Cholecalciferol and Formic Acid Synergistically Enhance Digestive Enzymes Activity and Bone Mineralization in Ctenopharyngodon idella Laiba Shafique 1 *, Mahroze Fatima 2 , Syed Zakir Hussain Shah 3 , Muhammad Afzal 4 , Huma Naz 5 , Saif ur Rehman 1 and Qingyou Liu 1 * 1 State Key Laboratory of Tropical Biological Resources Protection and Utilization, Guangxi University, Nanning, 530004, China 2 Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore, Pakistan 3 Department of Zoology, University of Gujrat, Gujrat, Pakistan 4 Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan 5 Department 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 (early access) Published 26 April 2022 Authors’ Contribution MA designed the experiment. LS executed the research. SZHS did statistical analysis. MF helped in compiling the data. HN, SUR and QL helped in writing the article. Key words C. idella, Formic acid, Vitamin D3, Bone mineralization, Digestive enzymes This study was planned to determine intestinal digestive enzymes activity and bone mineralization of Ctenopharyngodon idella. Four type food were prepared by using formic acid (%) and vitamin D3 (IU/Kg) in experiment viz, FD1, FD2, FD3 and FD4, respectively. Fish fed with formic acid showed considerable increase in calcium, magnesium potassium, however vitamin D 3 considerably increase iron and manganese contents (µg/g) in the bones of C. idella. Amylase and protease activity was maximum in fish intestine fed with formic acid followed by fornic acid × vitamin D3, vitamin D3 = control diet. Activity of lipase enzyme was reduced by formic acid supplementation as well as vitamin D 3 . The observed formic acid and vitamin D 3 (interactions) were synergistically bone mineralization of phosphorus, sodium, copper and also showed positive result for the activity of amylase, protease and lipase. Finally, formic acid and vitamin D 3 improves bone mineralization and digestive enzymes activity in the C. idella. 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 * Corresponding author: laibazoologist@gmail.com, qyliu- gene@gxu.edu.cn 0030-9923/2022/0004-1991 $ 9.00/0 Copyright 2022 by the authors. Licensee Zoological Society of Pakistan. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 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 anti- nutritional 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 ABSTRACT Pakistan J. Zool., vol. 54(4), pp 1991-1994, 2022 DOI: https://dx.doi.org/10.17582/journal.pjz/20190628110605 Short Communication