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Previous studies on probiotics in chickens have shown varying efficacy, and the use of phage as a supplement for improving chicken performance and health has gained interest. However, the combination of these supplements has not been thoroughly investigated. This study aimed to assess the effects of an Escherichia coli phage cocktail, both alone and in combination with probiotics, on specific gut populations, lipid profiles, heterophil-to-lymphocyte ratio, serum immunoglobulins, villus height to crypt depth ratio, and relative organ weights in chickens at day 21 and 35. The experiment followed a completely randomised design with factorial arrangements, consisting of three levels of phage cocktail dosage (0 g/kg, 1 g/kg, and 2 g/kg) and two levels of probiotics dosage (0 g/kg and 1 g/kg), with a total of 288 one-day-old male Cobb 500 broilers. Our study demonstrated that supplementing chicken diets with 1 g/kg phage cocktail and 1 g/kg probiotics resulted in significant (p < 0.05) improvements in growth performance and reduced Clostridium perfringens populations. It also reduced triglyceride levels and did not cause physiological stress, as indicated by the heterophil-to-lymphocyte ratio. Overall, this study provides important insights into the potential benefits of combining phages and probiotics in broiler chicken diets, which have not been extensively studied.

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p < .05) and T2 diets (p < .05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca.

Condensed tannin (CT) of varying molecular weights (MWs) may affect rumen microbial fermentation by shifting composition of fungal community. In this study the effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) from Leucaena leucocephala hybrid-Rendang (LLR) on the fungal mass and composition of fungal community were determined using molecular approaches. The results showed that the total fungi biomass decreased (p < .05) with the addition of higher-MWs CT fractions F1, F2 and F3 with values 35.3 µg/mL, 34.6 µg/mL and 39.3µg/mL, respectively, when compared to that of the control sample (without CTs) at 52.1 µg/mL. Sequencing of the polymorphic internal transcribed spacer 1 (ITS-1) by high-throughput sequencer, recovered mean of 109,190 sequences per-sample which comprised of a number of operation taxonomy units (OTUs) ranging from 22 to 38 and were classified into 7 genera. Piromyces 4 was the dominant genus in the control representing ∼70% of the sequences. Relative abundance of the Piromyces 4 increased significantly (p < .05) with increasing MWs of the CT fractions, however the second dominant Neocallimastigaceae was significantly (p < .05) reduced. This study demonstrated that CT fractions of different MWs could decrease the population mass as well as alter the rumen fungal community structure and diversity, and the effect was more pronounced for higher-MWs CTs. Hence, the shift in fungal diversity, accompanied by changes in population mass would influence fibre digestibility in the rumen in the presence of high MW CTs.

Phytase activity and growth of anaerobic rumen bacterium, Mitsuokella jalaludinii were investigated by semi-solid state fermentation. Carbon source (rice bran, yam and cassava), nitrogen sources (soya bean, offal meal, fish meal and feather meal) and growth factors (hemin, L-cysteine hydrochloride and minerals) were evaluated in a one-factor-at-a-time approach. Rice bran and fish meal produced better growth and phytase enzyme activity. The removal of L-cysteine hydrochloride and minerals significantly decreased (p<0.05) phytase activity from 1178.72 U to 446.99 U and 902.54 U, respectively. The response surface methods (RSM) was conducted to optimize the phytase production and the results showed the combination of 7.7% of rice bran and 3.7% of fish meal in semi-solid state fermentation gave the highest phytase activity. Maximum phytase production and optimum growth of bacteria were detected at 12 h incubation in both MF medium (control) and agro-medium. In this agro-medium, M. jalaludinii produced 2.5 fold higher phytase activity compared to MF medium.

Phytase is frequently used as a feed supplement to improve availability of P in poultry. Mitsuokella jalaludinii, isolated from the rumen of cattle showed high phytase activity capable of hydrolysing phytate compounds. In this study, the efficacy of freeze-dried M. jalaludinii phytase was evaluated as a feed supplement for broilers fed low-available P (aP) diet. The results showed that freeze-dried M. jalaludinii cells with functional phytase activity significantly enhanced (p<0.05) growth performance and feed conversion ratio of broilers fed low-aP diet and the apparent digestibility of dry matter and crude protein of the feed. Phytase supplementation also enhanced retention of P, Ca, Mn, Cu, and Zn as well as the Ca and Mn content in tibia of broilers. The Ca, Mn and Zn concentrations in plasma of broilers fed low-aP diet were not significantly different than those fed normal diet, but the levels of P and Cu were significantly lower (p<0.05) than the control.

Repetitive sequence-based PCR (rep-PCR) is a distinctive typing approach that is used to differentiate between bacterial strains. This method is also useful for studying bacterial diversity from different sources. In this study, four rep-PCR which are enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), BOX-PCR, repetitive extragenic palindromic PCR (REP-PCR) and polytrinucleotide (GTG)5-PCR were evaluated for differentiation of eighteen Escherichia coli isolates to correct source based on part of intestine and age. These isolates were recovered earlier from ileal and caecal mucosal contents of chickens at a different age. The purpose of this study was to investigate the efficacy of four rep-PCR methods and composite of rep-PCR patterns to differentiate E. coli isolates to original sources of part of intestines and age based on the D index (discriminatory power determined based on Simpson’s index of diversity calculated at similarity coefficient of 90%). The (GTG)5-PCR had the highest D index (0.9804) for part of intestine and age factors. The similar D index was observed in the composite of rep-PCR patterns. The lowest D index was observed in ERIC- and BOX-PCR at 0.9020 and 0.8039 for part of intestine and age factors, respectively. (GTG)5-PCR was also the most discriminative rep-PCR observed due to its ability to cluster 14I 3E and 14I 2X isolates, and 14C 1E and 14C 3E isolates correctly in part of intestine and age factors. It was concluded that (GTG)5-PCR is a promising tool for discriminating E. coli isolates extracted from chicken intestines.

Aims: To evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community in vitro. Methods and Results: The effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) on protozoal population and community were evaluated in vitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P < 0·05) decrease the number of ciliate protozoa. The real-time PCR analysis showed that the total protozoa was significantly (P < 0·05) lower in F0 and all CTs with fractions F1 and F2 having the lowest value. High-throughput sequencing of the partial 18S rRNA gene showed that the genus Entodinium significantly (P < 0·05) decreased with increasing MWs of CT, whereas Anoplodinium-Diplodinium were significantly (P < 0·05) increased. Inclusion of the highest MW CT fraction F1 decreased the relative abundance of the minor genera such as Eudiplodinium and Polyplastron compared to the control and CT fractions F2–F5. Conclusion: CTs of differing MWs could reduce and alter the rumen protozoa population in vitro. This effect was more pronounced for higher-MW CTs. Significance and Impact of the Study: The high MW CTs should be considered as a feed supplement in the ruminant diet to reduce the protozoal population which are known to be associated with methanogens as a means to mitigate methane production in the rumen.

The potential use of fed-batch cultivation (FBC) for improvement of the production of Lactobacillus salivarius I 24 biomass for subsequent use as probiotics was studied using a 2-L stirredtank bioreactor. Three different constant feeding rates (0.1, 0.05, and 0.033 L/h) were applied in FBCs and their effect on carbon metabolism was evaluated. The carbon flux for cell built-up with reduction in lactic acid synthesis was observed in the fed-batch as compared to the batch cultivation mode. The viable cell number obtained in the constant FBC (CFBC) operated at a feeding rate of 0.05 L/h was 8 times higher (10.7×1010 CFU/mL) than that recorded in the batch cultivation. This gave the viable cell yield based on glucose consumed for CFBC of 26 times higher (11.3×1012 CFU/gGlucose) than the batch cultivation. This study demonstrated CFBC, which is simple with minimal use of process control equipment, has an industrial potential for improvement of probiotic production.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1–F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3–F5. Although inclusion of F0 and CT fractions (F1–F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW.

A 16-wk feeding experiment was conducted to investigate the effects of a prebiotic, isomaltooligosaccharide (IMO), a probiotic, PrimaLac®, and their combination as a synbiotic on the chemical compositions of egg yolks and the egg quality of laying hens. One hundred and sixty 16-wk-old Hisex Brown pullets were randomly assigned to 4 dietary treatments: (i) basal diet (control), (ii) basal diet + 1% IMO (PRE), (iii) basal diet + 0.1% PrimaLac® (PRO), and (iv) basal diet + 1% IMO + 0.1% PrimaLac® (SYN). PRE, PRO, or SYN supplementation not only significantly (P < 0.05) decreased the egg yolk cholesterol (24- and 28-wk-old) and total saturated fatty acids (SFA; 28-, 32-, and 36-wk-old), but also significantly (P < 0.05) increased total unsaturated fatty acids (UFA; 28-, 32-, and 36-wk-old), total omega 6 and polyunsaturated fatty acids (PUFA), including linoleic and alpha-linolenic acid levels in the eggs (28-wk-old). However, the total lipids, carotenoids, and tocopherols in the egg yolks were similar among all dietary treatments in the 24-, 28-, 32-, and 36-wk-old hens. Egg quality (Haugh unit, relative weights of the albumen and yolk, specific gravity, shell thickness, and yolk color) was not affected by PRE, PRO, or SYN supplementation. The results indicate that supplementations with IMO and PrimaLac® alone or in combination as a synbiotic might be useful for improving the cholesterol content and modifying the fatty acid compositions of egg yolk without affecting the quality of eggs from laying hens between 24 and 36 wk of age.

Background: Chicken gut microbiota has paramount roles in host performance, health and immunity. Understanding the topological difference in gut microbial community composition is crucial to provide knowledge on the functions of each members of microbiota to the physiological maintenance of the host. The gut microbiota profiling of the chicken was commonly performed previously using culture-dependent and early culture-independent methods which had limited coverage and accuracy. Advances in technology based on next-generation sequencing (NGS), offers unparalleled coverage and depth in determining microbial gut dynamics. Thus, the aim of this study was to investigate the ileal and caecal microbiota development as chicken aged, which is important for future effective gut modulation. Material and methods: Ileal and caecal contents of broiler chicken were extracted from 7, 14, 21 and 42-day old chicken. Genomic DNA was then extracted and amplified based on V3 hyper-variable region of 16S rRNA. Bioinformatics, ecological and statistical analyses such as Principal Coordinate Analysis (PCoA) was performed in mothur software and plotted using PRIMER 6. Additional analyses for predicted metagenomes were performed through PICRUSt and STAMP software package based on Greengenes databases. Results: A distinctive difference in bacterial communities was observed between ilea and caeca as the chicken aged (P < 0.001). The microbial communities in the caeca were more diverse in comparison to the ilea communities. The potentially pathogenic bacteria such as Clostridium were elevated as the chicken aged and the population of beneficial microbe such as Lactobacillus was low at all intervals. On the other hand, based on predicted metagenomes analysed, clear distinction in functions and roles of gut microbiota such as gene pathways related to nutrient absorption (e.g. sugar and amino acid metabolism), and bacterial proliferation and colonization (e.g. bacterial motility proteins, two-comp

Normal gut microflora plays an important function in nutrient absorption, feed digestion and modulation of immune response in the birds. Earlier, gut microflora were studied based on traditional cultivation and molecular fingerprinting methods. However, low sensitivity, coverage, accuracy and reproducibility have shift researcher to high-throughput next-generation sequencing (HT-NGS) technology, which enable a better gut microflora study approach due to high coverages and depths of sequencing. Briefly, chicken gut microflora of the ilea and caeca were studied at various time points, day 7, 14, 21 and 42-day-old. DNA extraction was performed, amplified based on V3 hypervariable region of 16S rRNA, sequenced and analysed. The communities of broiler gut microflora in caeca were significantly different in comparison to ilea (P < 0.001). In addition, their communities subsequently became more diverse as chicken aged from day 7, 14, 21 until 42-day-old. The caeca had more diverse gut microflora compared to ilea, with low communities of Lactobacillus and high communities of Clostridium. Based on gene predictions study, gene pathways related to nutrient absorption (e.g. amino sugar nucleotide sugar metabolism), and bacterial proliferation and colonisation (e.g. two-component system, bacterial motility proteins and secretion system) were observed to be dominant in ilea and caeca, respectively (P < 0.05). In conclusion, gut microflora of ilea and caeca were unique in which they performed different roles and functions. The low presence of beneficial microbes (e.g. Lactobacillus) and high presence of potential pathogens (e.g. Clostridium) in the broiler intestines highlighted the needs of gut modulation to further improve the gut health.

This study investigated the effects of prebiotic, probiotic and synbiotic on the performance, cecal microbial diversity and yolk cholesterol content of laying hens. One hundred and sixty Hisex Brown hens, aged 20 weeks of age, were randomly allotted to four diets: basal diet (control), basal diet + 1% prebiotic isomaltooligosaccharide (IMO) (prebiotic diet), basal diet + 0.1% probiotic PrimaLac® (probiotic diet), and basal diet + 1% IMO + 0.1% PrimaLac® (synbiotic diet). Cecal microbial populations and community of hens were assessed by 16S rRNA gene sequencing and quantitative real-time PCR (qPCR) methods, respectively, at 36 weeks of age. Cholesterol content of egg yolk was analyzed by gas chromatography at 24, 28, 32 and 36 weeks of age. The results showed that the body weight gain, feed intake, feed conversion ratio (FCR), egg production, egg weight and egg mass were significantly (P<0.05) improved in hens fed prebiotic, probiotic or synbiotic diet from 20-36 weeks of age. Sequencing results revealed that the prebiotic, probiotic or synbiotic feeding increased the abundance of Lactobacillusand decreased the abundance of Enterobacteriaceae. The abundance of Bifidobacterium was increased in prebiotic- or synbiotic-supplemented hens. At 36 weeks of age, qPCR results showed that prebiotic, probiotic or synbiotic feeding significantly (P<0.05) increased the populations of Lactobacillus and Bifidobacterium, but decreased the populations of Enterobacteriaceae and Escherichia coli. Supplementation of prebiotic, probiotic or synbiotic had significantly (P<0.05) lowered the yolk cholesterol content of laying hens at 24 and 28 weeks of age, but not at 32 and 36 weeks of age. The results of the study indicate that prebiotic, probiotic or synbiotic supplementation had similar positive effects on the performance, cecal microbial diversity and cholesterol content of egg yolk in laying hens at 20-36 weeks of age.

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimized and characterized. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.