The use of antibiotic growth promoters (AGPs) in poultry feed has been a growing concern among the consumers due to the emergence of antibiotic-resistant strains of microorganisms1. The European Union has already restricted the use of antibiotic as growth promoters in poultry feed2. However, birds become more prone to get sick in the absence of in-feed antibiotic. Hence, alternatives to antibiotics are needed in the poultry industry to promote the performance of birds. In this quest, several alternatives, including probiotics, prebiotics and phytobiotic etc., have been suggested to be the potential alternative of AGPs in poultry diet3. Among the prebiotics, xylo-oligosaccharide (XOS) is one of the promising AGP replacers due to their beneficial effects on gut health maintenance4 and productive performance of broiler chicken5. XOS is a hydrolytic degradation product of arabinoxylans (plant or microorganism origin) that can be fermented by the gut microbiota. A previous study observed that XOS could promote the growth of beneficial bacteria, such as Bifidobacterium spp. and Lactobacillus spp. and subsequently improve the gut microbial ecology and intestinal health of poultry6 and pig7.
Supplementation of straw-derived XOS (5-20 g kg‾1) improved not only the serum triiodothyronine (T3), thyroxine (T4), insulin concentration but also the immune function and FCR of broilers8. Another study9 also reported that supplementation of XOS (2 g kg‾1) in the male broiler chicken diet increased the concentrations of acetate, propionate and the proportion of Lactobacillus spp. in the cecum. In contrast, several studies reported no effect of XOS on broiler performance10,11. Diet supplemented with 50 mg kg‾1 XOS showed no effect on broiler performance10.
Similarly, Suo et al.11 reported that there was no significant effect of XOS supplementation (50-100 mg kg‾1) on average daily gain (ADG), average daily feed intake (ADFI) on days 1-21, 22-42 and 1-42 and feed conversion rate on days 1-21.
Limited studies have been done so far on the beneficial effects of XOS on the overall gut microbiome and blood chemistry of broiler chickens. Besides, the effective dose of XOS in the broiler diet is yet to be explored. Therefore, the present study was carried out to investigate the effect of different levels of XOS on growth performance, blood biochemistry and intestinal bacterial count of broiler chickens.
MATERIALS AND METHODS
All experimental procedures were approved by the CVASU (Chattogram Veterinary and Animal Sciences University) Ethics Committee (EC) and the EC Approval NO is CVASU/Dir(R&E) EC/2019/94(6).
Dietary management: Four experimental diets were formulated with four levels; 0, 2.5, 5.0 and 7.5 g of XOS kg‾1 of diet, respectively. At first, a single batch of corn-soybean meal-based mashed diet (Table 1) was formulated according to the recommendation of the Cobb 500 Broiler performance and nutrition supplement guide to meet or exceed the nutrient requirements12. After that, the basal diet was divided into four aliquots according to the experimental diet arrangement. Each supplemental XOS level was mixed on top with each aliquot of the basal diet. The diets were made as mash and offered to the birds from day 13-26. The corn-derived XOS was purchased from Henan Heagreen Bio-technology Co., Ltd. (China) that contains <5% moisture, >95% XOS (as dry basis), <5% xylose, glucose and arabinose.
Birds’ managements and diets: A total of 96 Cobb 500 day-old broiler chicks (40±0.10 g) were purchased from a local hatchery and randomly distributed into four groups with four replicates per dietary group (6 birds/replicates). From day 1-12, the birds received a basal (control) diet (Table 1). The experimental diets were offered to the birds during 13-26 days of age. The birds were reared in cages with well-equipped feeders and drinkers. Birds had free access to feed and water. Standard vaccination schedules and management procedures were maintained throughout the trial period. Feed intake (FI) and live weight were recorded weekly. Mortality was recorded as it happened. Feed conversion ratio (FCR; feed intake/body weight gain) was corrected for mortality.
Sample collection and processing: On day 26, three birds from each replicate were sacrificed by cutting the jugular vein after 12 h of fasting. The blood sample was collected in a falcon tube for separation of serum by centrifugation at 5000 revolutions per minute. Harvested serum samples were taken into the 2 mL eppendorf tubes and stored at -20̊C in the laboratory for further analysis. The weight of the visceral organs (liver, spleen, bursa, breast, gizzard, intestine and abdominal fat content) was recorded after opening the abdominal cavity of the same birds. The ileal (from the duodenum to Meckel's diverticulum) and cecal content were collected by gently pressing and stored in a separate labeled container at -20̊C for further analysis. The weight of the different body parts of the dressed birds was recorded accordingly.
Culture and total viable count: The collected intestinal samples of three individual birds/replicates were mixed and pooled. Around 1 g of ileal and caecal content was taken into two separate labeled sterile test tubes containing 2 mL of 0.9% saline solution with a stick. A 10-fold serial dilution was done for each pooled sample (0.1 mL) from 10-1 to 10-10. MacConkey agar, Violet red bile agar and KF streptococcus agar were used to enumerate the Enterobacteriaceae, Streptococci and Enterococci, respectively. Baird Parker agar and Mannitol salt agar were used for the enumeration of Staphylococci. All the plates were incubated at 37̊C aerobically for 24-48 h and the number of colonies was counted accordingly13.
Chemical analysis: The protein, CF, ash and moisture percentage of the diet were analyzed by using the Association of Official Analytical Chemists method14. The nitrogen content of the samples was determined by the Kjeldahl method. The obtained nitrogen value was multiplied by 6.25 to convert it to crude protein. The weight difference methods were used to determine moisture and ash content levels. The crude fat of the diet was determined using the AOAC procedure with petroleum ether as a solvent. The serum glucose, triglyceride, total protein (TP), GPT (glutamic pyruvic transaminase), GOT (glutamic oxaloacetic transaminase), cholesterol, creatinine, T3 (triiodothyronine) and T4 (thyroxine) level was analyzed by using their respective standard assay kit (Randox Laboratories Ltd, UK) and semi-automated Humalyzer (Humalyzer 4000 Merck®, Germany).
Statistical analysis: Data was analyzed using one-way ANOVA. Differences between means were tested by the least significant difference (LSD) using SPSS v.16 statistical software (SPSS, Chicago, Illinois, USA) for windows. The linear and quadratic responses of dependent variables to dietary supplemental XOS levels were assessed by using orthogonal polynomials. The difference between means was considered significant at p≤0.05. RESULTS
Growth performance: The effects of different levels of XOS on the growth performance of broiler chickens are presented in Table 2. Supplementation of XOS had no (p>0.05) effect on BWG and FI of broiler chickens during 13-26 days of age. However, the FCR was significantly different among the treatment groups. Chicken received a diet with 2.5 g XOS kg‾1 showed better FCR than those of the birds fed diet with 7.5 g XOS kg‾1. A significant linear response was observed between supplementation of XOS and FCR. With the increased level of XOS supplementation, the FCR also linearly increased (p<0.021).
Serum biochemical profile: Supplementation of XOS had no significant effect on blood parameters except for blood glucose and T4 level of birds (Table 3). The serum glucose level of the birds fed diets containing 5 g XOS kg‾1 was higher than those of the birds fed diet with 0 or 2.5 g XOS kg‾1. Supplementation of 2.5g XOS kg‾1 of the diets increased (p<0.001) the serum T4 level compared to the other diet groups. Dietary XOS supplementation resulted in linear (p>0.37) and quadratic (p = 0.027) response on serum T4 concentration.
Total bacterial count in ileum and caecum: Table 4 shows the effect of different levels of XOS on the total bacterial count in the ileum and caecum. Birds that received diet containing 7.5 g XOS kg‾1 showed lower TVC count in both ileum (p<0.023) and caecum (p<0.012) than those of the birds fed on other diet groups.
Relative weight of the visceral organ: Table 5 shows the effect of different levels of XOS on visceral organ development in broiler chicken. Birds fed diet with 5.0g XOS kg‾1 showed greater (p<0.05) proventriculus than those of the birds fed diet containing 7.5g XOS kg‾1. The diet containing 7.5 g XOS kg‾1 reduced (p<0.05) the gizzard weight compared to diets without XOS. A linear response (p = 0.044) was observed between gizzard weight and XOS supplementation. The liver and pancreas weight was higher in the birds fed diets with 2.5 g XOS kg‾1 than those of the birds fed diet containing 5 or 7.5 g XOS kg‾1. Supplementation of XOS had no (p>0.05) effect on the relative weight of the small intestine, heart, spleen and bursa. Significant (p<0.05) quadratic response was observed between supplementation of XOS and development of visceral organs (proventriculus, liver, pancreas and heart).