids were significantly elevated as compared to cultured L. rohita in all the months. The occurrence of privileged quantity of total lipid content in cultured fish liver may be due to the consequences of feeding supplementary (having high quantity of saturated fatty acids) to the studied fish. Presence of highest amount of fatty acids in liver of captured species might be attributed to the voracious and omnivorous features of the presently studied fish or to a accessibility of food during diverse seasons which influences feeding of captured Labeo rohita. Thus, this can be concluded, processing waste (liver) of the captured and cultured, Labeo rohita (Hamilton) is a good resource of lipids and the essential FAs, the PUFAs collected from both the habitat, out of which the captured species are best as compared to cultured species in terms of fatty acid composition.

Statistical Analysis:
Values in all tables were given as Mean ± S.E (mean ± Standard error of mean). Statistical difference among the mean of lipid and fatty acids of captured and cultured and fish liver during individual month between captured and cultured fish were determined using one way and multifactor ANNOVA. The analysis was done using Microsoft Excel and STATGRAPHICS statistical packages.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

ETHICAL APPROVAL
As present work is on fish and fish is cultivable organism, hence, no ethical approval is required.

CONSENT
It is not applicable.