CC BY
0JWPR
© 2019, Scienceline Publication
J. World's Poult. Res. 9(1): 24-31, March 25, 2019
Journal Of W°rld s Research Paper, PII: S2322455X1900004-9
Poultry Research License: CC BY 4.0
DOI: https://dx.doi.org/10.36380/jwpr.2019.3
The Effect of Dietary Inclusion of Probiotics on Growth and Intestinal
Morphology of Broiler Chickens
Murat Gulmez:*a, Nurhayat Gulmez2, Seyitali Bingol3, Turgay Deprem4and Serap Koral Tasci4
'Asilcag Trading Ltd. Nicossia, Cyprus.
2Near East University, Faculty of Veterinary Medicine, Department of Histology and Embriology, Nicossia, North Cyprus 3Kafkas University, Faculty of Medicine, Department of Histology and Embriology, Kars, Turkey 4Kafkas University, Faculty of Veterinary Medicine, Department of Histology and Embriology, Kars, Turkey a Research support provided in part by a grant from the Scientific and Technological Research Council of Turkey, Ankara, Turkey
Corresponding author's Email: mgulmez@hotmail.com; ORCID: 0000-0003-3888-6815
Received: 07 Feb. 2019 Accepted: 15 Mar. 2019
ABSTRACT
Probiotics are currently under investing the most valuable substances alternative to antibiotic growth promoters in poultry breeding practice. This research was performed to evaluate the effect of supplementing broiler drinking water with probiotics (Pediococcus acidilactici and Bacillus subtilis) at a concentration of >108 CFU/ml during 42 days of feeding period on growth performance and gut health. A total of 144 one-day-old Ross 308 broiler chicks (mixed gender) with an average initial BW of 42.3 g were used. The chicks were allotted to pens with 12 birds per pen and six replications per treatment with food and water provided ad libitum. Feed intake of Probiotic group was 4134 g, 338 g less than that of control group. Live weight of probiotic group was 2537 g and a 113 g more than that of control group. The feed conversion ratio of probiotic group was 1.61, 0.22 less than that of control group. The crypt depth of probiotic group (1110.46 ± 224.016 ^m) was statistically deeper than that of control group (949.39 ± 114.166 ^m) in ileum. Continuously use of probiotics in drinking water of commercial poultry flocks appears to be alternative to AGPs. The results of this study provide a greater understanding of the impact of long-life use of probiotics on broiler health and growth performances. Key words: Broiler, Gut morphology, Growth performances, Probiotics
INTRODUCTION
Globally, antibiotic resistance is a growing public health issue (Gaggia et al., 2011). This emerging issue will likely lead to even greater challenges that will requisite innovative solutions in order to provide even greater public health protection. The goal is to enhance the knowledge base of alternative antimicrobials currently under investigation and to identify additional materials for potential strategic use in food animal production, leading to better protection of human health (Hume, 2011).
Antibiotics as growth promoters in animal feeds have been used in the member states of the European Union during the last 50 yr. However, the EU banned antimicrobial growth promoters (AGPs) in Jan 2006. So, the poultry industry has aimed to develop new strategies in order to maintain animal health and performance on a commercial scale (Castanon, 2007; Vahdatpour and Babazadeh, 2016). Accordingly, scientific efforts have been focused on the development of new non-antibiotic health and growth promoters for use in poultry breeding
(Griggs and Jacob, 2005; Nikpiran et al., 2014). Such as from AGPs, it is expected from non-AGPs that they should promote digestive tract health to increase disease resistance in chickens (Istiqamah et al., 2013; Mahdavi et al., 2013; Afsharmanesh and Sadaghi,2014; Li et al., 2014 and Chughtai et al., 2015).
As a source of non-AGPs, extracts of some medicinal or aromatic plants, probiotics, prebiotics, Competitive Exclusion (CE) cultures, organic acids, specifically fermented feeds or their extracts has been used (Lee et al., 2001 and Prukner-Radovcic and Grozdonic, 2003). Except for these attempts, intensive efforts have been spent in developing dietary fibers, bacteriocins, strain specific phages and vaccines to combat many significant enteric diseases (Heres et al., 2003; Vandeplas et al., 2008 and Latha et al., 2016).
Probiotics are live microbial complements that provide beneficial effects on the host due to improvement in the intestine's microbial equilibrium (Fuller, 1989 and Giannenas et al., 2014). Sharma et al. (2012) demonstrated
hisQaperB Gulmez M, Gulmez N , Bingol S, Deprem T and Koral Tasci S. (2019). Effect of dietary inclusion of probiotics on growth and intestinal morphology of broiler chickens. J. World's Poult. Res., 9 (1): 24-31.
them as "the emissaries of health from microbial world". The efficacy of these products is often due to specific microbial ecological factors that alter the competitive pressures experienced by the microbial population of the gut (Callaway et al., 2008 and Lutful Kabir, 2009). Many researchers have suggested that probiotics and CE cultures can reduce colonization of pathogenic bacteria in chickens' intestines (Lee et al., 2001; Wolfenden et. al., 2007 and Schneitz et al. 2016).
Feed efficiency, feed conversion ratio, survival rate and weight gain rate are considered as the main parameters for evaluating effectiveness of non-AGPs in scientific studies, either via comparison with AGPs or using them alone (Mehr et al, 2014; Olnood et al., 2015 and Zhang et al., 2015). Gut histopathology, blood and digestive parameters are used as comparison parameters in such studies (Mehr et al, 2014; Agboola, 2015 and §tef et al., 2015). Varieties of non-AGPs recommended for use in drinking water or in combination with poultry feed (Swiatkievicz, 2014 and Abu Akkada et al., 2015). Many studies have revealed the effectiveness of non-AGPs as growth promoters, providing alternative to AGPs to improve chicken growth indices (Landy and Kavyani, 2013). From the literature, it appears to be difficult to come to a consensus as to the best method to probiotic application for broiler chickens on an industrial scale. So, a liquid probiotic was used (Smart ProLive, a commercial preparation) in non-chlorinated drinking water once a day. So, it is aimed to investigate the effects of on growth performance and some histological changes in large intestines of broiler chicks during a period of 42 d of breeding.
MATERIALS AND METHODS
A total of 144 one dayold Ross 308 broiler chicks (mixed sexes) with an average initial BW of 42.3 g were allotted to pens with 12 birds per 1 m2 cages in a 20 m2 environmentally controlled room (32 to 24 °C and 65% relative humidity) flock. Six control (control group) and six experiment (probiotic group) cages were placed on the ground as two parallel lines. Four kg of wood shavings was used as bedding material for each cage. The light regime was 23 h light and one h darkness. The temperature in the flock was 32°C at the beginning of the experiment and was gradually reduced to 21°C at 21 d. Broiler chickens were vaccinated with live attenuated vaccines against Newcastle Disease Virus (NDV) Avinew© VG/GA strain (Merial-Lyon-France) at day 7 and day 26. No antimicrobial agent was applied to the birds. Birds
were only subjected to 3 routine vaccination applications at different intervals and vaccines were applied to all the birds via drinking water.
The birds were provided with ad libitum feed and drinking water during the entire experimental period. All diets were taken from a commercial Ross 308 broiler chicken breeding farm. Chickens were feed with four structures of compound feed according to the recommendations in the growth boom for Ross 308 hybrid, namely: pre-starter, starter, grower and finisher in the following sub-periods: from hatching to 10 d, from 11 d to 24, from 24 to 35 d and from 36 to 42 d. The feed compositions are given in table 1. Control group was fed with basal diet and de-chlorinated drinking water. Probiotic group was fed with basal diet and de-chlorinated drinking water with 0.1 % Smart ProLive. Live weight and feed consumption ratio (FCR) were recorded weekly until 42 d (slaughter d).
Table 1. Composition of basal diets (g/kg) used for feeding of broiler chickens.
Composition of basal diets (g/kg)
Feeding periods (day)
Ingredients 0-10 11-24 25-35 36-42
Maize 440 392.22 361.55 357.42
Soybean Meal (46%) 210 84 21 0
Soybean (Full fat) 150 200 270 267
Wheat 85.6 197 247 267
Maize gluten 54.5 24 0 0
Limestone 16.6 9.4 8.6 8.4
Soybean oil 15 5 5 3
CaHPO4 12.3 5.1 3.6 3.7
L-Lysine HCl 4.1 3.89 2.58 2.76
DL-Methionine 2.94 2.67 2.35 2.18
NaHCO3 2.44 3.4 3.14 3.85
NaCl 2.16 1 1.2 0.62
L-Threonine 1.13 0.78 0.78 0.85
Meat-bone meal 0 60 60 70
Vegetable oil 0 5 10 10
Vitamin premix a 0.2 0.2 0.2 0.2
Mineral premix b 2 2 2 2
Choline chloride 1 1 1 1
Nutrient level
Metabolizable energy (MJ/kg) c 3037 3140 3225 3220
Crude protein (%) 23.56 20.9 19.2 18.9
Lysine (g/kg) 1.45 1.3 1.15 1.13
Methionine (g/kg) 0.7 0.61 0.53 0.5
Calcium (g/kg) 1.25 0.96 0.92 0.95
Available phosphorus (g/kg) 0.68 0.67 0.64 0.65
a Vitamin premix provided 1 kg of diet with: vitamin A, 10.800 IU; vitamin D3. 2160 IU; vitamin E, 15 IU; Vitamin K3, 1.0 mg; vitamin B1, 4 mg; riboflavin, 5 mg; pantothenic acid, 10 mg; niacin 25 mg; vitamin B6, 8 mg; folic acid, 0,4 mg; vitamin B12, 0.08 mg; biotin, 0,15 mg. b Mineral premix provided 1 kg of diet with: I, 0,35 mg; Se, 0,15 mg; Zn, 40 mg; Cu, 8 mg; Fe, 80 mg; Mn, 100 mg. cMetabolizable energy was obtained by calculation.
Enumeration of total aerobic bacteria in the probiotic source: Smart ProLive sold in the form of 5 l plastic container (as seen on the label, it contains >1*10n CFU/ml probiotics as Pediococcus acidilactici and Bacillus subtilis) was analyzed for its total viable bacterial count. A 10 ml sample was mixed with 90 ml sterile saline solution (0.9% NaCl) and the 10-fold increment serial dilution technique was conducted according to Maturin and Peele (2001). One milliliter of the homogenized suspension was then transferred into 9 mL of 0.9% saline solution (NaCl) and serially diluted from 10-1 to 10-8 by using the same saline solution tubes. From the last three diluted samples, 0.1 mL each was plated on the appropriate agar medium for enumeration of live bacterial population. After colony count, bacterial load was calculated as CFU/ml.
Histological measurements
At the 42th d of the trial period, all the birds were weighed individually and sent to a local commercial broiler slaughterhouse for the routine slaughter process. Electrically stunned birds were slaughtered. For histological examination, fragments from duodenum, ileum and ceca were taken from the individuals of six experimental variants in each of Probiotic Group and Control Group after the commercial slaughtering process. The fragments of the intestine were fixed in neutral formalin (10%), then dehydrated in increasing ethylic alcohol solutions (70°, 80o, 90o, 100o) and clarified in two baths of benzene and put in paraffin. The sectioning of the paraffin blocks was carried out using a manually rotary microtome (§tef et al., 2015). The slides were stained with Periodic Acid Schiff (PAS) and Hematoxylin Eosin (HE) then examined by light microscopy. Micrometer in microscope was used for histometric measurements (Luna, 1968).
Statistical analysis
Data were analyzed using SPSS v.16.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistically significant differences between group means were determined by independent sample t test. Mean values were considered significantly different at P<0.05. Data are expressed as mean values ± SD (standard deviation).
Ethical approval
Direct collection of tissues and organs from freshly slaughtered birds was carried out in strict accordance with the recommendations of Kafkas University, Kars, Turkey for the care and use of laboratory animals. Also, slaughtered chickens were humanly handled.
RESULTS
There was no mortality or physical injury during the trial period of 42 d. After a 42 d of breeding period, each bird in probiotic group consumed 4134±112 g feed and it was 338 g less than that of control group (4472±137 g). Live weight of each bird in the probiotic group was 2537±62 g and it was 113 g more than that of Control Group (2424±67 g at 42nd d). The FCR of probiotic group was 1.61±0.007 and it was 0.22 less than that of control group (1.83±0.012). As seen in the table 2, feed consumption, weight gain and FCR results of probiotic group were superior to that of control group during all the breeding period. After total mesophilic aerobic count of the probiotic source, it is confirmed that it contained at the concentration of 1 x 1011 CFU/ml (data has not been shown).
The figures 1 and 2 represent the guts histological structures. There was no difference between probiotic group and control group aspect of histological structure, which were lymph follicles, goblet cells, crypt, submucosa and mucosa, in all parts of small intestine. The histometric differences between the two groups are given in table 3. Although the crypt depth in duodenum and in ceca of probiotic group and control group were statistically similar to each other, there was a significant difference between the two groups in ileum. The crypt depth of probiotic group (1110.46 ± 224.016 ^m) was statistically deeper than that of control group (949.39 ± 114.166 ^m) in ileum. Mucosa thickness of probiotic group in ceca and ileum appeared to be thicker than those of control group in those parts of the intestine (Table 3). There was no statistically significant difference between the two groups in the thickness of the duodenum mucosa (Table 3).
Table 2. Effect of probiotic (Smart ProLive) on growth parameters during 42 days of rearing period of broiler chickens (mean±SE).
1 2 3 4 5 6
Tests
Feed intake; 89 407 930 1734 2270 4134
Probiotic ±6 ±14 ±22 ±40 ±75* ±112*
Feed intake; 89 396 954 1780 2979 4472
Control ±6 ±24 ±30 ±72 ±76 ±137
Bodyweight gain; 121 369 722 1235 1879 2537
Probiotic ±6 ±12 ±15 ±27* ±47* ±62*
Bodyweight gain; 112 338 689 1175 1796 2424
Control ±6 ±18 ±21 ±45 ±47 ±67
Feed conversion 0.39 0.99 1.23 1.37 1.45 1.61
rate; Probiotic ±0.03 ±0.08 ±0.06 ±0.04* ±0.04* ±0.07*
Feed conversion 0.42 1.05 1.32 1.48 1.64 1.83
rate; Control ±0.01 ±0.02 0.05 0.05 0.01 0.01
*P<0.05.
Figure 1. a) General view of the cecum in the experimental group of broiler chickens after 42 days of rearing period, 4x.Arrows: Lymph follicles, tm: tunica muscularis, s: submucosa, m: mucosa. Haematoxylin and eosin stain (H&E) Bar: 1000 ^m; b) Cecum of the experimental group, 10x. Arrows: Crypt, L: lymph follicles. H&E. Bar: 200 ^m.
Table 3. Effect of probiotic on crypt depth and mucosa thickness in gut segments of broiler chickens after 42 days of rearing
Tissue Groups Crypt depth (^m) ± SD F Mucosa thicknes (^m) ± SD F
Doudenum Probiotic 1622.5 ± 347.0 0.666 1965.1 ± 333.4 0.37
Control 1445.7 ± 318.7 1721.6 ± 326.3
Ileum Probiotic 1110.5 ± 224.0 9.305* 1325.2 ± 222.8
Control 949.39 ± 114.2 1144.9 ± 129.0
Ceca Probiotic 306.8 ± 65.1 0.275 402.8 ± 109.5 4 73*
Control 273.8 ± 59.3 353.1 ± 63.1
*P < 0,05.
z&mmn f
Figure 2. Duodenum of the experimental group of broiler chickens after 42 days of rearing period. Arrows: Goblet cells, 40x. Periodic Acid Schiff (PAS). Bar: 50 ^m.
DISCUSSION
Antibiotics used in the field of veterinary application have been found to be responsible for the global antibiotic resistance problem. Currently, there is an alarming situation regarding antimicrobial resistance (Tellez et al.,
2012). In 2013, the G8 summit was dedicated this subject alone and at the end of the summit the conclusion was that the situation had demonstrated as "an alarm state". The G8 ministers released a joint statement on June 14, identifying antimicrobial drug resistance as a "major health security challenge of the 21st century" (Davies,
2013).
Competition of good and bad flora demonstrates the health of the digestive system, and accordingly health of the total body (Ghadban, 2002). AGPs are used to suppress bad flora and allowing good flora to dominate in the intestines. Ban of antibiotic growth promoters in feeds from 2006 in the EU countries, many other countries have gradually adapted their regulations. After the ban, a rapid search has started to find new growth promoters to replace AGPs (Hume, 2011). In last 20 years, many studies have been conducted on new natural gut health promoters such as probiotics, prebiotics, CE cultures, direct feed microbials, fermented feeds, organic acids, essential oils. Probiotic are defined as a live microbial feed supplement that beneficially affects the host animal by improving its microbial intestinal balance (Fuller, 1989). Given the lack and hazards of antibiotics, including reduction of microbiome diversity and antibiotic resistance, the use of probiotics instead of antibiotics is becoming increasingly more acceptable. (Nami et al., 2015). Probiotics, prebiotics and CE cultures have demonstrated to be the good natural digestive system promoters (Ghadban, 2002 and Nami et al., 2015). It is also pointed out by this study that probiotics can be used in broiler meat production as gut health enhancer and growth promoter.
Many studies have been conducted to determine, the efficacy of non-AGPs on the health and growth performances of meat chickens. Nevertheless, it is not easy to make a comparative evaluation on the subject (Applegate et al., 2010). Due to differences in breeding conditions, feed and water quality, and probiotic and CE culture type, confirmative results between scientific studies has not easily been demonstrated (Otutumi et al., 2012). Variations in the effects of probiotics on growth performance of broiler chickens may be attributed to differences in the strains of bacteria used as dietary supplements (Angel et al., 2005; Timmerman et al., 2006; O'Dea et al., 2006; Lutful Kabir, 2009; Blajman et al.,
2015 and Olnood et al., 2015). Differences between physical and environmental conditions of the trials may also bias the results from these studies (Olnood et al., 2015). Also, an accurate dosage of administration has not yet to be established despite the wide use of probiotics (Khan et al. 2013; Li et al., 2014; Abu-Akkada and Awad, 2015 and Getachew, 2016). A continuously giving the probiotics via drinking water to the broiler chicken at whole breeding period may be more trusted way for taking maximum profit from it.
Adding to feed is the most commonly used method for administering probiotic preparations to broiler chickens in poultry production. Nevertheless, feed-type probiotic products rarely produce optimum results in pelletized diets usually fed to broilers (MacDonald and Wang, 2011). Probiotic bacteria incorporated into crumbles have an increased lifespan than those in pelletized feed (Eckert et al., 2010). Only spore forming probiotic bacteria can successfully survive in pelletized feed. Thus, the best natural solution to challenge the stability non-spore forming probiotic bacteria may be used in drinking water. However, chlorinated water can decline viability of the organisms rapidly (Raevouri et al., 1978). It is also in agreement with the researchers who mentioned that the best way of the giving non-spore forming probiotics to the broiler chickens may be the rote of drinking water.
Nurmi and Rantala (1973) have demonstrated that intubation in to the crop is probably the most satisfactory method for delivering a gap precise dose of probiotics to the animal. However, this route is not an applicable way on an industrial scale. Blankenship (1992) suggested that spray application of probiotic cultures, either on the environment of the birds or on the litter material seems to be an effective way of administering probiotic cultures. This way can also be applied during the first d of life of the chickens in industrial production practices, and it appears not to be easy and practical to apply at the farm level during rearing period.
The results of researches available in literature involving probiotics are very variable, several factors can interfere with the results, such as the type of probiotic, its action mode, its interaction with the host and breeding environment. There are few studies that demonstrate the usefulness of probiotics or CE cultures on growth performances (§tef et al., 2015; Abu- Akkada and Awad, 2015 and Getachew, 2016). Almost all of the other studies have demonstrated at least one positive effect including growth promotion of probiotics on the broilers (Mehr et al., 2014; Ritzi et al., 2014; Agboola et al., 2015; Zhang et
al., 2015; Schneitz et al., 2016 and Erdogmu§ et al., 2018). In this study, probiotic use had less feed consumption (338 g), more weight gain (113 g) and less FCR (0.22) than Control group (Table 2). The results have demonstrated that an efficient result of continuous use of a fresh liquid probiotic source at appropriate dose via drinking water appears to be alternative to AGPs. The performance results were significantly affected by probiotic use.
The Figures 1 and 2 represent the gut morphological structures. No differences were determined or superiority between the groups when examined the duodenum samples. There was no difference between probiotic group and control group aspect of histological structure, which were lymph follicles, goblet cells, crypt, submucosa and mucosa, in duodenum. The histometric differences between the two groups are given in table 3. There was no statistical significance in the crypt depth of duodenum and ceca between the groups. But, crypt depth of probiotic group (1110.46 ± 224.016 ^m) was statistically deeper than that of Control Group (949.39 ± 114.166 ^m) in ileum. Mucosa thickness of probiotic group in ceca and ileum appeared to be thicker than those of Control Group (Table 3). Present results are in aggreement with many other researchers who mentioned positive effects of probiotics on the gut health and accordingly growth performances (Giannanes et al., 2014; §tef et al., 2015; Zhang et al., 2015 and Erdogmus et al., 2018). A good histological development in the ileum and ceca in the probiotic group chickens may contribute to understand the BWG and FCR efficiencies in the group compared to Control Group.
CONCLUSION
Based on the findings of the present study, it may be concluded that a continuously inclusion of a good blend of probiotics at 108 CFU/ml dose in drinking water may successively improve the performance and gut health of commercial broiler chicks. Therefore, under the conditions of the present study, it can be recommended that using a freshly produced liquid microbial growth promoter in the non-medicated and de-chlorinated water could prove highly beneficial for the local broiler producers. It could be suggested that further research work should be performed to comparatively evaluate the effectiveness of freshly produced liquid live microbial cultures with other powder forms both as applications in drinking water and rations as-post pellet applications. So, replacement of AGPs with non-AGP microbial cultures, of broiler meat
industry and public health safety issues could be more lessened.
DECLARATIONS
Acknowledgments
The authors appreciate the research support provided in part by Bakin Tarim Ltd, Ankara/Turkey and a grant from the Scientific and Technological Research Council of Turkey, Ankara/Turkey (Grant no. 7100168).
Author's contributions
All authors participated equally in making the design, support with sampling andinterpretation of results, and writing the paper.
Competing interests
The authors declare that there is no conflict of interest.
Consent to publish
All persons gave their informed consent prior to their inclusion in the study.
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