Evaluation of Probiotic Potential of Some Native Lactobacillus Strains on the Growth Performance and Serum Biochemical Parameters of Japanese Quails ( Coturnix Coturnix Japonica ) during Rearing Period

The objective of the present study was to evaluate the probiotic effects of different concentrations of four selected native Lactobacillus strains on the growth performance and serum biochemical parameters of Japanese quails. A completely randomized design (CRD) was applied, including seven probiotic treatments with four replicates of 20 quails each, totaling 560 quails. Treatments were applied for five weeks. Four native Lactobacillus strains were anaerobically grown in a 10-L batch fermenter and lyophilized (1010 CFU/g). Treatments were as follows: T1: control (basal diet); T2: commercial probiotic CP1; T3: commercial probiotic CP2; and T4, T5, T6, and T7: four native strains added at levels of 50, 100, 150, and 200 g/ton diet, respectively. The native probiotics significantly improved body weight gain (BWG) and feed conversion ratio (FCR) during the starter, finisher, and overall periods (35 days) (p<0.05), whereas no significant effect was observed on feed intake. The native strains significantly influenced the serum glucose, total protein, globulin, phosphorus, uric acid, low-density lipoprotein (LDL), LDL/HDL ratio, white blood cell counts (WBC), and mean corpuscular hemoglobin concentration (MCHC) of quails during the rearing period (p<0.05), whereas treatments had no influence (p>0.05) on blood cholesterol, calcium, high-density lipoprotein (HDL), or hemoglobin (HB) levels or on red blood cell counts (RBC). The cecal and small intestine samples of the quails fed the native Lactobacillus strains contained significantly higher Lactobacillus spp. and lower E. coli populations compared with the control diet and those supplemented with commercial probiotics. It was concluded that the use of the native Lactobacillus strains (150 g/ ton diet) promoted the best performance of Japanese quails.


ABStRACt
The objective of the present study was to evaluate the probiotic effects of different concentrations of four selected native Lactobacillus strains on the growth performance and serum biochemical parameters of Japanese quails.A completely randomized design (CRD) was applied, including seven probiotic treatments with four replicates of 20 quails each, totaling 560 quails.Treatments were applied for five weeks.Four native Lactobacillus strains were anaerobically grown in a 10-L batch fermenter and lyophilized (10 10 CFU/g).Treatments were as follows: T1: control (basal diet); T2: commercial probiotic CP1; T3: commercial probiotic CP2; and T4, T5, T6, and T7: four native strains added at levels of 50, 100, 150, and 200 g/ton diet, respectively.The native probiotics significantly improved body weight gain (BWG) and feed conversion ratio (FCR) during the starter, finisher, and overall periods (35 days) (p<0.05),whereas no significant effect was observed on feed intake.The native strains significantly influenced the serum glucose, total protein, globulin, phosphorus, uric acid, low-density lipoprotein (LDL), LDL/HDL ratio, white blood cell counts (WBC), and mean corpuscular hemoglobin concentration (MCHC) of quails during the rearing period (p<0.05),whereas treatments had no influence (p>0.05) on blood cholesterol, calcium, high-density lipoprotein (HDL), or hemoglobin (HB) levels or on red blood cell counts (RBC).The cecal and small intestine samples of the quails fed the native Lactobacillus strains contained significantly higher Lactobacillus spp.and lower E. coli populations compared with the control diet and those supplemented with commercial probiotics.It was concluded that the use of the native Lactobacillus strains (150 g/ ton diet) promoted the best performance of Japanese quails.

IntRoduCtIon
The term probiotic is etymologically derived from the Latin preposition pro ("for" or "in support of") and the Greek word (biotic), which literally means "for life".I n 1989, Fuller defined probiotics as live microbial food supplements that beneficially affect the host animal by improving the intestinal microbial balance (Fuller, 1989).There is a huge body of evidence that support the significant positive impacts of probiotics and bioactive compounds on poultry performance and health.Probiotic bacteria improve the economic indexes and resistance to pathogens of laying or meat-type chickens (Hippenstiel et al., 2011;Aazami et al., 2014;Cean et al., 2015;Mountzouris et al., 2007).Lactobacilli and Enterococci have been widely used as probiotics in the poultry industry (Kabir et al., 2004;Awad et al., 2009;Aazami et al., 2014;Mountzouris et al., 2007).The main postulated health benefits associated with probiotics include improving the gut microflora balance, stimulating the immune reaction, producing different antimicrobial substances, Siadati SA, Ebrahimnezhad Y, Salehi Jouzani G, Shayegh J

Evaluation of Probiotic Potential of Some Native Lactobacillus Strains on the Growth Performance and Serum Biochemical Parameters of Japanese Quails (Coturnix Coturnix Japonica) during Rearing Period
modulating the immune response, producing digestive enzymes, and reducing cholesterol levels (Ramirez-Chavarin et al., 2013;Smug et al., 2014).Recent studies have confirmed that the addition of probiotic, symbiotic, and medicinal plant additives to feeds enhance nutrient bioavailability, health and immune status, and carcass yield and quality of Japanese quails (Yalçın et al., 2000;Siriken et al., 2003;Chimote et al., 2009;Sharifi et al., 2011;Kasmani et al., 2012;Babazadeh et al., 2011;Kheiri et al., 2015).The main factors that affect the general probiotic effects are probiotic species, strain origin, and application levels (Mountzouris et al., 2007;Amerah et al., 2013).
In this context, exploring new strains with good probiotic properties and optimizing their concentrations in feed additives are important to achieve highly efficient application of probiotics.Our group previously isolated and characterized some potentially probiotic bacteria from Iranian native chickens (Aazami et al., 2014).The selected Lactobacillus strains showed high resistance to acidic gastric conditions (pH 2.5) and to bile salts (0.5% Oxgall), as well as to salinity (up to 15% NaCl) and low and high temperatures (between 4.5 and 45°C).The strains presented fast auto-aggregation rate (10 to 120 min) and high ability for adhesion to the Caco-2 epithelial cell line (up to 40 bacterial cells).High antimicrobial activities against different pathogens (Pseudomonas aeroginosa, E. coli, Streptococcus mutans, Clostridium difficile, Enterococcus hirea, Salmonella enterica, Staphylococcus aureus) were detected in vitro (Aazami et al., 2014).The aim of the present study was to evaluate the probiotic effects of these selected native Lactobacillus strains on the growth performance and serum biochemical parameters of Japanese quails.

Birds and experimental treatments
All the experiment procedures were approved by the Animal Care and Ethics Committee of the Tarbiat Modares University and complied with the Guidelines for the Care and Use of Animals in Research.
In total, 560 one-day-old Japanese quails of both sexes (equally divided) were distributed according to a completely randomized design into seven treatments with four replicates of 20 quails each.The experiment lasted five weeks.The day-old chicks were purchased from a commercial hatchery.Birds in each replicate was allocated to a floor pen (40x40 cm 2 ).Room temperature was maintained at 32°C during the first week and gradually decreased by 3°C weekly until 22°C, and then maintained constant until the end of the experiment (Khaksar et al., 2012).The quails were subjected to continuous lighting program of 23L:1D during the entire experiment (Kermanshahi et al., 2015).
The following treatments were applied: T1: basal diet (control diet without probiotics), T2: basal diet + CP1 (908 g/ton in the starter diet and 454 g/ton in the finisher diet), T3: basal diet + CP2 (150 g/ton in the starter diet and 100 g/ton in finisher phase), T4: basal diet + 50 g of the native strains/ton, T5: basal diet + 100 g of the native strains/ton, T6: basal diet + 150 g of the native strains/ton, and T7: basal diet + 200 g of the native strains/ton.The diets were supplied as mash, and formulated for the starter (1 to 21 d) and Salehi Jouzani G, Shayegh J

Evaluation of Probiotic Potential of Some Native Lactobacillus Strains on the Growth Performance and Serum Biochemical Parameters of Japanese Quails (Coturnix Coturnix Japonica) during Rearing Period
the finisher (22 to 35 d) rearing periods.The basal diets were based on corn and soybean meal basal and were formulated to supply the quails' nutrient requirements according to the recommendations of the National Research Council (NRC, 1994).The composition of the basal diet is shown in Table 1.The experimental diets were prepared every week, and stored in bags at 4ºC.

Growth performance parameters
Growth performance parameters, including body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were evaluated.Weight gain was determined for the starter (1 to 21 d), finisher (22 to 35d), and overall periods.FI, FCR, and BWG were calculated for the overall period of the experiment (Cengiz et al., 2015).

Blood biochemical analyses
Two quails per replicate (one male and one female) were randomly selected on day 35, and sacrificed by ventral cutting of the neck.As both male and female Japanese quails were reared together, their blood samples were pooled before the biochemical and hematological analyses, which allows reducing the cost and increasing the level of automation of the analyses.Samples of approximately 1 mL of whole blood were collected from eight quails per treatment by jugular vein puncture after sacrifice.Serum globulin, total protein, glucose, high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), cholesterol, LDL/HDL ratio, uric acid, phosphorus, and calcium contents were analyzed using an automatic biochemical analyzer (DiaSys, Diagnostic Systems, Germany), following the instructions of the corresponding reagent kits (Pars Azmon Co., Tehran, Iran).Blood (approximately 2 mL) was collected from the same birds and placed into tubes containing EDTA-Na 2 (an anticoagulant) for subsequent determination of the mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HB) level, and white blood cell (WBC) and red blood cell (RBC) counts using an automated hematology analyzer (Celltac E, MEK-7222 J/K; Nihon Kohden Co., Tokyo, Japan) within 2 h after collection.

Evaluation of the cecal and small intestine Lactobacilli and E. coli populations
At the end of the experiment, four birds per treatment were randomly selected and sacrificed by decapitation.The cecal and small intestine contents of each bird were transferred to a sterile glass vial for microbial enumeration.Cecal and small intestine digesta samples (1 g) were diluted at 1:10 with normal saline solution, which were then further diluted to 10 −3 , 10 -5 , 10 -7 , and 10 −9 , in normal saline solution, out of which 100 μL were plated on agar plates.In order to evaluate Lactobacillus spp.and E. coli populations, the diluted samples were seeded on MRS agar (Merck, Germany) and MacConkey agar (Merck, Germany), respectively, and incubated for 48 h at 37°C.Colony forming units (CFU) were expressed as log10 CFU per gram of cecal and small intestine content (Hashemi et al., 2012).

Statistical analysis
Data were analyzed by analysis of variance using the GLM procedure of SAS/STAT software (SAS Institute, 2003) as a completely randomized experimental design with replicates as experimental units.Statistically significant differences among treatments was tested by Duncan's multiple range test at p ≤0.05.Salehi Jouzani G, Shayegh J

Effect of probiotics on growth performance
The results (Tables 2 and 3) showed that dietary inclusion of probiotics (native strains and commercial products) significantly increased body weight gain (BWG) and improved feed conversion ratio (FCR) during the starter and overall periods (p<0.05)compared with the basal diet (T1).Feed intake was not influenced by the dietary treatments in none of the evaluated periods.
The highest and lowest BWG values were obtained with the native probiotic product at 150 g/ton (T6, 202.99 g) and with the control diet (T1, 176.41 g), respectively, during the overall period (Table 3).Interestingly, the BWG during the overall period of the birds fed the diet with native probiotic at 150 g/ ton (T6) was significantly higher than that of those fed CP1 (188.72 g) and CP2 (188.50 g) (p<0.05).This was also observed for both the starter and finisher periods.Values are expressed as means ± standard error of the mean.A-d Values followed by different letters on the same row indicate significant differences (p<0.05).
FI= Feed intake, BWG= Body weight gain, FCR= Feed conversion ratio, CP = commercial probiotic, NP = native probiotic.The FCR of the birds fed the diet with native probiotic at 150 g/ton (T6) was significantly lower than that of the T2 (CP1, 2.89), T3 (CP2, 2.91), and control treatments during the overall period, demonstrating the better efficiency of the native strains compared with the commercial products (Table 3).This pattern was also observed in the finisher period (p<0.05).However, in the starter period, the highest and the lowest FCR (p<0.05) were obtained with the control diet and the diet with native probiotic at 200 g/ton, respectively (Table 2).
These results are consistent with those of Pelicano et al. (2004) and Gunal et al. (2006) in broilers, and Cakir et al. (2008) and Babazadeh et al. (2011), who did not find any significant effect of probiotics on feed intake of Japanese quails, whereas Corrêa et al. (2003) and Bitterncourt et al. (2011) showed that dietary probiotics reduced the feed intake of broilers.Alkhalf et al. (2010) reported that a probiotic level of 0.8 g/kg feed enhanced the live performance of broilers compared with the control diet and that with 1.6 g probiotic/kg.This indicates that increasing the level of probiotics in the feed does not ensure better performance, which is in accordance with the result of the present study, as T6 (150 mg/kg) promoted better performance than T7 (200 mg/kg).Chimote et al. (2009) showed that significantly higher body weight gain and better feed conversion ratio in Japanese quails fed probiotics those fed a control group.Kalavathy et al. (2003) reported that the supplementation of probiotics in broiler diets improved their body weight gain and feed conversion ratio from 1 to 42 days of age.Yu et al. (2007) also showed that the supplementation of an intestinal Lactobacillus reuteri strain to a wheatbased diet increased body weight gain of broilers from 1 to 21 days of age.Moreover, Khaksefidi & Ghoorchi (2006) reported that the body weight gain and feed conversion ratio of broilers fed probiotics Siadati SA, Ebrahimnezhad Y, Salehi Jouzani G, Shayegh J

Evaluation of Probiotic Potential of Some Native Lactobacillus Strains on the Growth Performance and Serum Biochemical Parameters of Japanese Quails (Coturnix Coturnix Japonica) during Rearing Period
were significantly improved compared with those fed control diets.On the other hand, Ramarao et al. (2004) reported that the body weight gain of broilers was not influenced by the dietary supplementation with probiotics, as opposed to findings of this experiment.The results of the present study were contrary with the findings of Ergün et al. (2000) and Arslan (2004), who suggested that probiotic supplementation did not influence the feed efficiency ratio or body weight gain of broilers.
The reason for the improvements in body weight gain and feed conversion ratio of the Japanese quails fed probiotics in the present experiment was probably an increase in the population of beneficial intestinal bacteria and reduction of the population of pathogenic bacteria, and consequently, better nutrient digestibility and absorption.The results of the present experiment confirmed the probiotic potential of the selected strains, and were consistent with those obtained in other studies, showing that the presence of probiotic additives enhances the performance (FCR, BWG and BW) of broilers (Ashayerizadeh et al., 2009;Arslan & Saatci, 2004;Yalçın et al., 2000;Sharifi et al., 2011;Kasmani et al., 2012;Chimote et al., 2009;Babazadeh et al., 2011;Kheiri et al., 2015).

Effect of probiotic on blood parameters
The effects of different probiotics on serum biochemical parameters and blood parameters are shown in Tables 4, 5, and 6, respectively.The inclusion of native and commercial probiotics had a significant effect on serum glucose, total protein, globulin, phosphorus, uric acid, LDL and LDL/HDL ratio, WBC, and MCHC of Japanese quails (p<0.05).No differences (p>0.05) in cholesterol, calcium, HDL, RBC, and HB values were detected (1-35 d).The serum cholesterol results of the present study are in conflict with the findings of Panda et al. (2006), who reported that the probiotic L. sporogenes (100 mg/kg diet) significantly reduced total cholesterol levels in broilers.Dibaji et al. (2012) and Kalavathy et al. (2003) also found that feeding probiotics decreased serum LDL, but not HDL levels in broilers.
In the present experiment, feeding probiotics had no effect on serum calcium levels, in agreement with the findings of Sahin et al. (2008), who did not find any influence of feeding a probiotic on the serum calcium levels of quails.On the other hand, Scholz-Ahrens et al. (2007) explained that probiotics may increase the intestinal absorption of calcium, because the short-  chain fatty acids produced by some probiotic bacteria reduce gastrointestinal pH, thereby increasing calcium solubility and presumably, calcium absorption.Significant reductions were recorded in serum glucose values in the probiotic groups (native probiotic at 150 and 200 g/ton, and CP2) compared with the control group (p<0.05),but no differences between commercial and native probiotics were detected (p>0.05).The reduction of serum glucose levels in quails fed probiotics in the present study are in agreement with the findings of Al-Kassie et al. (2008) and Arslan & Saatci (2004) in broilers and quails respectively, whereas Abd-El-Rahman et al. (2012) reported that feeding probiotics increased serum glucose levels in broilers.These differences serum glucose levels among studies may be ascribed to dietary ingredients, nutrient composition, and probiotic effectiveness.
The Japanese quails fed native probiotic strains at 150 g/ton presented higher serum total protein level compared with those fed the commercial probiotics, which were not statistically different.In addition, the highest blood globulin level was obtained in the treatments containing native probiotic strains (150 g/ ton) and the commercial probiotic CP2 compared with native probiotic strains (50 g/ton) and the control group.The results of the present study are in agreement with the findings of Arslan & Saatci (2004) who showed that a probiotic-supplemented diet fed to Japanese quails increased serum total protein levels.However, Yalçın et al. (2000), Djouvinov et al. (2005), Sahin et al. (2008), andAlkhalf et al. (2010) did not detect any influence of dietary probiotic supplementation on total protein and globulin levels in the serum of quails, broilers, quails, and ducks, respectively.The higher serum total protein levels detected in the birds fed probiotics, except for those fed the native probiotic strains at 50 g/ton, compared with those fed the control diet in the present study may be due to the better protein digestion promoted by probiotics (El-Faham et al., 2014).
The highest and lowest phosphorus contents were observed in the quails fed the native probiotic strains at 150 g/ton and those of the control group, respectively.Significant increases in serum phosphors values were recorded in the native probiotic strains (100, 150 and 200 g/ton) and commercial probiotics (CP1 and CP2) compared with the native probiotic strain 50 g/ton and control group (p<0.05) .These findings are comparable with the results of Hosseini et al. (2013), who found higher serum phosphorus levels in broilers fed a probiotic-supplemented diet compared with those fed a control diet.Eizaguirre et al. (2002) reported that probiotics reduced intestinal pH in humans, improving the absorption of minerals by enhancing their solubility.
Serum uric acid levels were significantly lower in the quails fed the native probiotic at 100, 150 and 200 g/ton and the CP1 compared with the control diet, but those fed the diets containing CP2 and the native probiotic at 50g/ton presented intermediate levels, not statistically different from the other treatments.El-Faham et al. (2014) also reported lower uric acid levels in broilers fed a probiotic than those fed a control diet, and suggested that probiotics may improve kidney function and protein metabolism, and consequently, increase nitrogen utilization.Moreover, certain probiotic strains utilize urea, uric acid, and creatinine and other toxins as nutrients for growth (Salim et al., 2011).The lower uric acid levels detected in the quails fed probiotics are consistent with the findings of Newaj-Fyzul et al. (2007) and Tonekabon (2013) in rainbow trout.The observed reduction in serum uric acid level with the diets containing probiotics in our experiment may be due to better kidney function and protein metabolism and consequently, better nitrogen utilization.
The maximum total protein (5.20 g/dL), globulin (3.75 g/dL), phosphorous (6.77 mg/dL), and MCHC (37.92%) as well as minimum serum glucose (264 mg/dL), uric acid (2.75 mg/dL), LDL (60 mg/dL), and LDL/HDL (0.69) were observed in T6 (1.5 % native strains).The birds fed the native probiotic strains at 150 g/ton (T6) had the highest MCHC compared to the other treatments, and there was no statistically significant difference between the commercial and native probiotics.The highest and lowest white blood cell counts was observed in native probiotic strains (200 g/ton) and control treatment, respectively, and difference between the CP1 and CP2 with native probiotics was not significant.These results are consistent with those a previous study that showed that the dietary addition of a probiotic did not affect the blood constituents, including hemoglobin concentrations, of ducklings (Djouvinov et al., 2005).Thongsong & Chavananikul (2008) demonstrated that probiotics significantly increased red blood cell counts, mean hemoglobin concentration, and mean corpuscular hemoglobin concentration of broilers.The white blood cell counts obtained in the present study are in agreement with the findings of Zare et al. (2007) and Fathi (2013), who obtained significantly higher WBC counts in broilers fed probiotics than in those fed a control diet.The manipulation of intestinal microbiota via the utilization of probiotics influences the development of the immune response.The mechanisms that mediate the effects of probiotics on the immune system are not known.However, it was shown that probiotics stimulate several subsets of immune system cells to produce cytokines, which in turn play an important role in the regulation of the immune response (Kabir et al., 2009).Haller et al. (2000) observed that gut microflora imbalance activated the intestinal mucosal immune system, causing an inflammatory reaction in the gut.The results of the present study suggest gut microflora imbalance and its effects on immune stimulation may increase the number of white blood cells.In the present study, the quails fed the diets containing probiotics has WBC compared with the control group.This could be due to their increased serum globulin level.Globulin serves as precursors of immunoglobulins (antibodies).The immune system of birds is composed of various cells and soluble elements (proteins) that need work together to create a protective immune response (Okuneye et al., 2016).

Effect of probiotics on gut microbial population
As is shown in Table 7, all the probiotics fed in this study influenced (p<0.05) both Lactobacillus bacteria and E. coli populations in the cecal and small intestine content samples.The largest Lactobacillus bacterial population was observed in the cecal and small intestine contents of T6 birds (fed 150 g/ton native probiotics).Interestingly, the lowest E. coli populations were observed in the treatments containing different concentrations of the native probiotics (50, 150 and 200 g/ton), followed by T5 and commercial probiotics (CP1 and CP2).These results are in accordance with those of Kizerwetter-Swida & Binek (2009) who indicated that the Lactobacillus salivarius decreased the number of Clostridium perfringens and Salmonella Enteritidis in the group of chickens treated with Lactobacillus.Yu et al. (2007) evaluated the effects of probiotic Lactobacillus reuteri strain Pg4 on the performance and intestinal characteristics of broilers, and reported that the Lactobacillus populations in the ileum, crop, and cecum of the broilers fed probiotics were higher than those in the control group.Other studies have also shown the potential of probiotics to populate the gut microflora of broiler chickens with beneficial bacteria and to supress the growth potentially pathogenic bacteria (Vicente et al., 2008).Values are expressed as means ± standard error of the mean.A-d Values followed by different letters on the same row indicate significant differences (p<0.05).CP = commercial probiotic, NP = native probiotic.

ConCluSIon
It is concluded that the supplementation of the combination of four native Lactobacillus strains at a concentration of 150 g/ton feed effectively enhanced the growth performance (BWG and FCR) and blood biochemical parameters of Japanese quails during the starter, finisher, and overall rearing periods.The results show that the mixture of four Lactobacillus strains has a probiotic potential in Japanese quails.Salehi Jouzani G, Shayegh J

Table 2 -
Effects of commercial and native probiotics on the performance of Japanese quails

Table 3 -
Effects of commercial and native probiotics on the performance of Japanese quailsValues are expressed as means ± standard error of the mean.A-dValues followed by different letters on the same row indicate significant differences (p<0.05).FI= Feed intake, BWG= Body weight gain, FCR= Feed conversion ratio, CP = commercial probiotic, NP = native probiotic

Table 4 -
Effects of commercial and native probiotics on selected serum biochemical parameters of Japanese quailsValues are expressed as means ± standard error of the mean.A-dValues followed by different letters on the same row indicate significant differences (p<0.05).CP = commercial probiotic, NP = native probiotic

Table 5 -
Effects of commercial and native probiotics on selected serum biochemical parameters of Japanese quails A-d Values are Mean s ± S.E.M with different letters on the same row implies significant differences (p<0.05).HDL=High density lipoprotein, LDL=Low density lipoproteinSiadati SA,

Table 6 -
Effects of commercial and native probiotics on hematological parameters of Japanese quailsValues are expressed as means ± standard error of the mean.A-dValues followed by different letters on the same row indicate significant differences (p<0.05).RBC = red blood cells, WBC = white blood cells, MCHC = mean cell hemoglobin concentration, Hb = hemoglobin, CP = commercial probiotic, NP = native probiotic

Table 7 -
Effects of commercial and native probiotics on viable cell counts of microflora in the cecal and small intestine contents of Japanese quails