Livestock Research for Rural Development 24 (12) 2012 Guide for preparation of papers LRRD Newsletter

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Effect of probiotic containing Lactobacillus salivarius on the laying performance and egg quality of Japanese quails

U Kalsum, H Soetanto*, Achmanu* and O Sjofjan*

Faculty of Animal Husbandry, Islamic University of Malang, Malang, East Java
* Faculty of Animal Husbandry, Brawijaya University, Malang, East Java, Indonesia


The objective of this study was to investigate the potential of Lactobacillus salivarius, isolated from the intestine of Japanese quails (Coturnix japonica), as a probiotic product.  The influence of this probiotics preparation on the laying performance and egg quality of quails was evaluated in a 28-day experiment.  A fully randomized design was used with four experimental treatments, namely standard feed, standard feed with 2.45 106 cells/ml, standard feed with 2.64107 cells/ml L. salivarius and standard feed with 2.83108cells/ml L.salivarius.

The results showed that L.salivarius supplementation did not influence egg quality parameters (haugh unit, % egg albumen, % egg yolk and egg shell thickness) and egg weight, but significantly improved total egg production and lowered cholesterol content in egg yolk. In conclusion, L. salivarius may be used as a feed additive in Japanese quail diets to improve egg production and to lower egg cholesterol content. 

Keywords: Coturnix japonica, egg cholesterol, egg production, endogenous probiotic


Japanese quail (Coturnix japonica) is a kind of poultry able to produce 250 – 300 eggs per year. The weight of one quail egg is almost 7 % of the body weight of quail (Adams 2000; Abidin2002).In addition to being cheaper and delicious, quail eggs are rich in protein and good sources of folate, vitamin B12, pantothenic acid, iron, phosphorus, riboflavin and selenium (Bing 2011).However, quail egg is reported to be high in saturated fats and cholesterol (Anonymous 2011). It is therefore necessary to explore ways by which egg cholesterol levels can be lowered. The use of probiotics as feed additives is one effort to reduce the cholesterol levels.

In recent yearsconsumers are becoming increasingly concerned about the safety of poultry products. The major concerns are related to the presence of antibiotic residues in poultry products that can cause adverse effects on human health and the possible development of antibiotic resistant bacteria. One key strategy to replace the use of antibiotics in poultry diets is to feed microorganisms, which are known to exert beneficial effects in the gut, directly to the bird. Probiotics are live microorganisms which are supplemented to the feed in order to establish a beneficial gut microflora. Use of these microbes in diets for Japaneses quail may enhance the intestinal health by modifying the gut microflora. Probiotics not only deliver health benefits for the birds that consume them, but also for consumers who purchase the poultry products.In our laboratory, several strains of microbes have been successfully isolated from the intestinal tract of quails and characterised (Kalsumet al 2008). From these microbial strains, the genus of Bacillus was selected for evaluation as a probiotic.  This genus is known to produce several enzymes such as protease (Libertina et al 2009) and amylase (Wardhani et al 2009) and therefore it potentially can be exploited as a probiotic. The bacteria belonging to this genus had several dominant species of interest. Of these,Lactobacillus salivarius was identified as promising and considered for further evaluation as a probiotic (Kalsum et al 2008). The spore-forming lactic acid bacteria belonging to this genus are stable, resistant to high temperatures and tolerant to bile salts.

Kalsum et al (2010) reported that the supplementation of L.salivarius probiotic, isolated from quail intestine, improved the bacterial profile and health of the digestive tract of quail. This was due to the fact that L.salivarius can produce anti-bacterial compounds that could inhibit the growth of microbial pathogens such as Salmonella typhimurium and Eschericia coli.Such beneficial effects on gut flora may be expected to improve the production performance of quails.

Using lactic acid bacteria as probiotics is an alternative to the use of in-feed antibiotics as prophylactic and growth-promoting agents in poultry production. Previous studies on probiotics with laying hens have demonstrated positive responses with dietary supplementation (Li et al 2006; Kalavathyet al 2009), but no studies to date have investigated endogenous probiotics in diets for laying quails.

The objective of the present study was to examine effects of the addition of a probiotic bacteria L. salivarius, isolated from quail intestine, on performance of laying quails, egg quality characteristics and egg yolk cholesterol content 

Materials and Methods

Location of the study area

The experiment was carried out in Microbiology laboratory Faculty of Scienceand the experimental farm ofFaculty of Animal Husbandry, Brawijaya University, Malang, East Java, Indonesia. The experimental protocol was approved by the Brawijaya University Animal ethics and welfare Committee.

Preparation of probiotics

L. salivarius was isolated from the intestine of Japanese quails and cultured in agar based on the formulations of deMan, Rogosa and Sharpe (MRS) for 48 hours at 37 C. This medium supports luxuriant growth of lactobacilli from oral, fecal, dairy, and other sources (Murray et al 1995). Then  a series dilutions up to 10-7 in MRS liquid was performed, followed by spreading on MRS agar that containing 60 mg/kg Bromocresol purple (BCP) as the pH indicator and incubated for 24 hours at 37 C. Lactic acid bacteria (LAB) colony count was determined based on the change of media to be pale yellow at the location of the growth of bacterial colonies.  The characteristic test confirmed that the isolates are LAB, gram positive, catalase negative and negative endospores. Isolation of the species was confirmed with the API 50 CH test Medium Kit (bioMerieux2001;Abegaz2006) and API 50 CHL medium. To determine it’s feasibility as a probiotic, the candidate was then evaluated using several tests, including pH test, pathogen test and bile salt test.

Birds, experimental design and treatments

A total of 160 laying quails, 64 days old, were randomly assigned to 16 cages (40 x 40 x 25 cm) of 10 birds each and then the cages were allocated to four treatment groups with four replications per treatment. The birds were housed in individual wooden battery cages, in a temperature controlled room at 73F. All birds had free access to feed and water. The photoperiod was 16 hours of light per day throughout the experimental period according to Vatsalya and Kashmiri (2011). Prior to the introduction of experimental diets, all birds were fed ad libitum a standard diet (Table 1) for 10 days and egg production was monitored. During this period, it was found that the egg production was similar in the different groups and  that the coefficient of variation did not differ.

The four experimental diets were as follows:
SFC, a standard feed (control diet);
SFMP, standard feed with million probiotics (2.45 106 cells/ml L.salivarius);
SFTP, standard feed with ten million probiotics (2.64 107 cells/ml L.salivarius); and
SFHP, standard feed with Hundred million probiotics (2.83 108 cells/ml L.salivarius).

After confirming the cell counts in the laboratory using haemocytometer according to Henderson (2010), the probiotic was first mixed with soybean meal and then added to the diet.  Diets containing the probiotic were mixed at weekly intervals. The standard feed was formulated to meet or exceed the nutrient recommendations by National Standard Indonesia (SNI 2006) and the diet composition is shown in Table 1.

Table 1. Composition (%, as fed) of the standard feed



Yellow corn


Soybean meal


Rice bran


Wheat pollard


Coconut cake


Fish meal




Vitamin-trace mineral premix1




DL Methionin




Chemical composition


Dry matter

Crude protein



Crude Fat


Crude fiber




Metabolizable energy (kcal/kg)


1 Gram of premix contained: 2000 IUretinol, 400 IU cholecalciferol, 2 mg α-tocopherol, 1.2 mg riboflavin, 2 g cobalamin, 200 g pyridoxine, 100 g vitamin K3, 1.5 mg calcium D-pantothenate, 5 mg choline chloride, 50 mg DL-Methionine, 3 mg nicotinic acid, 6.4 mg iron, 1.6 mg copper, 10 mg magnesium, 8 mg zinc, 6 mg manganese, 20 g cobalt, 20 g Iodine, antioxidant. 

Measurement and data collection

Feed intake, egg production and egg weights were recorded daily during the experimental periodthat lasted 28 days.Egg mass was calculated by multiplied of egg production by egg weight according to Olgun et al(2009). Feed conversion ratio (FCR) is a number indicating the amount of feed (grams) required by quail to produces one gram of egg. FCR was calculated by dividing the feed intake by egg mass. Egg quality parameters were measured on the Saturday of each week. Egg weight, egg yolk and albumen weight percentages were determined in 10 eggs per replication according to the procedures of Christaki et al (2011). Egg shell thickness was measured by micrometer according to the method of Balkan et al (2006). Haugh unit was calculated from the logarithm of albumen height and transformed into a correction value of the function of egg weight based on Roberts et al (2004). The height of albumen was measured by a spirometer. Yolk extracts were collected from 10 eggs per replication and yolk cholesterol was analyzed according to the method of Subekti et al (2006).

Statistical analysis

Data were statistically analysed using the analysis of variance (ANOVA) program in the Minitab (2003) software (version 14). Significant differences between treatment means were separated using Duncan's multiple range test according to Steel and Torrie (1992).

Results and Discussion

The endogenous probiotic as feed additive that was used in this study was the lactic acid bacteria identified as Lactobacillus salivarius. The potential of this bacterial species as a probiotic was supported by Jans (2007). Most of the probiotics used as feed additive in broiler diets belong to Lactobacillus bacteria and Bifidobacterium.

Feed intake and egg mass did not differ between treatments with probiotic L.salivarius (Table 2), due to the metabolic energy content of the diets was the same. According to Ferket and Gernat(2006) metabolic energy requirements depend on the energy needs required for basic living and growth or production. Similar findings have been reported by akiret al (2006) who reported no effect of dietary supplementation of probiotic-prebiotic, organic acid and avilamycin on the growth performance, feed intake, feed efficiency and proportional organ weights of growing Japanese quails. In contrast, Zeweil et al (2006) found that the supplementation of 1.0 or 2.0 g/kg probiotic (Lacto-Sac) significantly improved egg production, egg mass, egg weight and feed conversion ratio as compared to control laying Japanese quail hens.

Table 2. The effect of L.salivarius administration on the performance of laying quails1









Feed intake (g)







Egg production, %







Egg mass, g/flock/d














a,b Different superscripts in a column denote significant difference (P< 0.05).

1 Each value represents the mean of four replicates (10 birds/ replicate). 

Probiotic supplementation did not affect feed conversion ratio, but improved egg production by 18.7 %. Egg production was highest in birds fed the standard feed containing the high dose of probiotics (2.83 108 cells/ml) and this was higher than those receiving the standard feed control diet. The differences in egg production can be explained mainly by the differences in numbers of cells probiotic (Figure 1). This observation highlights the potential of L.salivarius to improve egg production and this may be due to its role in improving the apparent metabolizable energy and digestibility of protein (Wu et al 2004; Centenoet al 2007).

Figure 1. Relationship between egg production and numbers of cells of L. salivarius

Table 3.The effect of L. salivarius administration on  egg quality parameters1









Egg weight,g







Egg yolk, %







Egg albumen, %







Egg shell, mm







Haugh unit







Egg yolk cholesterol, mg/100g







a,b,c,d Different superscripts in a column denote significant difference (P< 0.01).

1 Each value represents the mean of 40 observation. 

The data presented in Table 3 show that the quality of quail eggs did not differ between addition of L.salivarius to quail diets, except for egg yolk cholesterol levels which were decreased by 9.25 % in the probiotic diets. These findings are consistent with those of Gl (2011) that the use of probiotics did not have a significant impact on egg weight, albumen index, yolk index, and haugh unit. Similarly, Haddadinet al(1996) reported that the inclusion of Lactobacillus acidophilus in laying hen diets at levels up to four million viable cells per gram feed decreased cholesterol values in egg yolk by 18.8%.

Mahdavi et al (2005) also reported that the use of Bacillus subtilis and Bacillus licheniformis decreased egg cholesterol of laying hens. Kankaanpaa et al (2004) speculated that probiotics can increase the secretion of enzymes in the digestive tract that can destroy a specific substrate or anti-nutrient in the feed. Another mechanism is by inhibiting reactions that produce carcinogens, stimulating the increase of detoxification reactions and synthesizing the essential ingredients. Abdulrahimet al (1996) observed that Lactobacillus acidophilus improved egg production, feed conversion and reduced the cholesterol content in eggs. Nahashon et al (1994) also found that Lactobacillus acidophilus has the ability to break down simple carbohydrates into lactic acid. As lactic acid increased, pH of the gut environment is decreased and the growth of pathogenic microorganisms is restricted. Sultan and Abdul-Rahman (2011) reported that the probiotic treatment significantly decreased serum triglyceride levels and increased serum uric acid levels.



We wish to acknowledge the support financed from Department  of Higher Education, Ministry of Education and Culture of Republic of Indonesia.  Gratitude is expressed to the students who helped to carry out this study and to the Department of Animal Husbandry and Microbiology, Brawijaya University for access to research facilities.


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Received 27 August 2012; Accepted 1 November 2012; Published 2 December 2012

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