PJZ_57_4_1619-1625

Impact of Supplementation of Poultry Feed with Locally Characterized Recombinant Thermostable Xylanase on the Growth Performance of Broiler Chicks

Asma Waris1, Ali Raza Awan1, Sehrish Firyal1, Naeem Rashid2,

Abu Seed Hashmi3, Muhammad Wasim1, Shagufta Saeed1 and Muhammad Tayyab1*

1Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences, Abdul Qadir Jillani (Outfall) Road, Lahore, Punjab, Pakistan.

2School of Biological Sciences, Faculty of Sciences, University of The Punjab, Quaid e Azam Campus, Lahore, Punjab, Pakistan.

3Riphah International University, Raiwind Road Campus, Lahore, Pakistan

ABSTRACT

The current study evaluated the efficiency of locally characterized recombinant thermostable xylanase (XYLTN) from Thermotoga naphthophila in broiler chicks. The XYLTN was produced using BL21 Codon Plus (DE3) cells having pET-21a containing xylanase gene from Thermotoga naphthophila and was used for the supplementation of poultry feed. For the poultry trail, a total of 150 day old broiler chicks were divided into five groups having 30 birds each. Group A served as negative control while groups B, C, and D were experimental groups and fed on a basal diet supplemented with 1000, 1500, and 2000 IU/Kg of locally produced XYLTN, respectively, whereas group E served as positive control and was fed on diet supplemented with 1500 IU/Kg of commercially available xylanase. The supplementation of poultry feed with XYLTN revealed, a maximum weight gain of 1681.25g, feed intake of 2810g, and feed conversion ratio of 1.67 when the feed was supplemented with 2000 IU/Kg of XYLTN. Locally produced XYLTN exhibited promising outcomes compared to positive control which is being utilized currently for the supplementation of poultry feed in the industry. The weight gain, feed intake, and feed conversion ratio of 1681.25g, 2810g, and 1.67 for experimental group D were comparable to 1610.38g, 2830g, and 1.75 for positive control. The ability of enzyme to enhance weight gain, feed consumption, and feed conversion ratio in poultry chicks makes it a strong candidate for replacement of its commercial counterpart being imported for the poultry industry, and its domestic production will contribute to the economic availability of this xylanase for the poultry feed industry.

Article Information

Received 24 October 2023

Revised 12 February 2024

Accepted 27 February 2024

Available online 23 April 2024

(early access)

Published 09 June 2025

Authors’ Contribution

AW performed experimental work. MT planned and supervised the study, and guided for manuscript write-up and editing. ASH, SF and SS facilitated the conduction of experiments. MW and ARA helped in data analysis. NR supported in proofreading of manuscript.

Key words

Recombinant thermostable xylanase, XYLTN, Thermotoga naphthophila, Poultry trail, weight gain, feed conversion ratio

DOI: https://dx.doi.org/10.17582/journal.pjz/20231024054103

* Corresponding author: [email protected]

0030-9923/2025/0004-1619 $ 9.00/00

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/).

INTRODUCTION

Poultry is one of the most organized and fast-growing agro-based industries in Pakistan and is playing a key role in the fulfilment of the needs for the animal protein of the nation. The high growth rate and feed efficacy are two major considerations of the poultry feed industry. However, the poultry industry is facing a lot of challenges in developing countries and one of the main problems is the expense of feed ingredients which is about 70% of the total production cost (Alagawany and Attia, 2015). The highest production cost of this industry is attributed to the scarcity of cereal grains due to the global increase in demand for these grains, especially corn for ethanol production (Donohue and Cunningham, 2009; O’Nell at al., 2012). Thus, aggravated prices of poultry feed enforce the producers to explore alternative, cheaper, and non-conventional feed sources (Ncobela and Chimonyo, 2015).

For poultry feed formulation, protein is one of the main nutrients. Animal protein sources are more expensive whereas plant protein sources are not only cost-effective but also available in abundance, as by-products of the oil seed industry (Beski et al., 2015). Hence, the major ingredients of the poultry feed are derived from plant sources which are composed of non-starch polysaccharide (NSPs) fibre like cellulose, glucan, mannan, and xylan. However, the limitation in the use of plant-based feed sources is the presence of a high level of NSPs fibres which are not digested by poultry. The presence of dietary NSPs fibre in the poultry feedstuff acts as an anti-nutritional factor and can be accumulated in the form of a gel-like substance in the animal gut which decreases the digestion and absorption of nutrients. Moreover, these SNPs can facilitate intestinal fermentation through modulating intestinal micro-flora that might be harmful to the chicken digestion process (Fathima et al., 2022; Choct et al., 1996, 1999). Thus, poor digestion and absorption of nutrients lead to the bad performance of birds (Kidd et al., 2001; Graham et al., 2002; Bedford and Morgan, 1996).

The poultry birds are mono-gastric animals and do not produce endogenous enzymes for the digestion of these fibres. Due to the unavailability of these endogenous enzymes including xylanase, phytase, and cellulase in the animal body, NSPs are being excreted with the manure (Lie and Porress, 2013). To overcome this problem, the diet is supplemented with suitable exogenous enzymes. The usage of exogenous enzymes in poultry feed has been largely studied during the last decades (Bedford and Schulse, 1998). Thus, various types of exogenous enzymes have been recognized for targeting such types of substances, and their effect may be variable depending upon the factors like birds’ age, feed quality, and type (Acamovic, 2001; Bedford, 2000). Supplementation of NSPs cleaving enzymes enhances the digestibility of nutrients by decreasing the intestinal viscosity, eliminating the anti-nutritive effect of these NSPs fibre, and also releasing certain bound nutrients which eventually improve the bird performance (Dongare et al., 2017). Xylanases are the main enzymes that are involved in the breakdown of xylan by hydrolysing the 1, 4-beta-D-xylosidic linkage between the xylose residues randomly (Mendes et al., 2013). The supplementation of feed with xylanase results in the hydrolysis of xylan in feed to simple sugar which improves the nutritive values of the poultry diet that put a positive impact on the growth performance of the poultry birds (Hosseini and Afshar, 2017). Thus, the use of exogenous enzymes like xylanase is one of the key components of the poultry diet for the efficient utilization of feed ingredients and downgrading the production cost (Hahn-Didde and Purdum, 2014).

Thermostability is highly concerned when animal feed is supplemented with exogenous enzymes because, during the pelleting steps, feed is exposed to a very high temperature that denatures mesophilic enzymes (Svihus et al., 2005). Thermophilic or hyper-thermophilic microbes can provide thermostable enzymes which can withstand high-temperature conditions without denaturation (Chesson, 1993). The enzymes available in the market are mostly from fungal strains however, the bacterial enzymes have got attention recently because of their specific activity, broad pH range, and higher thermostability (Maki et al., 2009). The production cost of these enzymes is high enough, thus there is a need to develop a mechanism for low-cost availability of such thermostable enzymes for industry (Klein et al., 2012).

In Pakistan, various groups worked on the production of these enzymes but unfortunately there is no single enzyme available in the market that can be used for the fulfilment of the local industrial demand. The production of the thermostable enzyme by recombinant DNA technology is the most appropriate tool for the economic availability of thermostable enzymes in the market (Kulkami et al., 1999; Hough and Danson, 1999).

The current study was designed for the evaluation of the efficacy of locally characterized recombinant thermostable xylanase for the growth of poultry birds and its suitability for the poultry feed industry.

MATERIALS AND METHODS

Chemicals and reagents

All the chemicals and reagents used in the present study were of purified grade and were purchased from Sigma Aldrich (USA) and Merck (Germany).

Production of recombinant xylanase

Recently recombinant thermostable xylanase from Thermotoga naphthophila has been characterized. This recombinant xylanase was produced using E. coli BL21 CodonPlus cells (DE3) cells having pET-21a containing the xylanase gene from T. naphthophila. The microbial culture was maintained on LB agar plates containing ampicillin (100 µg/mL). For the production of recombinant thermostable xylanase under pre-optimized conditions, the LB medium was sterilized in an autoclave (SH-AC-60; SAMHEUNG, Sout Korea). Regarding the production of xylanase, a white colony of overnight grown recombinant BL21 CodonPlus (DE3) cells were diluted to 1% with the fresh LB medium followed by incubation at 37 ºC under shaking conditions until OD reached 0.4. Cells were induced with 0.4 mM of isopropyl β D-1-thiogalactopyranoside and were further incubated under the same conditions. The cells were harvested and re-suspended in 50 mM Phosphate buffer (pH 7) (Tayyab et al., 2011).

Cell disruption

The cells were lysed by sonication through the ultrasonic processor (Sonics, Newtron, USA). The sonicated cells were centrifuged (Z326 K, Hermle Laboratory, Germany) at about 12,000 rpm for 5 min at 4°C (Sabir at al., 2017). The supernatant was stored at 4°C for determination of xylanase activity and, for supplementation of the poultry feed.

Xylanase activity assay

Xylanase activity was examined by the dinitrosalisylic acid (DNS) method (Miller, 1959). The total 1000 µL reaction mixture comprised of 50 µL enzyme, 100 µL 1% beech-wood xylan as substrate and 850 µL 50 mM phosphate buffer of pH 7. The enzyme activity assay mixture was incubated for 10 min at 90 °C. The reaction was stopped with the addition of 1mL of DNS reagent followed by boiling for 10 min, cooled for color stabilization and finally the absorbance was recorded at 540 nm. One unit of xylanase activity was defined as the amount of enzyme required to produce one µmol of xylose per min under assay conditions. Xylanase activity units were determined using the standard curve of xylose (Bhalla et al., 2015).

Experimental treatment formulation

The soluble portion of sonicated cells was utilized for supplementation of the poultry feed. The feed was formulated in an automated unit at Nizami Feed (Pvt. Ltd), 25 km Sheikhupura, Punjab, Pakistan. The soya bean meal and corn-based feed were prepared for the fulfillment of the nutritional requirements of the poultry chicks, as recommended by the National Research Council (NRC, 1994), and are being used by the poultry industry. The basic composition of feed was the same as being used in commercial feed available for the poultry forms shown in Table I. Five different types of feeds designated as A, B, C, D, and E were formulated, according to the composition given in Table I. Diet A served as negative control and was not supplemented with enzyme whereas diets B, C, and D were supplemented with 1000, 1500, and 2000 IU/Kg of locally produced recombinant xylanase, respectively, while diet E was supplemented with 1500 IU/Kg of commercially available xylanase, Econase XT by AB Vista Animal Nutrition Technology Company (Woodstock, Marlborough, United Kingdom) which served as a positive control. The enzyme concentrations were selected based on literature that indicated the positive response of poultry feed enzymes (Hu et al., 2019).

Feeding trials on broiler chicks

The Feeding trials were conducted in the controlled environment at the Dua poultry farms, Niaz Koot, Kala Shah Kaku, tehsil Shahdara, district Lahore, Punjab, Pakistan with the collaboration of Crescent Feeds and Allied Products, Sundar Sharif, Lahore, Punjab, Pakistan. A total of 150-day old broiler chicks of commercial strains were divided into 5 groups each comprises of 3 replicates of 10 birds. Each group was assigned a unique diet as discussed above. The feeding trial was conducted for five weeks (35 days) under controlled conditions and with easy access to feed and water. During the trial, initial, weekly and final body weight, total feed consumed, feed efficacy, feed intake, weight gain, and feed conversion ratio were calculated (Sabir et al., 2018).

Table I. Composition of poultry feed.

Feed composition	A	B	C	D	E
Corn	45	45	45	45	45
Rice polish	8.58	8.58	8.58	8.58	8.58
SBM	20	20	20	20	20
SFM	4	4	4	4	4
Oil	3	3	3	3	3
Bone ash	2	2	2	2	2
Canola	12	12	12	12	12
Wheat bran	2	2	2	2	2
Isoleucine	0.01	0.01	0.01	0.01	0.01
Threonine	0.07	0.07	0.07	0.07	0.07
Calcium carbonate	0.23	0.23	0.23	0.23	0.23
L-HCL	0.57	0.57	0.57	0.57	0.57
DCP	1.86	1.86	1.86	1.86	1.86
NaHCO3	0.18	0.18	0.18	0.18	0.18
DLM	0.2	0.2	0.2	0.2	0.2
Premix vitamin	0.3	0.3	0.3	0.3	0.3
Xylanase (IU/Kg feed)	-	1000	1500	2000	1500 (Econase XT)
Sum (kg)	100	100	100	100	100

SBM, soybeans meal; SFM, sunflower meal; Oil, vegetable oil; L-HCl, L-lysine hydrochloride; DCP, Dicalcium phosphate; NaHCO3, sodium bicarbonate; DLM, DL-methionine.

Statistical design

The collected data was analyzed using the SPSS software and one-way ANOVA. The obtained results were represented as the significance of differences between means calculated by the least significant difference test and differences were significant at P ≤0.05 (Sabir et al., 2018; Steel et al., 1996).

RESULTS AND DISCUSSION

The purpose of the study was to evaluate the impact of supplementation of locally characterized recombinant thermostable xylanase on the growth performance of broiler chicks. The role of xylanase in improving the nutritive values of the poultry diet has been reported previously (Papadopoulos et al., 2022; Bedford, 2000; Bedford and Morgan, 1996; Annison and Choct, 1991). Xylanases are applied as a feed additive to improve the nutritional value of feed resulting in improvement of feed intake, weight gain, and feed conversion of animals (Baker et al., 2021; Fisher and Petersson, 2008). The supplementation of poultry feed with locally characterized xylanase showed a growth-enhancing impact in poultry birds and resulted in increased weight gain, feed consumption, and improved FCR. At the end of the first two weeks of trials, a significant impact on bird growth was recorded. The supplementation of feed with 1500 IU/Kg of locally produced xylanase (group C) or 2000 IU/Kg (group D) could enhance the weight of birds from 1050.37 to 1108.32 g or 1270.13 g at the end of the 4th week and from 1410.19 to 1572.31 g or 1681.25 g at the end of 5th week of the trial (Table II) when compared with control. The weight gain data comparison at the end of the trial showed a clear difference in weight gain as compared to the control. The supplementation of feed with locally characterized xylanase with 1000, 1500, and 2000 IU/Kg of feed enhanced the weight gain from 1410.19g for negative control to 1450.16, 1572.31, and 1681.25g for group B, C, and D. The feed supplemented with locally characterized xylanase in group D showed better weight gain 1681.25g as compared to 1610.38g for group E supplemented with 1500 IU/Kg of Econase XT commercially available xylanase. The weight gain of 1681.25g in group D is significantly high as compared to 1610.38g for group E supplemented with commercial Econase XT shown in Table II. These results are in agreement with previous findings regarding improvement in body weight gain in the broilers fed on a corn-soybean-based diet supplemented with xylanase (Rao et al., 2021; Hu et al., 2019; Olukosi and Bedford, 2019; Dongar et al., 2017; Olukosi et al., 2007) or wheat-based diet supplemented with xylanase (Pirgozliev et al., 2023; Zhang et al., 2014; Esmaeilipour et al., 2012). However, a greater impact in the wheat-based diet was recorded as compared to the corn-based diet due to the higher amount of soluble NSPs in wheat-based feed (Anwar et al., 2023; Nian et al., 2011; Hajati, 2010; Mathlouthi et al., 2002).

Similarly, the FCR value was improved from 1.88 to 1.67 (group D) which is quite high as compared to 1.75 for Econase XT (group E) which served as a positive control (Table III). The present study revealed the positive effect of locally characterized xylanase supplementation of feed for poultry birds. Thus, FCR was enhanced by the addition of xylanase, which is similar to previous reports (Nian et al., 2011; Goa et al., 2008). The improved performance of the birds in the present study was might be due to the reduction in the anti-nutritive effect of NSPs due to improved digestion (Kocher et al., 2003; Singh et al., 2012; Stefanello et al., 2015; Vandeplas et al., 2010; Malathi and Devegowda, 2001).

CONCLUSION

The locally characterized recombinant thermostable xylanase has strong potential for NSPS digestion and for enhancement of weight gain, feed consumption and improved FCR value in broiler chicks. The weight gain data clearly showed a significant impact of locally characterized xylanase as compared to Econase XT, xylanase being utilized currently in poultry feed. Domestic production of locally characterized xylanase will save the huge foreign exchange for import of this enzyme. The local production of this enzyme will result in the economic availability of xylanase that can replace the imported counterpart being utilized currently in the poultry feed industry.

Table II. Effect of xylanase on weight gain data of poultry trials.

Groups	Week 1 P= (0.000***)	Week 2 P= (0.000***)	Week 3 P= (0.000***)	Week 4 P= (0.000***)	Week 5 P= (0.000***)
A (Negative control)	160.87±0.81	407.73±0.59	741.61±0.24	1050.37±0.61	1410.19±0.42
B (1000 IU/Kg)	166.67±0.59	416.89±0.45	757.16±0.57	1087.15±0.37	1450.16±0.64
C (1500 IU/Kg)	177.60±0.51	427.25±0.89	766.51±0.42	1108.32±0.27	1572.31±0.33
D (2000 IU/Kg)	181.53±0.41	477.92±0.80	799.22±0.74	1270.13±0.71	1681.25±0.56
E (1500 IU/Kg) (Econase XT)	180.12±0.33	431.83±0.45	784.72±0.47	1140.17±0.29	1610.38±0.89

Table III. Efficacy of xylanase in weight gain, feed intake and feed conversion ratio.

Groups	A negative control	B 1000 IU/Kg	C 1500 IU/Kg	D 2000 IU/Kg	E 1500 IU/Kg (Econase XT)
Average feed intake (g)	2650	2700	2770	2810	2830
Overall weight gain (g)	1410±0.42	1472±0.33	1572±0.56	1681±0.56	1610 ±0.89
FCR	1.88	1.84	1.76	1.67	1.75

ACKNOWLEDGEMENT

The authors are thankful to Nizami Feed (Pvt. Ltd), Sheikhupura, Punjab, Pakistan for all their support in the conduction of trails.

Funding

The study received no external funding.

IRB approval

This study was approved by the Advanced Studies Research Board of University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan

Ethical statement

The experiments were conducted with the prior approval of Animal Ethics Committee, University of Veterinary and Animal Sciences Lahore, and with Pakistan code for the care and the use of animals for scientific purposes.

Statement of conflict of interest

The authors have declared no conflict of interest.

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