The restriction on use of antibiotic as growth promoter in commercial poultry farming has instigated search for newer and feasible alternatives for maintaining poultry health1. Probiotics are one such alternative and can be defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”2.
Many probiotic products are available for commercial use in poultry where use of Bacillus subtilis spores have been predominant due to their capacity to resist harsh environmental conditions, thriving pelletizing process, tolerance to wide pH variation, dehydration, bile salts and long shelf life3-5. Their use primarily targets displacement of enteric pathogen like Clostridium perfringens, the causative organism of necrotic enteritis in chicken6.
Earlier reports of probiotic application in poultry has shown that multi-strain probiotics enhanced performance more than the single strain probiotic7-9. In search of other potential alternative strains for probiotic application in poultry, it has shown that spore forming obligatory anaerobe like C. butyricum promotes growth performance, immune function and benefits the balance of the intestinal microflora in broiler chickens10.
With this background, the present study was conducted to compare the effects of dietary supplementation of a single and a multi-strain probiotic, the latter at two different levels of inclusion, on intestinal histomorphology and growth performance of broiler chickens.
MATERIALS AND METHODS
Ethical approval: Experiments were carried out in accordance with the guidelines laid down by the Institute of Animal Ethics Committee for the use of poultry birds.
Probiotic: The multi-strain probiotic (MSP) (ImprovalTM BFS) used in the present study, is a proprietary preparation manufactured by Zydus Animal Health (A Div. of Cadila Healthcare Ltd., Ahmedabad, India). The preparation is composed of spore-forming bacteria, containing 0.5×109 viable spores g1 of B. subtilis, B. coagulans, B. licheniformis and Clostridium butyricum HJCB998. The single strain probiotic, a commercially available preparation (the name of which is kept masked to avoid commercial complications) contains 2×109 viable spores g1 of Bacillus subtilis PB6 (BSPB6).
General bird husbandry and treatments: A total of 400 one-day-old Vencobb broiler chicks (initial mean body weight 42.6±0.5 g) of mixed sex were distributed into 4 treatment groups according to the experimental design described in Table 1. Distribution of the chicks between the groups and within the groups between the pens was done following a completely randomized block design. Each treatment group consisted of 10 replicate pens (each pen measured 1.2 m×1.2 m) and there were 10 chicks in a single pen (n = 100 in a group). The chicks were raised on litter composed of saw dust and paddy straw. The birds were vaccinated at 5 and 20 day against Newcastle disease (Nobilis® ND Clone 30, MSD Animal Health, Kenilworth, NJ, USA) and infectious bursal disease (Nobilis® Gumboro 228E, MSD Animal Health, Kenilworth, NJ, USA) at 12 day of age. The lighting schedule involved 24 h light during the first week and 20 h of light up to 5th week and 24 h light for 6th week. The test facility, pens and birds were observed twice daily for general flock condition, lighting, water, feed, ventilation and unanticipated events and records were maintained from the beginning whenever any bird was found dead, culled or sacrificed due to any reason. All the mortalities were subjected to necropsy to determine the probable cause of death.
Diets and chemical composition: Diet and drinking water were offered ad libitum. The birds were fed with a pre-starter (1-14 day), starter (15-28 day) and a finisher diet prepared fresh at the beginning of each feeding period with raw materials of same lot. All diets were formulated following the ideal protein ratio using standardized ileal digestible (SID) amino acid requirement for broiler chickens11.
Accordingly, the digestible lysine content was maintained at 1.22, 1.10 and 1.00% and the rest of the amino acids were fixed using the SID ratio in the starter, grower and finisher diets respectively. The metabolizable energy values were maintained at 2900, 3000 and 3150 kcal kg1 respectively in the starter, grower and finisher diets. The ingredients and calculated chemical composition of the experimental diets were as per the breed standard and are presented in Table 2 and 3 respectively.
Growth performance parameters: The body weights (BW) of individual bird were recorded at weekly interval and average daily body weight gain (ADG) was calculated during 1-14, 15-28, 29-42 and 1-42 day. Feed consumption of birds of each replicate was recorded at weekly intervals and feed consumption per bird per week was calculated which were used to derive data on average daily feed intake (ADFI) during 1-14, 15-28, 29-42 and 1-42 day. Feed conversion ratio (FCR) was calculated as a ratio between feed intake over body weight during corresponding growth periods as detailed above. Mortality, if any, was recorded as it occurred and the data was used to adjust subsequent measurements. Finally, European performance efficiency factor (EPEF), as suggested by Huff et al.13, was calculated using following formula:
EPEF = BW (kg) ×% liveability×100/FCR×trial duration (day)
Histology of the small intestine: The histological study of the small intestine (SI) was performed to evaluate the effects of the trial diets on the histomorphology and integrity of gut. At 42 day of age one male bird was taken from each pen at random (10 birds from each dietary group, 40 birds in total) and they were slaughtered by exsanguinations. The SI was removed and washed with sterile phosphate buffered saline (PBS) and the contents were removed. Segments measuring 2 cm in length from the mid-points of the jejunum were cut and fixed in 10% buffered formalin. The tissue samples were later embedded in paraffin and a 2 μm section of each sample was placed on a glass slide and stained with haematoxylin and eosin. Histological sections were examined with a phase contrast microscope coupled with an integrated digital imaging analysis system (Olympus Corporation, Tokyo, Japan). The variables measured were villus height, crypt depth and thickness of the lamina propria, tunica muscularis and tunica serosa. Villus height was measured from the tip of the villus to the top of the lamina propria and the crypt depth was measured from the base up to the region of transition between the crypt and villus. Ten measurements were taken per bird for each variable and the average of these values was used for statistical analysis14.
Statistical analysis: Data was analysed according to one-way analyses of variance using the diets as the grouping factor in a SPSS (version 17.0) processor. The pens were the experimental units for all the parameters except for histology of small intestine where it was the single observations which were used as experimental units. The results were expressed as means and pooled standard error of means. Probability values of p<0.05 were expressed as statistically significant and in case the means were found to be significantly different then the means were separated by Turkey’s B test.
Growth performance: Data on BW and ADG of the birds in different experimental groups are presented in Table 4. At 42 day, the birds fed with MSP 0.5 and MSP 1.0 diets had numerically greater (p = 0.230) BW and ADG as compared with the NC and the BSPB6 groups of birds. There were non-significant (p>0.05) variations between the dietary groups with regard to BW and ADG of the birds at different period of experiment. Numerical difference (p>0.05) in BW between groups were observed on 21, 28, 35 and 42 day where highest BW was recorded in BSPB6 group (909 g) on 21 day, MSP 0.5 group (1245.3 g) on 28 day, MSP 1.0 group (1687.9 g) on 35 day and MSP 0.5 group (2252.5 g) on 42 day. The NC group had numerically lesser BW (p>0.05) than either of the treatment groups (BSPB6, MSP 0.5 and MSP 1.0) during these periods.
Data on FCR and EPEF are presented in Table 5. Overall, there was no effect of dietary treatments on the FCR of the experimental birds (p>0.05). Feed conversion ratio (FCR) during 1-14 day of age was numerically better (p = 0.431) in broiler fed MSP 1.0 diet as compared with the rest of the dietary treatments. During 15-28 and 1-28 day period, FCR was numerically poorer (p = 0.501) in the NC group and was relatively better (p = 0.501) in the MSP 1.0 group. Similar trend in FCR was observed when data was pooled from 1-42 day of period where it was superior in MSP 1.0 group (1.655) and poorer in NC group (1.674). EPEF values (p = 0.340) were found numerically better in treatment groups as compared with the NC group where highest value was recorded in the MSP 1.0 group (316.01).
Differences with regard to feed intake were observed between the groups (p>0.05) only when measured at different periods of the experiment (data not shown). Mortality was negligible and was non-specific in nature (data not shown). Hence, liveability was not affected by dietary treatments.
Intestinal histomorphology: The data related to the height of the villus, depth of the crypts and villus height to crypt depth ratio (VH/CDare presented in Table 6. No significant differences (p = 0.773) were observed between the groups supplemented with different dietary treatments. Numerically, the villi were longer (p = 0.638) in birds fed BSPB6, MSP 0.5 and MSP 1.0 diets as compared with the NC group where it was superior in birds fed MSP 0.5 diet.