Effect of Dietary Supplementation of Aspergillus Xylanase on Broiler Chickens Performance

Document Type: Research Articles

Authors

1 Department of Livestock and Pasture Science, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa

2 Department of Animal Science, Faculty of Agriculture and Natural Science, University of Mpumalanga, Private Bag X11283, Mbombela 1200, South Africa

3 Department of Animal Science, School of Agricultural Science, North-West University, Private Bag X2046, Mmabatho 2735, South Africa

Abstract

The effect of Aspergillus xylanase (ASXYL) supplementation to maize-soybean diets on serum aspartate aminotransferase, serum alanine aminotransferase, microbial examination, growth traits, carcass characteristics and meat quality traits of broiler chickens was investigated. Three hundred one-day-old mixed sex “Cobb 500” broiler chicks were allotted to 5 dietary treatments with 5 replicates of 12 birds each. The treatments include, ASXYL0 (0 g/kg), ASXYL10 (1 g/kg), ASXYL15 (1.5 g/kg), ASXYL20 (2 g/kg) and ASXYL25 (2.5 g/kg). Birds fed ASXYL20 had the highest (P<0.05) body weight with an improved feed conversion ratio (FCR) and a higher values for thigh, breast, wing and carcass yields. Neck weight was high (P<0.05) for fed birds ASXYL0, ASXYL15 and ASXYL20. Drumstick recorded higher (P<0.05) value for birds fed ASXYL20 though, similar to ASXYL10, ASXYL15 and ASXYL25. Drip and cooking loss decreased (P<0.05) with increased supplemental levels of ASXY while shear force increased (P<0.05) as ASXYL supplementation increased. Urea, aspartate aminotransferase and alanine aminotransferase decreased (P<0.05) with increased supplemental levels of ASXYL while glucose level increased (P<0.05) with increased levels of ASXYL supplementation. Supplementary ASXYL influenced (P<0.05) the proliferation of Lactobacillus counts in ileum and caecum while no difference (P>0.05) was observed on the population of Bifidobacteria and Escherichia coli in both ileum and caecum of broilers at the end of the feeding trial. It was concluded that dietary ASXYL20 produced a much improved body weight and retail cut yields. Again, the bio-markers showed that ASXYLimproved the health status of broiler chicken and the tested enzyme influenced a positive intestinal environment.

Keywords


INTRODUCTION

Poultry farming is one of the most important sectors in the global agribusiness. This sector increased significantly after inclusion in the production chain in the 1980 s mainly as a result of increased adoption of technology in the sector. The mechanisms of technological advances in nutrition with the inclusion of exogenous enzymes in broiler diets allow greater assimilation of nutrients resulting in increased meat yield as a result of improved body weight. Research studies have reported the beneficial impact of exogenous xylanase enzymes on the performance and nutrient digestibility of broiler birds fed non-conventional feedstuffs (Mathlouthi et al. 2002; Oyeagu et al. 2016). Broiler ration in South Africa and most part of the world are almost entirely formulated from two basic ingredients; maize, which is an excellent energy source and soybean meal, which contributes a high-quality protein and with great amino acid availability (Opalinski et al. 2006). However, it is known that the nutrients originated from these ingredients are not absorbed properly, mainly due to the presence of anti-nutritional factors such as non-starch polysaccharides (NSPs) which are inherent in the plant cell wall (Oyeagu et al. 2015). Due to the chemical structure of the plant cell wall matrix, NSPs degrading enzymes has been recommended to enhance poultry performance. Exogenous xylanase enzyme supplementation has been documented to be effective in breaking polymeric chains of NSPs and hence improve the nutritive value of feedstuffs (Giraldo et al. 2008). Therefore adding NSPs – degrading enzymes in poultry diet has increased considerably in recent years. Birds do not produce enzymes such as xylanase for the digestion of NSPs. Supplementation of xylanase enzymes may not only reduce the anti-nutritive effects of NSPs, but also releases some nutrients from these, which could be utilized by birds (Oyeagu et al. 2016). Again, these NSPs – degrading enzymes has been reported to improve Lactobacillus and Bifidobacteria counts in caecum of broilers fed corn-soybean meal based diet (Nian et al. 2011). Exogenous xylanase enzyme supplementation can change the nutritional status which may regulate the metabolism and functioning of the growth related endocrine system that will improve carcass yield of broiler chickens (Hajati et al. 2009). Nutritional status is an important factor in the regulation of plasma biomarkers in broiler chickens (Buyse et al. 2002; Swennen et al. 2005; Gao et al. 2008). Evaluation of plasma biochemistry in birds allows for the identification of metabolic alterations as a result of management conditions such as diets (Alonso-Alvarez and Ferrer, 2001). The use of blood examination as a way of assessing the health status of animals has been documented (Muhammed et al. 2000; Owoyele et al. 2003). Haematobiochemical examination plays a vital role in the physiological, nutritional and pathological status of organisms (Muhammed et al. 2000). They range from giving the level of the blood to detecting ailment or disorders through them. It had been reported that biochemical changes as a result of toxins have effects on blood parameters (Karnish, 2003). The effect of different diets (barley-based, wheat-based, and non-conventional feed) supplemented with different mono-enzymes or multi-enzymes has been evaluated on the blood parameters of broiler (Muhammed et al. 2000; Owoyele et al. 2003; Oyeagu et al. 2016; Oyeagu et al. 2019a), but there is little information on serum biomarkers, ileum and caecal micro-flora, carcass characteristics and meat quality traits of broiler chickens fed Aspergillus xylanase. The present study, therefore, sought to examine the effect of Aspergillus xylanase on serum biomarkers, ileum and caecal micro-flora, growth traits, carcass characteristics and meat quality traits in broiler chickens fed maize-soybean meal diets.

 

MATERIALS AND METHODS

Ethical consideration

Ethical principles were taken into consideration during the study to adhere to the national and international standards governing research of this nature with regards to the use of research animals. Ethical approval was obtained from the Ethical Clearance Committee of University of Fort Hare, Alice, South Africa.

 

Study site

This study was conducted at the poultry unit of North-West University experimental farm (Molelwane), in the North West province of South Africa. The study area is located at 25.80˚ S and 25.50˚ E and experiences summer climate from August to March with temperatures ranging from 22 to 35 ˚C and average annual rainfall ranging from 200 to 450 mm per annum. The study site experiences winter from May to July, with sunny dry days and cool nights with average minimum and maximum temperatures of 2 and 20 ˚C, respectively. The study lasted for six weeks.

 

Enzyme characteristics

The tested Aspergillus xylanase enzyme (RONOZYME® WX (CT), DSM Nutritional Products Johannesburg South Africa) is a granulated heat stable endo-xylanase. The active substance in the enzyme is endo-1, 4-β-xylanase (IUB No. 3.2.1.8) which is produced by a genetically modified strain of Aspergillus oryzae micro-organism (Aquilina, 2016). This strain is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) with the accession number DSM 26372 (Aquilina, 2010b). According to the manufacturer (Aquilina, 2010a), the bulk density is approximately 1.1 g/ml and the average particle size is approximately 600 µm with enzyme activity of 1000 FXU/t (1000 g/t).

 

Experimental diets

The feeding strategy consisted of starter (0 to 21 d) and finisher (22 to 42 d) basal diets (Tables 1 and 2), which were formulated to meet the birds’ dietary nutrient requirements (NRC, 1994). At each feeding phase (starter and finisher), five dietary treatments of iso-nitrogenous and iso-caloric were formulated through the addition of Aspergillus xylanase (ASXYL) enzyme at five different levels.

 

Table 1 Ingredient (%) and chemical composition (g/kg DM unless otherwise stated) of experimental diets for broiler chicks at the starter phase (0-3 weeks)

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

1 Vitamin and mineral premix (2.5 kg of vitamin premix) contained: Retinal: 2700 mg; Calcidiol: 400 mg; Tocopheryl acetate: 18 g; Menadione: 2000 mg; Thiamine: 1800 mg; Riboflavin: 6600 mg; Niacin: 10 g; Calcium pantothenate: 30 g; Pyridoxine: 3 g; folic acid: 1 g; Cobalamin: 15 mg; Choline chloride: 250 g; Biotin: 100 mg; Mn: 100 g; Fe: 50 g; Zn: 100 g; Cu: 10 g; I: 1 g and Se: 200 mg.

2 1000 g of Maxiban contained: Narasin: 80 g/kg and Nicarbazin: 80 g/kg.

3 1000g of Surmax contained: Avilamycin: 100 g/kg.

 

The five experimental diets formulated were, ASXYL0 (only basal diet; BD), ASXYL10 (BD+1 g ASXYL/kg feed), ASXYL15 (BD+1.5 g ASXYL/kg feed), ASXYL20 (BD+2 g ASXY/kg feed) and ASXYL25 (BD+2.5 g ASXY/kg feed) for both starter and finisher phases. The ingredient and chemical composition of the five experimental diets for starter and finisher phases are presented in Tables 1 and 2, respectively. The chemical (proximate) composition of the experimental diets was analyzed according to AOAC (2006) methods with average crude protein (CP) and metabolizable energy (ME) of 23.70 CP and 12.60 MJ of ME/kg respectively for starter chicks while an average of 19.70 CP and 13.00 MJ of ME/kg was recorded for finisher birds.

 

Experimental birds and management

A total of three hundred, one day old, non-sexed broiler birds (Cobb 500®) were used in the present study. Sixty birds (five replication of twelve birds in each replicate per treatment group) were assigned randomly to one of the five experimental diets (ASXYL0, ASXYL10, ASXYL15, ASXYL20 and ASXYL25). Each experimental diet was replicated into five experimental pens measuring 2.5 m length × 2.5 m width × 2.5 m height with twelve birds each. The birds were housed in cages with wood shavings as litter. General flock prophylactic management and routine vaccinations were administered as follows; day 1 – intra ocular New castle disease vaccine, week 2 – Gumboro disease vaccine, week 3 – Lasota (New castle disease vaccine), week 4 – Gumboro disease vaccine, and week 5 – fowl pox vaccine. A stress pack was administered to the birds via drinking water at 100 g/50 liters (according to manufacturer’s recommendation) to boost appetite and energy supply. Dietary treatments and clean water were provided ad libitum in a six-week feeding trial.

 

Serum biochemical profile

Before blood collection, birds were feed-fasted for 4 h in an attempt to allow for the stabilization of the various plasma constituents.

 

Table 2 Ingredient (%) and chemical composition (g/kg DM unless otherwise stated) of experimental diets for broilers at the finisher phase (4-6 weeks)

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

1 Vitamin and mineral premix (2.5 kg of vitamin premix) contained: Retinal: 2700 mg; Calcidiol: 400 mg; Tocopheryl acetate: 18 g; Menadione: 2000 mg; Thiamine: 1800 mg; Riboflavin: 6600 mg; Niacin: 10 g; Calcium pantothenate: 30 g; Pyridoxine: 3 g; folic acid: 1 g; Cobalamin: 15 mg; Choline chloride: 250 g; Biotin: 100 mg; Mn: 100 g; Fe: 50 g; Zn: 100 g; Cu: 10 g; I: 1 g and Se: 200 mg.

2 1000 g of Maxiban contained: Narasin: 80 g/kg and Nicarbazin: 80 g/kg.

3 1000g of Surmax contained: Avilamycin: 100 g/kg.

 

Blood was collected in the morning to further reduce the variability of the measured plasma constituents. At 42 days of age, five birds were chosen randomly from each experimental pen and 2 mL of blood was collected from the wing vein using a sterile syringe and needles. Blood collected was emptied into a labelled treated vacutainer tubes. Red-top tubes without anticoagulant were used for serum biochemical analysis. The blood was stored for 10 minutes at room temperature. After centrifugation (20 minutes, 1500 rpm), the serum was collected into 0.5 mL centrifuge tube and stored at -20 ˚C pending determination of serum metabolites. All analyses were conducted within 48 h after collection. The serum metabolites considered are alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein, albumin, urea and cholesterol were assayed using automated Idexx Vet Test Chemistry Analyser (IDEXX Laboratories, Inc) in the Animal Health Laboratory at the Centre for Animal Health Studies North-West University, Marfikeng.

 

Growth performance

Average daily feed intake (ADFI) per bird was measured from day 1 to day 42 of age by subtracting the weight of the feed left over from that of the feed offered, and dividing the difference by the total number of birds in the pen. Average live-weight was measured weekly by weighing all the birds in each pen using a 10100 g capacity precision weighing balance with model, A and D Weighing GF-10 K industrial balance, made in Japan. The feed conversion ratio (FCR) was calculated as follows: FCR= feed intake/weight gain.

 

Slaughter procedure

At 42 days of age, all chickens were taken to Rooigrond poultry abattoir (North-West province, South Africa) for slaughter. The chickens were starved for 12 hours before slaughter. All the chickens were humanely gas stunned by exposing them to relatively low concentrations of carbon dioxide (<40% by volume in air), and then, once they were unconscious, exposed to a higher concentration (approximately 80% to 90% by volume in air). At the abattoir, all the chickens were hung onto a movable metal rack that holds them upside down by their feet. Chickens were then slaughtered by cutting the jugular vein with a sharp knife and they were left hanging until bleeding stopped.

 

Carcass characteristics

Five birds per replicate were randomly selected for determination of carcass characteristics and meat quality. Immediately after slaughter, the feathers were plucked and the gastro intestinal tract (GIT) was removed. The carcasses were then weighed to obtain the carcass weight of the birds. For the measurement of carcass cuts, head and shanks were removed close to the scull and at hock joint, respectively. Wings were removed by cutting at the humeoscapular joint, the cuts were made through the head to the shoulder girdle, and the vertebrae was then removed intact by pulling outwardly. The breast muscle, neck, wings, shank, thighs, drumsticks and vertebrae (back) were each weighed separately.

 

Meat pH and temperature measurements

Meat pH and temperature were recorded immediately after slaughter and 24 h post slaughter on the breast muscle (central area of the breast) using a Corning Model 4 pH-temperature meter (Corning Glass Works, Medfield, MA) equipped with an Ingold spear-type electrode (Ingold Messtechnik AG, Udorf, Switzerland) according to Stanford et al. (2003). After every 20 measurements, the pH meter was calibrated with pH 4, pH 7 and pH 10 standard solutions (Ingold Messtechnik AG, Udorf, Switzerland).

 

Meat colour

Colour of the meat (L*=Lightness, a*=Redness and b*=Yellowness) was determined 24 hours after slaughter, using a Minolta colour-guide (BYK-Gardener GmbH, Geretsried, Germany), on a 20 mm diameter measurement area and illuminant D65-day light, 10 degree observation angle. The colour meter was calibrated using the green standard before measurements. Colour recording was done on the surface of the breastmuscle, which was allowed to bloom for 1 hour on a polystyrene tray at 4 ˚C.

 

Water holding capacity (WHC)

The pressure method as described by Delezie et al. (2007) was used to determine the WHC. About 100 g meat sample from pectoral major (PM) was cut and weighed to obtain the initial weight using a digital scale sensitive up to 0.01 g. The meat sample was then placed in between 2 filter papers, placed on a flat surface and approximately 60 kg weight was applied on the sample for 5 min. Thereafter, the meat sample was re-weighed. The WHC was calculated as the ratio of the amount of water retained over the initial sample weight.

 

Drip loss

Approximately 30 g meat strips were sampled from the breast muscle parallel to the fibre direction then weighed to get the wet sample using a digital scale sensitive to 0.01 g. The samples were suspended inside a plastic container and sealed under atmospheric pressure. The samples were then held at 2 ˚C for 72 hours after which they were removed from the container. The samples were blotted with paper towels to remove excess surface moisture and were then re-weighed. The drip loss was then calculated by subtracting the blotted sample weight from the initial sample weight. The drip loss was expressed as a percentage of the initial sample weight (Honikel and Hamm, 1994).

 

Cooking loss measurement

Raw meat cubes were cut from the breast muscle, weighed in natura, then placed in a plastic bag and cooked in a water bath at 75 ˚C for 45 minutes (Rizz et al. 2007). The samples were then cooled in running water for 15 min, dried with soft tissue and weighed (Sanka and Mbaga, 2014). Cooking loss was calculated as percentage loss of weight during cooking relative to the weight of raw muscle (Petracci and Baéza, 2009) according to the following formula:

Cooking loss (%)= ((weight before cooking–weight after cooking)/(weight before cooking)) × 100

 

Tenderness

Breast muscles were wrapped in aluminum foil and baked to reach an internal temperature of 85 ˚C, which was maintained for 30 minutes. Warner Bratzler shear device mounted on an Universal Instron apparatus (cross head speed=200 mm/minute, one shear in the centre of each core). Smaller samples were then cut parallel to the muscle fibres with the aid of a Meullenet - Owens Razor Shear Blade (A/MORS) with a diameter of 1.2 cm. The reported value represented the average peak force measurements of each sample in Newtons. The shear force was recorded using the Texture analyser (TA XT plus).

 

Cecal and ileum micro flora composition

Five birds per treatment at the age of 42 days were killed by severing the jugular vein. The abdominal cavity was opened, and the entire gastro intestinal tract was removed aseptically. All digesta contents of ileum, caecum and colon were collected immediately under aseptic conditions into sterile glass bags and put on ice before they were transported to the laboratory for enumeration of microbial populations. Ceacal and ileum digesta contents were emptied aseptically in a new sterile bag and were immediately diluted 10-fold (ie 10% wt/vol) with sterile ice-cold anoxic PBS (0.1 m; pH 7.0) and subsequently homogenized for 3 min in a stomacher (Bagmixer 100 Minimix, Interscience, Arpents, France). Each ceacal and ileum digesta homogenate was serially diluted from 10-1 to 10-7. Dilutions were subsequently plated on duplicate selective agar media for enumeration of target bacterial groups. In particular, E. Coli, Lactobacillus spp. and Bifidobacterium spp. were enumerated using VRB agar (MERCK, 1.01406), Rogosa agar (MERCK, 1.10660), and Beerens agar respectively according to Tuohy et al. (2002). Plates were incubated at 39 ˚C for 48 to 120 hours anaerobically (Beerens, Rogosa agars) or 24 to 48 hours anaerobically at 37 ˚C (VRB agar). The bacterial colonies were enumerated, and the average number of live bacteria was calculated based on the weight of original ileum and caecum contents. All quantitative data were converted into logarithmic colony forming units (cfu/g), Koc et al. (2010).

 

Statistical design and analysis

Data collected during the study were subjected to analysis of variance (ANOVA) for completely randomized design (CRD) as described by Steel and Torrie (1980)using general linear model Procedure of SAS (2010). The statistical model used to test the effects of treatment on growth traits, meat quality traits, carcass characteristics, serum biochemical profile, and gut micro-flora was:

Yij= µ + Ai + Eij

Where:

Yij: observed value of a dependent variable.

µ: overall mean.

Ai: effect of different levels of dietary Aspergillus xylanase enzyme.

Eij: residual error.

The differences between means were tested for significance at P < 0.05 using least significant difference (LSD) range test.

 

RESULTS AND DISCUSSION

Serum biochemical profile

Table 3 represents the serum biochemical traits of broiler chickens fed different inclusion levels of Aspergillus xylanase (ASXYL). The addition of ASXYL altered (P<0.05) the concentrations of serum glucose, urea and serum enzymes (aspartate aminotransferase (AST) and alanine aminotransferase (ALT) while total protein, cholesterol and alkaline phosphatase (ALP) did not differ (P>0.05). Urea, AST and ALT values decreased (P<0.05) as the levels of ASXYL enzyme increased. Again, glucose level increased (P<0.05) with increased inclusion levels of ASXY enzyme.

 

Gut micro-flora composition

The population of ileum and caecum microbes of broilers fed maize-soybean meal diet with different inclusion levels ofASXYL is presented in Table 4.ASXYL supplementation had no influence (P>0.05) on the population of Bifidobacteria and Escherichia coli in both ileum and caecum of broilers at 42 days of age. However, supplementing ASXYL enzyme increased (P<0.05) the counts of Lactobacillus in the ileum and caecum of broiler birds compared to their counterparts that received XYL0 (control diet) that do not contain Aspergillus xylanase.

 

Growth performance

Different phases in growth performance of broiler birds fed maize-soybean meal based diets supplemented with ASXYL are presented in Table 5. All the growth traits considered in this study were affected (P<0.05) by the inclusion of dietaryASXYL except for body weight gain (BWG) during finisher phase. Birds fed ASXYL20 consumed less feed at starter phase, finisher phase and overall feeding trial when compared with birds fed other levels of ASXYL. Average daily feed intake of birds fed ASXYL20 was lower but similar to birds fed ASXYL25 when compared with other treatments used in the study. Daily weight gain and BWG were highest (P<0.05) for birds fed ASXYL20 during the starter phase and overall phase (entire feeding period) but similar to birds fed ASXYL25. Birds fed ASXYL20 recorded an improved FCR during the starter and the overall feeding period compared with other treatments used in the study.

 

Carcass characteristics of broiler chickens

The meat yield traits of broiler birds fed maize-soybean meal diet with different supplemental levels ofASXYL enzyme is presented in Table 6. The weights of carcass, neck, wing, drumstick, thigh, breast and head were affected (P<0.05) by the addition of ASXYL while shank and vertebrate weights did not differ (P>0.05). Birds fed ASXYL20 had the highest (P<0.05) values for thigh, breast, wing as well as carcass yields compared with birds fed other treatments used in the present study. Neck weight was highest (P<0.05) for ASXYL20 fed birds, though statistically similar to birds fed ASXYL0 and ASXYL15. Drumstick recorded an improved (P<0.05) value for birds fed ASXYL20, though similar to their counterparts that received dietary ASXYL10, ASXYL15 and ASXYL25.

 

Table 3 The effect of dietary Aspergillus xylanase supplementation on serum biochemical (SB) metabolites of broiler birds

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

AST: aspartate aminotransferase; ALT: alanine aminotransferase and ALP: alkaline phosphatase.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 4The effect of dietary Aspergillus xylanase supplementation on the counts of bacteria in the ileum and caecum (log10 cfu/g) of broilers fed corn-soybean meal diets

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

AST: aspartate aminotransferase; ALT: alanine aminotransferase and ALP: alkaline phosphatase.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 5 The effect of dietary xylanase supplementation on feed intake (FI), weight gain (WG) and feed conversion ratio (FCR) of broiler birds

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Again, head weight was lowest (P<0.05) for birds fed ASXYL0 and ASXYL25 while those that received ASXYL20 recorded the highest head weight value, though statistically similar to their counterparts that received ASXYL10 and ASXYL15. Generally, birds fed ASXYL20 were observed to have an improved (P<0.05) retail cut yields of neck, wing, drumstick, thigh, breast, head as well as carcass when compared with birds fed other ASXYL levels.

 

Meat quality attributes

The meat quality traits of broiler chickens fed different in clusion levels ofASXY are presented in Table 7. Different levels of ASXYL supplementation to maize-soybean meal diet had an influence (P<0.05) on cooking loss, shear force and drip loss while colour, water holding capacity, pHo, pHu and temperature were not affected (P>0.05). Cooking loss value of chicken breast meat was significantly (P<0.05) higher for birds fed ASXYL0, though similar to their counterparts that received ASXYL10. The cooking loss of chicken breast meat recorded the lowest (P<0.05) value for birds fed higher levels of the tested enzyme (ASXYL15, ASXYL20 and ASXYL25). Shear force values of the breast meat increased (P<0.05) as the levels of the tested enzyme increased. The lowest (P<0.05) shear force value was recorded for control birds (ASXYL0) while ASXYL15, ASXYL20 and ASXYL25 fed birds had the highest (P<0.05) shear force values, though similar to birds fed ASXYL10. The addition of ASXYL enzyme influenced (P<0.05) the reduction of drip loss value, whereas the breast meat of birds fed ASXYL0 and ASXYL10 had the highest (P<0.05) values of drip loss, though statistically similar to their counterparts that received ASXYL15. The lowest (P<0.05) drip loss value was recorded for birds fed ASXYL20 and AS XYL25.

 

Serum biochemical profile

Blood biochemical parameters can reflect the physiological state of the body. In the present study, we observed that broilers had a higher concentration of blood glucose with increased supplemental levels ofASXYL. Shouqing et al. (2015) opined that lactose can be degraded into glucose and galactose by lactase which is mainly synthesized and secreted by the small intestine epithelia cells of piglets. In mammals, glucose is not the only host energy sources, but it promotes the development of brain and neuron, again, excess of the glucose is deposited in the tissue (flesh), hence, developing the retail cut yields (Bano, 2013). The result of the present study did not conform to those of Balamurgan and Chandrasekaran (2010), who found no effect of supplemental multi-enzyme (xylanase and protease) in blood glucose of broiler birds. Chauhan et al. (2002) found that no effect was detected on blood glucose when they fed dietary phytase enzyme to broilers. Blood glucose level did not change in the findings of Mithu et al. (2017) who supplemented xylanase enzyme to high fibre diet (wheat). Exogenous enzymes used in the diets of broiler birds are very beneficial in improving nutrient digestibility by supplying external enzymes that the birds cannot produce in sufficient quantity by itself, and even though the birds can produce enough quantity of an enzyme by itself, exogenous enzyme may reduce the requirement for the endogenous enzyme, thus making more nutrients and energy available for growth of the birds (Olukosi et al. 2007; Oyeagu et al. 2019a), hence, this may be the reason for increased blood glucose concentration recorded in the present study at 42 days of age. As the inclusion levels of ASXYL enzyme increased, the concentration of urea decreased. The lowest urea value was recorded for birds fed ASXYL20and ASXYL25. Although, urea concentration may not have much value or importance in detecting renal diseases in many avian species (including broilers), but it could be used as a sensitive indicator for dehydration (Lumeij, 1998). However, high urea plasma levels has been linked to conditions such as dehydration and ureteral obstructions that caused low urine flow (Oloruntola et al. 2016). In the present study, birds fed ASXYL0 and ASXYL10 recorded more concentration of blood urea compared with birds fed other levels of ASXYL enzyme. A decreased urea value was reported to be associated with reduced urea synthesis (build-up), reduced mal-absorption, reduced protein malnutrition and reduced advanced liver disease (Peter and Susan, 1991; Oloruntola et al. 2016). The result of the present study is in line with the report of Obikaonu et al. (2012) who observed a decrease in urea level in broiler birds when the dietary inclusion of neem leaf meal increased from 0% to 10%. The progressive reduction in the concentration of ALT and AST of broiler birds with increased supplemental levels of ASXYL enzyme indicates a safety inclusion of the tested enzyme in broiler ration at the rates used in this study. Measurement of ALT and AST activities are indicative of liver damage in broiler chicks and is therefore a valuable tool for determination of a safe inclusion rate for feed additives, since diets may influence serum enzymes (Mohamed et al. 2014). Some authors reported that the higher serum AST and ALT concentrations indicate the release of aminotransferase from cytoplasm to blood stream probably due to damage liver or different other tissues (Ahmad et al. 2013; Abida et al. 2017). The reduction of the serum enzymes (ALT and AST) with the inclusion of ASXYL highlights the possibility of reducing cases of myocardial problem in broilers as a result of ASXYL enzyme inclusion in the diets. Oloruntola et al. (2016) also share the same view. Aspartate aminotransferase and alanine aminotransferase are enzymes found in cardiac muscle, skeletal muscle, kidney, brain, liver and red blood cell, also they are regarded as a biochemical markers for the diagnosis of acute myocardial infection (Gaze, 2007).

 

Table 6 The effect of dietary Aspergillus xylanase supplementation on carcass characteristics of broiler birds at 42 day of age

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 7 The effect of dietary Aspergillus xylanase supplementation on meat quality attributes of broiler birds at 42 day of age

 

ASXYL0: basal diet (without Aspergillus xylanase (XYL)); ASXYL10: basal diet + 1 g XYL/kg feed; ASXYL15: basal diet + 1.5 g XYL/kg feed; ASXYL20: basal diet + 2 g XYL/kg feed and ASXYL25: basal diet + 2.5 g XYL/kg feed.

 The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

The myocardial degradation has been identified as a major cause of sudden death and serious losses in broiler industry (Kwada et al. 1994; Oloruntola et al. 2016) which in most cases is the resultant effect of burden imposed on heart due to procedures adopted for growing chickens to produce a fast body weight gain of about 2.5 kg to 3 kg in 42 days (Oloruntola et al. 2016).

 

Gut micro-flora composition

The gut micro-flora has an important role in animal health and production. They positively influence the hosts’ gastro-intestinal development, digestion, metabolism, pathogen exclusion, immune stimulation and vitamin synthesis (Torok et al. 2008; Albazaz and Buyukunal Bal, 2014). In poultry, the micro-organism that colonizes the gastro-intestinal tract during the early post-hatch period forms a synergistic relationship with their host. According to Munyaka et al. (2016), the gut micro-biota plays an important role in host nutrition and health by promoting digestion and absorption of nutrients, preventing pathogen’s colonization, and shaping and maintaining normal mucosal immunity. Bacterial richness and evenness are the major parameters for defining microbial structure and diversity and it is generally expected that dietary manipulations would influence the intestinal microbial structure and diversity (Kiarie et al. (2014). Masey et al. (2014) had earlier reported that feed enzymes can influence microbial populations due to the changes imposed on the lumen contents.In the present study, the supplementation of ASXYL enzyme increased the total number of Lactobacillus spp. in both ileum and caecum. Lactobacilli have a number of biochemical properties, including, the production of antibacterial compounds (Stern et al. 2006) and bile salt hydrolase compounds (Knarreborg et al. 2002), in addition to their known probiotic roles that help to maintain gut integrity.On the other hand, even though the E. coli counts was not influenced by inclusion levels of ASXYL enzyme, there was a progressive drop/decrease in the numerical values reported for E. coli as the inclusion levels of ASXYL increased. The result of the present study is in agreement with the findings of Nian et al. (2011), who revealed that inclusion of xylanase enzyme in wheat-based diet reduced the number of Coliform and Salmonella and increased the number of Lactobacillus in the ileum. Some studies have shown that xylanase addition could inhibit the proliferation of potentially pathogenic microbial populations in the intestine and improve animal health (Nian et al. 2011; Sugiharto, 2016). It is possible that, enzyme supplementation resulted in multiplication of beneficial bacteria in the present study. In this case, exogenous enzyme acts not only by lowering intestinal viscosity, but also leads to development of more competitive bacterial communities with higher intra-bacterial competition and thus additionally limits bacterial interference with nutrient absorption, and may have the effect of reducing potentially pathogenic population (Jozefiak et al. 2010). Dietary factors can also influence microbial populations in birds. Some authors (Rodriguez et al. 2012) observed higher number of Escherichia coli and Lactobacilli in the digesta of broilers fed wheat and barley, relative to those fed corn diets. Changes in the intestinal micro-biota of chickens can alter the mucosal structure and thereby influence nutrient absorptive capacity (Munyaka et al. 2016).According to Bedford (2000a), xylanase enzymes can change the microbial populations indirectly within the gastrointestinal tract through their utilization of the substrates that bacteria use as a carbon source. The composition of diet affects the gastro-intestinal micro-flora in broilers. The presence of viscous polysaccharides has been shown to increase the intestinal microbial activities associated with poor broiler performance (Hubner et al. 2002). Exogenous enzyme supplementation can significantly influence microbial populations in the intestine. NSP – degrading enzymes such as xylanase are hypothesized to work in two steps which are described as an ileal phase and ceacal phase (Bedford, 2000b). During the ileal phase, enzymes remove fermentable substrates. During the ceacal phase, degradation products of sugars such as xylose and xylo-oligomers are fermented by ceacal bacteria, thus stimulating the production of volatile fatty acid and the growth of specific beneficial bacteria (Bedford, 2000b). The study of Engberg et al. (2004) revealed that xylanase supplementation to wheat-based diets stimulated growth of lactic acid bacteria in the ileum which was confirmed by high lactic acid concentrations. However, there was a significant difference in Lactobacillus counts in both ileum and caecum while the population of E. coli and Bifidobacteria were not influenced. The results of the present study is slightly contrary with those of Gao et al. (2008) who reported that xylanase addition in wheat-based diet had no significant change on the Lactobacillus, Bifidobacteria and E. coli counts in the caecum and ileum of broilers. Our result also did not agree with those of Dingyuan et al. (2009) who found no influence of xylanase addition to wheat-based diets on the counts of Lactobacillus, E. coli and total aerobes in ileum and caecum. However, it should be noted from the present study that the increased supplemental levels of ASXYL allowed the proliferation of Lactobacillus in both caecum and ileum.

 

Growth performance

The dietary ASXYL20 (2 g/kg feed) improved weight gain, FCR and reduced FI throughout the feeding trial (Table 5). This May be the optimum/maximum bio-activity of the tested enzyme was achieved at ASXYL20 level of inclusion compared with other treatments which may have affected the variations in feeding and body weight pattern of the birds. This is in agreement with other researchers who found that the increase in BWG may be due to the use of carbohydrases (Mathlouthi et al. 2002; Francesch and Geraert, 2009; Hajati, 2010). Previous reports (Gao et al. 2008; Esmaeilipour et al. 2011; Zhang et al. 2014) showed that the supplementation of Bacillus xylanase in wheat-based diets significantly increased BWG and improved FCR of broilers. Feed conversion ratio is a good indicator of how efficient a feed or a feeding strategy can be. The improved growth performance recorded for birds fed ASXYL20 may be that the peak bio-activities of the ASXYL on the substrate are at 2 g Aspergillus xylanase per kg diet (ASXYL20). It was reported that the dietary fiber is responsible for accelerating digesta passage rate (Wilfarta et al. 2007; Al-Harthi, 2014; Sayehban et al. 2015). However, due to the low fiber content of the dietary ASXYL20 as a result of the maximum enzyme activities, broilers that received ASXYL20 may have experienced lower digesta passage rate than those fed other dietary treatments. Previous studies (Sayehban et al. 2016) reported that the longer the low fibre digesta in the intestinal tract, the greater the chances for better feed digestion which improves growth performance. According to Engberg et al. (2004) and Selle et al. (2010), whole wheat (high fibre) feeding reduces the activities of amylase in the pancreatic tissue while the addition of xylanase increased chymotrypsin and lipase activities. Meanwhile, the data in the present study were not in agreement with those of Wang et al. (2005), Gao et al. (2008) and Dingyuan et al. (2009) who found no difference in FCR of broilers supplemented with dietary xylanase. Some studies have shown that addition of xylanase resulted in improved broiler performance (Gracia et al. 2003; Jiang et al. 2008; Williams et al. 2014). However, other authors found no effects on growth performance after supplementing exogenous xylanase enzyme (Kocher et al. 2003; Singh et al. 2012). However, the improved performance of birds fed ASXYL20 recorded in the present study may be due to the reduction of anti-nutritive effects of NSPs and the release of some nutrients. Some authors share the same opinion (Giraldo et al. 2008; Oyeagu et al. 2016). Xylanase may increase access to entrapped nutrient components by digesting some fractions of the plant cell walls of grains, allowing α-amylase access to starch fractions and prevent “resistant starch” (Leslie et al. 2007; Stefanello et al. 2015). The reduction in the viscosity of the digestion is the primary effect of xylanase with the release of sugars being a secondary effect (Barekatain et al. 2013). Cereals contain up to 15% NSPs in their cell walls, which is made up of soluble and insoluble NSP (Diebold et al. 2004). The insoluble component of NSPs comprises the bulk of the total fibre in the diets and they act to become a wall or cage that keep nutrients trapped inside and it has an adverse effect on nutrient digestion and utilization in non-ruminant animals (Hetland et al. 2004). On the other hand, the soluble component of NSPs, mainly arabinoxylans, can only act as physical obstacles to nutrient digestion and absorption by increasing gut viscosity. These components also alter the digestive tract functions by modifying secretion of endogenous digestive enzymes, water and electrolytes as well as elevating fermentation in small intestine (Zhang et al. 2014). A study by Barekatain et al. (2013) showed that diets containing sorghum brewer’s dried grains with xylanase supplementation significantly reduced the concentration of insoluble NSPs and increased the concentration of free sugars (arabinose and xylose). They reported that the availability of these free sugars may have provided nutrients to birds which resulted in improved FCR and BWG (Oyeagu et al. 2019a).

 

Carcass characteristics of broiler chickens

Carcass and cut yield traits determine the purchase decision of chicken meat consumers. The carcass yield is closely linked to adequate food and nutrition of broilers (Cardoso et al. 2011). After all, animals with adequate supply of nutrients will deposit effectively muscle. The increased proportional weight of retail cut yields of broiler chickens (including the breast meat) fed 2 g Aspergillus xylanase per kg diet (ASXYL20) may have occurred due to the fact that this level provides a better digestibility of the ingredients and, therefore, increases the amount of nutrients available for improved muscle (tissue) development and breast growth, since the breast cutting represents about 40% of the total carcass yield (Dalólio et al. 2015). It was noticed from the results of the present study that every other treatment enzyme level over or lower than ASXYL20 did not have the same response as ASXYL20 which may be due to lack of substrate available after the addition of an amount of enzymes greater than or lower than ASXYL20 without considering the nutritional energy matrix and,or, the diet proteins.The result of this study supports earlier report of Ogunsipe et al. (2014) who revealed that rich fibre diets reduces slaughter yield in rabbits and broilers. Again, Moharrery and Mohammad (2005) reported that higher fibre diets disrupts intestinal micro-villus and depresses nutrient absorption which may hinder muscle (tissue) development. The dietary supplementation of exogenous enzymes enhanced diet digestibility possibly because it promoted an increase in the activities of the digestive enzymes by increasing the availability of substrates which will increase nutrient absorption for enhanced muscle (tissue) development (Oyeagu et al. 2019b). When adding exogenous amylase, xylanase or protease to broiler diets, researchers reported higher activities of pancreatic and intestinal enzymes measured in 14- and 42-d-old broilers (Horvatovic et al. 2015). Diets based on barley, maize, sorghum, and wheat without enzymes negatively affected the activity of the intestinal enzymes, while enzyme supplementation did not (Shakouri et al. 2008). According to another study (Zdunczyk et al. 2009), maltase and sucrase activities was reduced when turkeys were fed diets with high fiber levels. It was reported that xylanase enzyme supplementation to fibrous diets improved the growth rate which increased the carcass and retail cut yields (Wang et al. 2005). The results of the present research are in line with the findings of Alam et al. (2003); Wang et al. (2005) and Hajati (2010). They reported an increased carcass yield by the addition of exogenous enzymes in wheat-based diets and they attributed it to higher tissue (muscle) development in carcass and also breast muscle. On the other hand, the reports of the present study contradicts the findings of Nadeem et al. (2005) and Pattaniak et al. (2011) who revealed that carcass yield and retail cuts had no significant difference among xylanase enzyme supplemental diets.

 

Meat quality attributes

According to Petracci and Baéza (2011), broiler meat has some important intrinsic attribute such as, appearance, texture, succulence and flavor. Again, the functionality of broiler meat is the coloring which is the most important factor that affects the choice of consumers (Werner et al. 2009). The results of the present study showed that there was no change or distortion in the chicken meat colour. It was reported that meat quality parameters depends directly from the management conditions, especially the type of feed provided to the birds (Neves et al. 2014). The addition of exogenous enzymes in the feed of broilers, probably, improves nutrient digestibility efficiency and promotes greater carcass yield and meat deposition (Allouche et al. 2015). The increase in protein deposition may change the type and shape of fiber to be deposited on the substrate, and may therefore change the meat quality parameters (Fatufe et al. 2004; Dalólio et al. 2016). The results of the present study did not agree with the studies of Tabook et al. (2006), Werner et al. (2009), Hana et al. (2010) and Dalolio et al. (2016), who found no effect on meat quality traits such as, pH, water holding capacity, shear force among others when they expose chickens to different dietary multi-enzyme (xylanase and protease) supplementation. The results of the present study showed that, there was a progressive increase in the numerical values of water holding capacity as the inclusion levels of the tested enzyme increased. The higher values of cooking loss and drip loss of chicken breast meat recorded for birds fed ASXYL0 showed that reasonable amount of nutrient in the meat are lost due to increased water loss (exudate). Omojola et al. (2014) also shared the same view, they opined that meat with higher nutrient loss as a result of increased exudate (water loss) should be referred to as poor quality meat. This suggests that higher levels of ASXYL enzyme supplementation in the present study reduced nutrient loss as well as increased the cooking yield. This may be due to the specific hydrolyzing activity of ASXYL (from Aspergillus strain E A.1) used in this study because microbial xylanase (wild or modified) from nonpathogenic and safe microorganisms improves meat quality (Alaa et al. 2014), unlike the traditional plant xylanases that tend to have broad specificities and indiscriminately break down connective tissue and myofibrillar proteins (Foegeding and Larick, 1986; Miller et al. 1989; Ashie et al. 2002). Meanwhile, Gorsuch and Alvarado (2010) stated that cooking loss and / or drip loss is a measurement of water holding capacity of the muscle. They recorded a shear force of 1.7 to 3.7 in their study and they considered the meat as a tender meat. However, shear force is an indication of the degree of toughness or tenderness. In the present study, the shear force values ranges between 5.39 and 7.98 and the highest value was recorded when the supplemental levels of ASXYL increased. This is an indication that increased force was required to shear through the breast meat of birds fed higher supplemental levels of ASXYL. Gajana et al. (2013) recorded a similar result and they attributed it to the pre-slaughter glycogen depletion which may have resulted in meat with a higher ultimate pH (pHu) (Kannan et al. 2002) and therefore tougher meat that will require more force to shear it. Meat tenderness is related to ultimate pH (pHu) value and meat colour (Byrne et al. 2000; Strydom et al. 2000; Vestergaard et al. 2000), although there are some cases where such relationship may not be significant (Muchenje et al. 2008). In the present study, the ultimate pH (pHu) did not differ even when there was a change in the shear force. According to Muchenje et al. (2008), an increase in pHu does not necessarily result in tougher meat as other parameters with regards to meat tenderness. Meat tenderness varies, mainly as a result of changes to the myofibrillar protein structure of muscle in the period between animal slaughter and meat consumption (Muir et al. 2000; Muchenje et al. 2009). Gajana et al. (2013) revealed that the positive correlation between cooking loss or evaporation loss and shear force values may imply that increased evaporation loss or cooking loss has the influence of decreasing tenderness and juiciness in meat as it results in tough muscles (tissue) that may be difficult to chew. Some researchers found that increase in cooking loss may lead to more nutrient loss (Omojola et al. 2014) from the meat and more protein toughening due to denaturation (Barbut et al. 2005).

 

CONCLUSION

From the results of the present study, it can be concluded that dietary ASXYL20 improved the growth traits, retail-cut yields as well as carcass yield of broiler chickens compared with the control (ASXYL0) group. Moreover,ASXYL supplementation in maize-soybean meal diet did not show any detrimental effect on gut health and liver or kidney functions, while it also showed some positive potential on meat quality traits of chicken meat.

 

ACKNOWLEDGEMENT

Many thanks to the Department of Animal Science, North-West University, Mafikeng Campus, South Africa for providing facilities for this research trial. Again, the authors wish to acknowledge Govan Mbeki Research and Development Centre (GMRDC) University of Fort Hare, Alice, South Africa for their financial support.

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