Cholecalciferol and Formic Acid Synergistically Enhance Digestive Enzymes Activity and Bone Mineralization in Ctenopharyngodon idella

Laiba Shafique1*, Mahroze Fatima2, Syed Zakir Hussain Shah3, Muhammad Afzal4, Huma Naz5, Saif ur Rehman1 and Qingyou Liu1* 1State Key Laboratory of Tropical Biological Resources Protection and Utilization, Guangxi University, Nanning, 530004, China 2Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore, Pakistan 3Department of Zoology, University of Gujrat, Gujrat, Pakistan 4Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan 5Department of Zoology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan Article Information Received 28 June 2019 Revised 30 July 2019 Accepted 20 February 2020 Available online 04 November 2021

F ish meat is highly palatable, delicious and providing high quality proteins as well as source of vitamins and minerals (Shaikh et al., 2011). Many prebiotics and supplements are used in aquaculture farming which progresses the growth and fish survival rate (Shaikh et al., 2011). Prebiotics which are used in fish diet has been banned throughout the world for the well-being of humans. Many investigators are paying attention towards the use of supplements which increases the survival and growth rate in aquaculture industry (Luckstadt, 2006). Fish supplements contain amino acids, fatty acids and various minerals which are used in fish diet (Zhou et al., 2004). But due to its high demand and cost, it is compulsory to find alternate sources of protein (Toko et al., 2008;Correll, 1999). Instead of it, plant based proteins such as cereals are recommended as fish meal which are cost effective as well as low phosphorus content (Dalsgaard et al., 2009). The presence of phytate in fish meal reduces the digestibility as it contain indigestible carbohydrates and various antinutritional factors (Laining et al., 2010). However, fish feed supplemented with organic acid hydrolyzes phytate and enhances its solubility and absorption of minerals (Hossain et al., 2007;Zyla et al., 1995;Wood and Serfaty-Lacrosinere, 1992).
Fish meal supplemented with organic acid has following advantages: it reduces the microbial activity in the gut of fish and also inhibited the toxicity; it increases the absorption of nutrients by the proliferation of mucosal epithelium of intestine; it reduces the release of phosphorus ultimately decreases the water pollution, and increases the mineral (phosphorous, megnisum and calcium) absorption in fish as investigated by Baruah et al. (2008). Formic acid increases the absorption of minerals in fish (Vielma and Lall, 1997). Organic compounds including vitamins, minerals play important part in fish growth and fitness.

O n l i n e F i r s t A r t i c l e
As these are essential compounds so must be consumed by fish meal. Vitamin D is a fat-soluble vitamin having steroid-based chemistry. Naturally, there are two main sources of vitamin D, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) (Anthony, 1979). The primary role of cholecalciferol is the uptake and utilization of calcium and phosphorus as well as it maintains the normal concentration of calcium and phosphorus in blood to enhance the bone development as well as calcification (Li, 1994). The aim of present research work was to find out the effect of dietary formic acid and vitamin D3 supplementation on intestinal digestive enzyme activities and bone mineralization of Ctenopharyngodon idella.

Materials and methods
The present research work was conducted in Fisheries research Farmstead, University of Agriculture, Faisalabad. Four earthen ponds (19m×6m×1.5 m) were covered well with mesh net to avoid numerous undesired animals to enter in the specific ponds. Ponds were filled by tube well water. C. idella (grass carp) (n=25) was added to each pond after measuring the weight and length. In each earthen pond, inorganic compost containing urea and superphosphate was added on weekly basis for one month. During this one month duration, no fertilizer was added to ponds. Fish were then nourished with about 2 % body weight of fishes twice a day. In each pond, water level was kept constant throughout the study period. After duration of fourteen days, sampling was done and feed ratio was set accordingly. Finally, after feeding duration of 90 days, the fishes were harvested.
Four different experimental diets (FD1, FD2, FD3 and FD4) comprising formic acid (%) and vitamin D 3 (IU/ Kg) by following the levels 0, 0; 2, 0; 0, 500 and 500, 2, respectively. The time period for the whole experiment was about three months. For the formulation of experimental diets, instruments like, cereal grinding machine (FFC-45, JIMO, China) was used for the purpose of screening (0.05 mm) as well as grinding the diet ingredients. After electrically grinding the ingredients, mixture of mineral samples of fish oil and vitamins were added. For better texturing of dough, good quantity of watter was used for pellet construction, and also the experimental extruder (Model SYSLG30-IV) was used for formulation of pellets (3.5 mm). Pellets were dried with about 10% of moisture contents and stored at 20°C during the whole nourishing trial and then packed in plastic bags.
For bone mineralization, bone samples were digested in boiling nitric acid and perchloric acid mixture (3:1) for the estimation of minerals. Calicum, magnesium, zinc, iron, copper and manganese were measured by atomic absorption spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan) after appropriate dilution. Phosphorus content was analyzed at 720 nm wavelength using UV/VIS spectrophotometer (U-2001, Hitachi). Flame-photometer (Jenway PEP-7, UK) (AOAC, 1995) was used for sodium and potassium estimation. For digestive enzymes assays, eight fishes were randomly collected, anaesthetized and dissected from each replicate for the collection of intestines. The tissue homogenizer (Wisd HG-15D, DAIHAN Scientific) was used for homogenization of sample in cold sucrose (0.25 M) solution. The homogenate were centrifuged at 5,000 × g for 15 min in a cooling centrifuge (5ºC), supernatant was collected, frozen in sample vials, and stored at -20ºC until use. The dinitrosalicylic acid (DNS) methods were used for the estimation of amylase activity (Rick and Stegbauer, 1974). In reaction mixture starch (0.5Ml) and enzyme extract (0.5mL) were incubated at 37ºC for half an hour. For stopping reaction dinitrosalicylic acid was added and placed in boiling water for five minutes. After cooling, distilled water was added for dilution and absorbance were taken at 540nm. Activity were observed by maltose standard curve. Kunitz (1947) describes the casein digestion method for analyzing the protease activity. In reaction mixture casein as substrate (1%), phosphate buffer (pH 7.5) and tissue homogenate were added and kept at 37ºC for fifteen minutes. Reaction was stopped after addition of trichloroacetic acid (5%). The reaction mixture was filtered and obsorbance taken at 280nm. The activity of lipase was analysed by using p-nitrophenylpalmitate as the substrate (Mahadik et al., 2002). In reaction mixture p-nitrophenylpalmitate solution (0.9 mL), phosphate buffer (0.5 M) and enzyme extract were incubated at 37°C for 30 min. p-nitrophenol values were calculated at 410 nm expressed as μmol/min (one unit of activity). Two-way analysis of variance under RCBD was applied for data analysis (Steel et al., 1996). For comparison of means Student-Newman-Keuls test was used for determining significant differences (Snedecor and Cochran, 1991). For analyzing statistical data CoStat computer package (Version 6.303, PMB320 Monterey, CA, 93940 USA) was used.

Results
The fish group fed with formic acid significant showed increase in calcium, magnesium and potassium; however vitamin D 3 significantly increased the iron and manganese contents (µg/g) in the bones of C. idella fingerlings (Table  II). Table III shows the intestinal digestive enzyme activities of C. idella fingerlings. The amylase and protease activities were maximum in intestine of fish fed with formic acid followed by fornic acid: vitamin D3 (500:2), vitamin D3 (control diet). The activity of lipase enzyme was reduced by supplementation of formic acid as well as vitamin D 3 . The observed formic acid and vitamin D 3 (interactions) were synergistically enhanced bone mineralization of phosphorous (13.13±0.04a), sodium (2.11±0.03a), copper (14.3±0.04) and also showed enhanced results for the activity of amylase (2.75±0.00), protease(0.95±0.01) and lipase (0.30±0.01).

Discussion
In the present research work, the supplementation of formic acid exhibited noteworthy effect on calcium, potassium and manganese absorption during the development of bones of C. idella. The phytate hydrolysis alters the physiological condition which enhances the utilization of minerals (Sugiura et al., 2006). Similar results were reported by Khajepour and Hosseini (2010) who described that phosphorous and calcium content were enhanced in muscle tissue by the supplementation of 2% and 3% citric acid in the diet of beluga. In our research work the vitamin D3 supplementation significantly affected the iron and manganese in bones of C. idella. Likewise, Zhu et al. (2015) stated that addition of vitamin D3 in fish diet considerably enhanced the retention of sodium, potassium, phosphorous, calcium, magnesium and zinc in whole body of yellow catfish. Connell and Gatlin (1994) described that blue tilapia fed with dietary vitamin D3 considerably condensed the mineralization in hard tissues as compared to soft tissue, because dietary vitamin D had partial participation in calcium metabolism and mineralization of the hard tissues of tilapia. In the current research work, it was concluded that the activity of amylase, lipase and protease considerably improved in the intestine of C. idella fed with formic acid supplemented diets. The activity of enzymes was perhaps due to release of trace element by supplementation of organic acid in fish diet (Shah et al., 2015). The results are in line with those of Shah et al. (2015) who stated that supplementation of 3% citric acid in the diet of Labeo rohita fingerlings improved the activities of protease, lipase and amylase. Likewise, the tilapia fed with 1% citric acid supplemented diet improved the activity of amylase. When citric acid was added in diet, metal ion present in diet increases the activity of amylase in intestine of tilapia (Li et al., 2009). Furthermore, according to Agouz et al.

O n l i n e F i r s t A r t i c l e
(2015) supplementation of organic salts (malic+oxalic acids blend and Na-acetate+Ca-lactate blend) in the diet of Nile tilapia considerably increased the activity of protease enzyme. The conclusion is dietary formic acid and vitamin D 3 improves the bone mineralization and digestive enzymes activity in the C. idella juveniles.