CC BY
0JWPR
Journal of World's Poultry Research
© 2016, Scienceline Publication
J. World Poult. Res. 6(4): 199-204, December 25, 2016
Research Paper, PII: S2322455X1600024-6 License: CC BY 4.0
Effect of Probiotic Lactobacillus Species Supplementation on Productive Traits of White Leghorn Chicken
Tarekegn Getachew*1, Estifanos Hawaz2, Negassi Ameha1 and Teklemariam Guesh2
1Haramaya University, School of Animal and Range Sciences, P.O.Box 138 Dire Dawa, Ethiopia 2Haramaya University, Department of Biology, P.O. Box 138 Dire Dawa, Ethiopia Corresponding author's Email: targech23@gmail.com
Received: 24 Oct. 2016 Accepted: 24 Dec. 2016
ABSTRACT
Probiotics are live microbial food ingredients that have a beneficial effect on human health. Intake of probiotics improves feed intake, egg production and egg quality in laying breeds. The objective of this study was to evaluate the effect of the probiotic lactobacillus species supplementation on productive traits of White Leghorn chicken. For this purpose, 30 samples of cow milk were collected from Haramaya university dairy farm during the period from May to August 2015. The probiotic properties of each isolates were confirmed by simulating gastrointestinal tract conditions. Based on physiological and biochemical tests Lactobacillus acidophilus and Lactobacillus plantarum were isolated. The experimental design used in this experiment was single-factor Completely Randomized Design (CRD) with treatments basal feed (control), supplementation of L. acidophilus (T2), L. plantarum (T3) and their combination (T4) and a 5% (P<0.05) level significance was used. Supplementation of Lactobacillus species improved the Feed Intake (FI), Hen Day Egg production (HDEP) and egg weight. The FI recorded were 98.9 g/day/hen, 99.8 g/day/hen, 101.8 g/day/hen and 105.0 g/day/hen in control, T1, T2 and T3 respectively. HDEP of 0.31%, 0.33%, 0.33% and 0.34% were recorded at control, T1, T2 and T3 respectively. The egg weight of the control treatment, T1, T2 and T3 were 50.8g, 51.4 g, 51.4g and 51 9g respectively. Probiotic Lactobacillus species (L. acidophilus and L. plantarum) improves the productive traits of the laying flock. Chicken received the combination of probiotic lactobacillus species significantly perform best in FI, HDEP and egg weight.
Key words: Chicken, Lactobacillus, Probiotic, Productive trait, Supplement
Probiotics are defined as live microbial food/feed a supplement which beneficially affects the host animal by improving its intestinal balance that prevent from the growth of pathogenic bacteria, help the growth, multiplication and establishment of beneficial microflora in the intestinal environment (Fuller, 1989). Feeding viable Lactobacillus improves feed consumption, size of egg, and mineral retentions and decreases intestinal length from 7 to 59 weeks of age (Nahanshon et al., 1996).
Probiotics supplementation into poultry diets improves feed intake and growth performance in poultry breeds (Sarangi et al., 2016). Similarly, inclusion of probiotics significantly influences feed conversion ratio, egg production performance and egg quality in laying strains (Lei et al., 2013;Inatomi, 2016).Commonly used microorganisms as probiotics in animal feed are mainly bacteria strains belonging to different genera, e.g. Lactobacillus, Enterococcus,
Pediococcus, Bacillus and microcopic fungi, including Saccharomyces yeasts (Guillot, 2009). Feeding viable Lactobacillus species increased daily feed consumption, egg size, and nitrogen and calcium retentions in laying breeds (Nahashon et al., 1996). Probiotics improve feed intake and body weight gain in chicken fed with probiotics compared with that in control group fed basal diet (Zhang and Kim, 2014).
Moreover, probiotics have several beneficial impacts, including stimulating appetite, improving intestinal microbial balance, stimulating the immune system, producing digestive enzymes and utilizing indigestible carbohydrates (Prins, 1977; Nahanshon et al., 1992; Nahanshon et al., 1993; Fuller, 1989; toms and Powrie, 2001; Gilliland and Kim, 1984; Saarela et al., 2000). The objective of this study was to evaluate the effect of the probiotic lactobacillus species supplementation on productive traits of White Leghorn chicken.
MATERIALS AND METHODS
Study area and sample collection
The experiment was conducted at Haramaya university poultry farm, Ethiopia (Effect of probiotic supplementation) and microbiology laboratory (isolation, characterization and testing Lactobacillus species). A total of 30 samples of raw cow milk were collected from Haramaya university dairy farm during the study period May to August 2015. The raw cow milk samples were collected using sterile bottles and transported to the microbiology laboratory in icebox for analysis. Aseptic sampling was followed as described by the Health Protection Agency (HPA, 2014) and the Food and Drug Administration (FDA, 2003). After arrival at the laboratory, samples were kept at temperatures below 4oC and were analyzed within 48 hours of collection.
Ethical approval
This research did not involve feeding of birds with pathogenic microorganisms, introduction of any intervention in/on birds, or direct collection of cells, tissues or any material from birds.
Isolation of lactic acid bacteria lactic acid bacteria
Lactic acid bacteria (LAB) were isolated from raw cow milk. A 0.1 ml of different dilution (10-2 to 10-8) of samples was inoculated on De Man Ragosa Sharpe (MRS) agar medium (pH 6.2) plates and incubated at 37oC for 24-36 hours anaerobically. The presence of acetate, citrate and tween-80 in MRS agar allows selective isolation of LAB, at the same time ensuring the removal of most fastidious organisms.
Physiological and biochemical characterization of lactic acid bacteria
Phenotypic properties of LAB such as cell morphology of all isolates were determined using a microscope by Gram staining (Bergey et al., 1989). Isolates were further tested for different tests including catalase test, CO2 production form glucose, growth at different temperatures (15, 37 and 45oC) as well as the ability to grow in different concentrations of sodium chloride, antibiotic resistance and pH in MRS agar. Sugar fermentation patterns of LAB isolates were determined using different sugars.
Feasibility tests of Lactobacillus probiotics
Feasibility tests of Lactobacillus was carried out using Gastrointestinal Tract (GIT) conditions of chicken including, antibiotic resistance, resistance to low pH, resistance to bile salt, bile salt hydrolysis and antimicrobial activity against pathogens were done using standard procedures.
Experimental animal management and design
A total of 120 White leghorn layers were used for the study. The feed ingredients used in the experiment were according to standard layers diet (basal diet) and probiotic bacteria were supplemented. Before the commencement of the actual experiment and placing the experimental animals in the pen, watering troughs, feeding troughs, laying nests and the pen itself were cleaned thoroughly, disinfected and sprayed. The birds were vaccinated for the common diseases.
The chickens were randomly distributed into the pens each having the capacity of 10 hens. The birds were fed in a group providing feed twice a day at 8:00 and 16:00 hours. Each pen was provided with laying nest, feeders and watering point. A regular 16 hours light was provided throughout the experimental period of 84 days (12 weeks). The birds were acclimatized for one week for the new feed treatment.
A completely randomized design with four treatments was used as in table 1. T1 was control without probiotic bacteria supplementation, T2 was supplementation of Lactobacillus acidophilus in the diet, T3 was supplementation of Lactobacillus plantarum in the diet and T4 was supplementation of both Lactobacillus acidophilus and Lactobacillus plantarum in the ration. Each treatment was replicated three times having 10 layers each replica. The probiotic bacteria used for the study were the isolated, characterized and cultivated probiotic bacteria in the Haramaya University, microbiology laboratory.
Response criteria
The parameters employed in this experiment were: Feed Intake (FI), Hen Day Egg Production (HDEP), egg weight and egg size. FI was calculated by subtracting the amount of feed refusal from the amount of feed offered/day. HDEP was calculated as the ratio of the number of eggs collected/day with the number of birds in the pen. Eggs collected during the experiment categorized as jumbo, extra-large, large, medium, small and pee wee based their size (table 2).
Data analysis
Collected data were analyzed using of SAS 9.1.3 and data on production and egg quality parameters were stratified into the main factor (probiotics). A 5% (P<0.05) level of significance was used to determine statistical significance.
RESULTS AND DISCUSSION
Isolation, testing and characterization of lactobacillus probiotic
Probiotic Lactobacilli species including
lactobacillus acidophilus (hudf8) and lactobacillus
plantarum (hudf20) were the candidates of LAB species from raw unpasteurized cow milk samples (Table 3, 4 and 5).
Effects of probiotic lactobacillus species on productive traits
The effects of probiotic L. acidophilus and L. plantarum on FI, HDEP, egg weight and egg size are presented in (Table 6). Supplementation of probiotic lactobacillus species improved the FI, HDEP and Egg weight. However, there was no significant effect on egg size in layers supplemented with probiotic. Significantly higher FI, HDEP and egg weight was recorded at chicken supplemented the combination of the lactobacillus species (L. acidophilus and L. plantarum).
In this experiment, improvement in FI was recorded as a result of probiotic supplementation. Raka et al. (2014) reported a rise in feed and water consumption in laying hens fed with Liquid Probiotics Mixed Culture (LPMC) containing two type microorganisms, Lactobacillus and Bacillus species which is in agreement with the current study. Similarly, Nahashon et al. 1996, feeding viable Lactobacillus at 1100 mg kg-1(4.4 xio7 colony forming unit kg-1) increased daily feed consumption, egg size, nitrogen and calcium retentions. Another study by Zhang and Kim (2014) reported an increase body in FI in chicken fed with multi-strain probiotics compared with that in control group fed basal diet. Similar results were observed with studies by Lei et al. (2013), Inatomi (2016) and Sarangi et al. (2016) in that Probiotics supplementation into poultry diets improves feed intake and growth performance in laying flocks. However, Inclusion of probiotic caused no significant increase in feed consumption, egg production and egg weight (P>0.05) (Mahdavi et al., 2005). Another study, Saadia and Nagla (2010) reported FI values of different treated groups were approximately similar and lacked significance with layer flock that fed with probiotics.
The study shows an increase in HDEP and average egg weight due to probiotic supplementation. Raka et al. (2014) reported the highest HDP and egg
weight in layers supplemented with LPMC containing two type microorganisms, Lactobacillus and Bacillus species. Similarly, Yoruk et al. (2004) reported that egg production in Hisex Brown layers fed with probiotics contained L. plantarum and L. acidophilus, showed greater egg production than the group fed with basal diet. Moreover, there were linear increases in egg production with increased supplemental probiotic. Moreover, significant improvement in egg production was observed in hens supplemented with a mixed culture of L. acidophilus and L. casei (Haddadin et al., 1996).
Figure 1.effect of Lactobacillus acidophilus, Lactobacillus plantarum and their combination on hen day egg production and egg weight in White Leghorn hens during the study period
A study by Davis and Anderson (2002) found no significant improvement in egg production of hens supplemented with Prima Lac, a commercial product containing Lactobacillus species. Similarly, Addition of probiotic had no significant effect (P>0.05) on shell hardness and shell thickness and these were expected which have already been reported (Haddadin et al., 1996 and Mohan et al., 1995). The same result was reported by Ramasamy et al. (2008) in which, supplementation of Lactobacillus cultures did not influence the egg production of hens throughout the experimental period and no significant difference in egg weight in hens fed with L. acidophilus.
Table1. Layout of the experiment on effect of probiotic lactobacillus species on productive traits in White Leghorn chicken during the study period_
Treatments Number of replication Supplementation of lactic acid probiotic bacteria Number of birds per replica Total number of birds per treatment
T1 3 No probiotic bacteria (control) 10 30
T2 3 Lactobacillus acidophilus 10 30
T3 3 Lactobacillus plantarum 10 30
T4 3 Lactobacillus acidophilus and Lactobacillus plantarum 10 30
T1 : treatment 1; T2: treatment 2; T3 : treatment 3 and T4: treatment 4
Chicken. J. World Poult. Res. 6(4): 199-204. ^^^^^^^^^H:http://jwpr.science-line.com/
Table 2. Modern egg size chart for adult laying chicken used from May to August 2015
Size Minimum weight (g)
Jumbo 70
Extra-large 63
Large 56
Medium 49
Small 42
Pee wee <42
Table 3. Physiological and biochemical characteristics of Lactobacillus strains isolated from fresh cow milk
, . ,. Isolates
Characteristic
Lactobacillus acidophilus (hudf8)
Lactobacillus plantarum(Hudf20)
Gas from glucose + -
Cell shape bacillus bacillus
Ammonia from arginine - -
Motility - -
Catalase test - -
Aerobicity f.a f.a
Growth at different temperature 10oC
15oC + -
45oC v +
Growth at different pH 2.0
4.0 - +
5.0 + +
Growth in the presence of NaCl
2% + -
4% + +
6.5% - -
Carbohydrate fermentation
Lactose + +
Maltose + +
Glucose + +
Galactose + +
Mannose + +
Mannitol + -
Melezitose + -
Salicin - -
Melibiose - -
Cellulose + -
Rhamnose - -
Sucrose - +
Ribose - -
v=variable reaction; f.a=facultative anaerobic; n=2
Table 4.Probiotic feasibility test of Lactobacillus strains simulating under gastrointestinal tract conditions of adult layers
Isolates
Characteristics
Lactobacillus acidophilus (hudf8)
Lactobacillus plantarum(hudf20)
Resistance to low pH
2.0 1.03±0.02a 0.98±0.00a
3.0 1.25±0.00a 1.05±0.01a
4.0 1.31±0.00a 1.32±0.00a
Resistance to bile acids 0.3 % (w/v)
0hr 1.23±0.03a 1.23±0.00a
1hr 1.01±0.01a 1.13±0.01a
2hr 0.93±0.00a 0.98±0.02a
3hr 0.87±0.00a 0.89±0.00a
Antibiotic resistance
Streptomycin R R
Gentamycin R R
Tetracycline R R
Heamolytic test - -
aMeans bearing similar superscripts in the same column differs insignificantly (p>0.05); R=resistant; -=negative reaction, n=2
Chicken. J. World Poult. Res. 6(4): 199-204. ^^^^^^^^^H:http://jwpr.science-line.com/
Table 5. Antimicrobial activity of Lactobacillus isolates from fresh cow milk from May to August 2015
Lactobacillus isolates Means zone of inhibition zone (mm)
Streptococcus aureus Klebsiella pneumonia Escherichia coli Pseudomonas aeruginosa
L. acidophilushudfl 8±0.00b 11±0.00b 12±0.00b 8±0.00b
L. plantarumhudf3 9±0.01b 12±0.03b 9±0.00b 11±0.00b
L. acidophilushudfl 21±0.02a 18±0.00a 17±0.02a 17±0.00a
L. acidophilushudfl 2 11±0.02b 10±0.00b 13±0.00b 11±0.00b
L. plantarumhudf5 12±0.00b 10±0.01b 8±0.00b 9±0.00b
L. acidophilushudf6 11±0.00b 11±0.03b 6±0.03b 10±0.00b
L.plantarumhudf20 19±0.03a 20±0.03a 18±0.00a 20±0.00a
ab Means bearing different superscripts in the same column differ significantly (p<0.05); n=2
Table 6.effect of Lactobacillus acidophilus, Lactobacillus plantarum and their combination on productive traits in White Leghorn hens during May to August 2015_
Parameter
Control
Lactobacillus acidophilus
Lactobacillus plantarum
Combination
FI (g/day/hen) HDEP(%) Egg weight (G) Egg size (%) Jumbo Extra-large large Medium Small Pee wee
98.9 ± 1.16 0.31 ± 0.01 50.8 ± 0.40
11.7 ± 0.76 22.7 ± 1.06 44.4 ± 1.24 17.0 ± 0.35 4.0 ± 1.00
99.8 ± 0.47 0.33 ± 0.01 51.4 ± 0.35
12.8 ± 0.20 23.3 ± 0.46 44.8 ± 0.59 15.2 ± 0.96 3.97 ± 0.15
101.8 ± 2.12 0.33 ± 0.01 51.4 ± 0.25
13.1 ± 0.25 23.9 ± 0.25 44.4 ± 0.40 14.8 ± 0.40 3.8 ± 0.10
105.0 1.00 0.34 0.01 51.9 0.15
13.5 ± 0.15 24.5 ± 0.50 44.7 ± 0.47 13.9 ± 0.36 3.3 ± 0.21
CONCLUSION
Supplementation of probiotics into layers diet improves their production performance. In this study, supplementation of probiotics significantly improves FI, HDEP and egg weight. Mixture of probiotics (L. acidophilus and L. plantarum) is recommended as it significantly improves FI, HDEP and egg weight. However, there was no significant effect of probiotic supplementation on egg size. Despite the improvements in productive traits, further investigation is recommended to establish the optimum dosage and mode of inclusion for different classes of poultry.
Acknowledgement
The authors are grateful to Haramaya University for the study support.
Competing Interests
The authors declare that they have no competing interests.
REFERENCES
Bergey D, Sneath P and John H (1984). Bergey's manual of systematic bacteriology. Williams and Wilkins, Baltimore. Vol. I section 5. Davis GS and Anderson KE (2002). Theeffects of feeding the direct-fed microbial, PrimaLac, on
growth parameters and egg production in single white leghorn hens. Poultry Sciences, 81:755759.
FDA (2003). Food sampling and preparation of sample homogenate. Bacteriological analytical manual on-line, 8thEdition.Food and Drug Administration (FDA), USA.
Fuller R (1989). Probiotics in man and animals. The Journal of Applied Bacteriology, 66(5): 365-378.
Gilliland SE and Kim HS (1984). Effect of viable starter culture bacteria in yoghurt on lactose utilization in humans. Journal of Dairy science 67:1-6.
Guillot JF (2009). Consequences of probiotics release in the intestines of animals. Universite de tours-IUT, reve du pant. 3:70-82.
Haddadin MSY, Abdulrahim SM, Hashlamoun EAR and Robinson RK (1996). The Effect of Lactobacillus acidophiluson the Production and Chemical Composition of Hen's Eggs. Poultry Science, 75(4): 491-494.
HPA (2004). Preparation of samples and decimal dilutions. National Standard Method D1. Issue No. 2. Health Protection Agency (HPA), UK.
Inatomi T (2016). Laying performance, immunity and digestive health of layer chickens fed diets containing a combination of three probiotics. Science Postprint 1(2).
Lei K, Li YL, Yu DY, Rajput IR, Li WF (2013). Influence of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens. Poultry Science 92(9): 2389-2395.
Mahdavi AH, Rahman HR and Pourreza J (2005). Effect of probiotic supplements on egg quality and laying hen's performance. International Journal of Poultry Science, 4(7): 488-492.
Mohan B, Kadirvel R, Bhaskaran M and Natarajan A (1995). Effect of probiotic supplementation on serum/yolk cholesterol and on egg shell thickness in layers. British Poultry Science, 36(5): 799-803.
Nahashon SN, Nakaue HS and Mirosh LW (1996). Performance of Single Comb White Leghorn fed a diet supplemented with a live microbial during the growth and egg laying phases. Animal Feed Science and Technology, 57(1-2): 25-38.
Nahanshon SN, Nakaue HS and Mirosh LW (1993). Performance of single comb white leghorn fed a diet supplemented with a live microbial during the growth and egg laying phases. Journal of Animal Feed Science Technology 57:25-38.
Nahanshon SN, Nakaue HS and Mirosh LW (1992). Effect of direct fed microbials on nutrient retension and production parameters of laying pullets. Poultry Science, 7(1): 111.
Prins RA (1977). Biochemical activities of gut microorganisms. In: Clarke. R.T.J. Bouchop T. (eds), Microbial ecology of the gut. Academic press, London.73-183.
RakaPambuka S, Sjofjan O and EkaRadiati L (2014). Effect of Liquid Probiotics Mixed Culture Supplements through Drinking Water on Laying Hens Performance and Yolk Cholesterol. Journal of World's Poultry Research, 4(1): 05-09.
Ramasamy K, Abdullah N, Wong MC, Karuthan C and Ho YW (2010). Bile salt deconjugation and cholesterol removal from media by Lactobacillus strains used as probiotics in chickens. Journal of the Science of Food and Agriculture, 90(1): 6569.
Saadia MH and Nagla KS (2010). Effect of Probiotic (Saccharomyces cerevisiae) Adding to Diets on Intestinal Microflora and Performance of Hy-Line Layers Hens. Journal of American Science, 6(11): 159-169.
Saarela M, Mogensen G, Fondrin R, Mottu J and MattilaSandholm T (2000). Probiotic bacteria: safety, functional microflora and performance of Hy-line layers hens. Journal of American Science. 6(11): 159-169).
Sarangi N, Babu L, Kumar A, Pradhan C and Pati P (2016). Effect of Dietary supplementation of prsbiotic, probiotic, and Synbiotic on growth performance and carcass
characteristics of broiler chickens.Veterinary world. 9 (3): 31-39.
Toms C and Powrie F (2001). Control of intestinal inflammation by regulatory T cells. Microbes infect. 3:929-935.
Yoruk MA, Gul M, Hayirli A and Macit M (2004). The effects of supplementation of humate and probiotic on egg production and quality parameters during the late laying period in hens. Poultry Science 83(1): 84-88.
Zhang ZF and Kim IH (2014). Effects of multistrain probiotics on growth performance, apparent ileal nutrient digestibility, blood characteristics, cecal microbial shedding, and excreta odor contents in broilers. Poultry science, 93(2): 364-370.
