Comparative study of in vivo and in vitro methods in determining protein digestibility of feeds for bagrid catfish, mystus nemurus

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Date
2008-04
Authors
Syed Muhammad, Sharifah Rahmah
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Abstract
The reliability study of the in vitro methods in relation to the in vivo method was conducted to assess the protein digestibility of experimental diets formulated at 35% protein and 15% lipid with 0 to 60% increasing protein substitution of fishmeal by soybean meal for bagrid catfish, Mystus nemurus. As a part of this study, biochemical assays showed optimum protease activity at pH 2.0 and 9.0 in the stomach and intestiness respectively. Protease activity of post feeding study in the stomach and intestines were highest at 2 and 5 hours accordingly. Pepsin was observed in the stomach while serine proteases were more dominant in the intestines, followed by trypsin, metalloproteases and chymotrypsin. Characterization of crude intestinal extract of M nemurus via SDS-PAGE using commercial specific protease inhibitors revealed eight alkaline proteases with molecular weights between 8.4 and 56.9 kDa. Prior to formulation of experimental diets, in vitro protein digestibility of feedstuffs was assessed using pH stat, pH drop, spectrophotometric assay and SDS-PAGE methods. pH stat and pH drop methods revealed a decreasing order of respective Degree of Hydrolysis (DH) and Relative Protein Digestibility (RPD) with fishmeal (FM) > soybean meal (SBM) > squid meal (SM) > raw soybean meal (RSBM) using all enzyme systems. However, RPD for pH drop method using Lazo 1-enzyme system showed FM > SM > SBM > RSBM. Saterlee 4-enzyme system revealed the highest DH and RPD values for all feedstuffs followed by the crude intestinal enzyme extract of M nemurus, Hsu 3-enzyme and Lazo 1-enzyme system. In vitro spectrophotometric assay and SDSPAGE demonstrated that crude intestinal enzyme extracts of M nemurus were inhibited the highest by RSBM followed by SM, SBM and FM. Zymogram from SDS-PAGE showed that preincubation of RSBM with crude intestinal enzyme extract of M nemurus resulted in 3 reduced and 5 disappeared protease bands. Overall, protein digestibility of experimental diets using in vivo and in vitro methods decreased as SBM protein substitution level increased while percentage of inhibition increased as SBM protein substitution level increased. The highest tolerance level of SBM protein substitution for M nemurus was up to 1 0% which was determined using the in vivo method. pH stat and pH drop methods showed the highest DH and RPD using Saterlee 4-enzyme system followed by crude intestinal enzyme extract of M nemurus, Hsu 3- enzyme and Lazo 1-enzyme system. The highest correlation of the in vitro methods with the in vivo method was obtained using spectrophotometric assay followed by pH stat, SDS-PAGE and pH drop although no significant differences were observed among the in vitro methods.
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Keywords
Determining protein digestibility , Bagrid catfish, mystus nemurus
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