ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2023, 28(1): 47–61 DOI: 10.53541/jams.vol28iss1pp47-61 Received 20 July 2022 Accepted 22 August 2022 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis Mai A S Al-Balushi1, Yasmin El Tahir1, Muhammad N Asi1, Hani M. El-Zaiat1,2, Mohammed A Al-Abri1, Kaadhia Al-Kharousi1 and Waleed Al-Marzooqi1,* Waleed Al-Marzooqi1,*( ) walmar@squ.edu.om,1Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Sultanate of Oman, 2De- partment of Animal Production, Faculty of Agriculture, University of Alexandria, Alexandria, Egypt. Introduction Local chicken production has contributed signifi-cantly as sources of protein, food security and source of income in communities with limited re- sources (Al-Jumaili et al., 2020). Local chicken produc- tion is among the farming activities in the rural com- munities of Oman that provides opportunities for food security and income for many rural families (MAF, 2013). The Cobb 500 broiler chickens are the world’s foremost broiler breed, adapting well to warmer weathers; has the best growth rate and an ability to thrive on low density and less costly diets (Dessie et al., 2017). However, Cobb 500 broiler chickens has become very popular in Oman where feed costs are inflated saving poultry farmers a fortune. An improvement in local chicken productivity would be highly valuable in the enhancement of the so- cioeconomic and nutritional status of farmers. Several studies have shown the beneficial effect of the microbial community in the gastrointestinal tract of the host and their important contributions in many roles such as in nutrient absorption, feed digestion, and immune system (Gong et al., 2002). Chickens’ gastrointestinal (GI) tracts are home to a rich and complex microbiota that aids in digestion and nutrition absorption, as well as immune system devel- opment and pathogen exclusion (Shang et al., 2018). The symbiotic relationship between the host and the micro- biota is critical for the health and production of chickens. The age of the chickens and their position in the diges- tive tract have a big impact on the diversity of their gut تقييم اجملتمع البكتريي املعوي للدجاج العماين احمللي ودجاج الكوب 500 الالحم 16S rDNA الذي مت تربيته يف منزل مفتوح ابستخدام التحليل القائم على مي أ س البلوشي1، ايمسني الطاهر1، حممد ن عاصي1، هاين م الزايت1، 2، حممد العربي1، كاذية اخلروصي1، وليد املرزوقي1،* Abstract. Little is known about how the intestinal bacterial microbiota differs among different strains of chickens raised in an open sided house, predominantly those with lower growth rates, such as Indigenous chickens. Ninety-one- day-old chicks of each strain of chickens were raised in an open-sided house system and fed a conventional corn-soy- bean meal diet from Day 0–35 days of age. The objective of this study was to assess the relative abundance of bacteria microbiota identified in the intestinal tract of local Omani and Cobb 500 broiler chickens raised in an open-sided house system using 16S rDNA-based analysis. The results obtained showed the diversity of bacterial populations in different intestinal regions of two chicken strains. Bacilli were found in higher numbers and reached 98.8% of the bacteria in the duodenum on Day 5 in Cobb 500 versus 72.5% in the Omani chickens. Local Omani chickens had significantly higher numbers of Clostridia at an early age period. On Day 5 Clostridia comprised 13.1% of the bacteria in the duodenum of local Omani chickens, versus only 0.062% in the Cobb 500. The relative abundance of the bacterial microbiota differed significantly (p <0.05) across different intestinal segments of the two strains of chickens, suggesting that each region generated its bacterial community with different relative abundances. Keywords: Omani, chicken, 16S rDNA, intestine, microbiota, house. امللخــص: ال يعــرف ســوى القليــل عــن كيفيــة اختــاف امليكــروابت البكترييــة املعويــة بــني ســاالت خمتلفــة مــن الدجــاج الــي تــرىب يف منــزل مفتــوح ، يف الغالــب تلــك الــي لديهــا معــدالت منــو أقــل ، مثــل الدجــاج احمللــي. مت تربيــة واحــدا وتســعني مــن الكتاكيــت البالغــة مــن العمــر يومــا مــن كل ســالة مــن الدجــاج يف نظــام منــزل مفتــوح اجلانــب وتغذيــة نظــام غذائــي تقليــدي لوجبــة الــذرة وفــول الصــواي مــن اليــوم 0-35 يومــا مــن العمــر. كان اهلــدف مــن هــذه الدراســة هــو تقييــم الوفــرة النســبية للميكــروابت البكترييــة الــي مت حتديدهــا يف األمعــاء للدجــاج العمــاين احمللــي ودجــاج Cobb 500 الاحــم الــذي مت تربيتــه يف نظــام منــزل مفتــوح اجلانــب ابســتخدام التحليــل القائــم علــى 16S rDNA. أظهــرت النتائــج الــي مت احلصــول عليهــا تنــوع اجملموعــات البكترييــة يف مناطــق معويــة خمتلفــة مــن ســالتني مــن الدجــاج. مت العثــور علــى العصيــات أبعــداد أكــرب ووصلــت إىل ٪98.8 مــن البكتــرياي يف االثــي عشــر يف اليــوم 5 يف كــوب 500 مقابــل ٪72.5 يف الدجــاج العمــاين. كان لــدى الدجــاج العمــاين احمللــي أعــداد أكــرب بكثــري مــن كلوســريداي يف فــرة مبكــرة مــن العمــر. يف اليــوم اخلامــس ، شــكلت كلوســريداي ٪13.1 مــن البكتــرياي يف االثــي عشــر مــن الدجــاج العمــاين احمللــي ، مقابــل ٪0.062 فقــط يف Cobb 500. اختلفــت الوفــرة النســبية للميكــروابت البكترييــة اختافــا كبــريا )p <0.05( عــرب شــرائح معويــة خمتلفــة مــن ســالتني مــن الدجــاج ، ممــا يشــري إىل أن كل منطقــة لديهــا جمتمعهــا البكتــريي اخلــاص هبــا بوفــرة نســبية خمتلفــة. الكلمات املفتاحية: عماين، دجاج، 16S rDNA، أمعاء، ميكروبيوات، منزل. 48 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis microbiota. Normal gut morphology and integrity are important in the maintenance of intestinal microbial ho- meostasis in the prevention of infection and promoting digestion and absorption of nutrients (Jimoh et al., 2017). Evaluation of the microbial diversity and intestinal development of different strains of chickens has become widely recognized (Glendinning et al., 2019; Richards-Ri- os et al., 2020). However, there is a limited information about the effect of house type on the composition and succession of intestinal microbial communities in birds raised in open-sided housing systems. The majority of small and medium scale farmers in Middle East coun- tries, such as Oman uses an open-sided housing system to grow their chickens. A greater understanding of the chicken gut function and microbiology can provide an opportunity for the improvement of chicken’s health and production especially those with various growth rates, such as the local Omani chickens. The use of modern high-throughput sequencing ap- proaches, that involve analyzing the structure of bac- terial communities by determining the characteristic features of the microbial DNA extracted from the com- munity samples, are a powerful tool that has led to im- portant new insights into the biological and ecological roles of the GI Microbiota (Shang et al., 2018; McLaren et al., 2019). The objective of this study was to assess the relative abundance of bacteria microbiota identified in the intestinal tract of local Omani and Cobb 500 broil- er chickens raised in an open-sided house system from 0 to 35 days of age using 16S rDNA-based analysis. Materials and Methods Ethical Approval All experimental work was conducted at the Poultry Research Unit at the Agricultural Experiment Station in accordance with the experimental unit policy on ani- mal welfare and the requirements of the procedures in- volving animals/birds and their care were conducted in conformity with international laws and policies at Sultan Qaboos University. Birds Housing, Diets and Sample Collection One hundred and fifty 1-day-old chicks of two strains of chickens: local Omani and Cobb 500 broiler chicken were obtained from commercial hatcheries at Barka. On arrival, all chicks were scrutinized to ensure that they were free of abnormalities and early signs of sickness. Before the trial, open-sided house unit, cages, feeders, and drinkers were disinfected through fumigation. In addition, strict hygiene and biosecurity measurements were implemented. The open-sided house was built from a galvanized iron shed with profiled steel shed roofing which was naturally ventilated. Chicken mesh panels were put on all sides, along with one-meter-high block work protection. To assist circulate the air, four sets of electric wall fans were used. Shade cloth were used to screen direct sunrays during midday. There were 15 rep- licates for each strain of chicken with each replicate cage containing six birds (a total of 90 birds/strain). Birds per replicate combinations were randomly allocated. Chicks of both strains were fed a nonmedicated convention- al corn-soybean meal diet from Day 0–35 days of age. The composition of experimental diet is as described by Al-Marzooqi et al. (2019). Feed was available ad libitum. The house temperature maintained at 33 °C on day 1 and reduced by 3 °C each week to reach a constant 22 °C. The lighting program was 23L: 1D. At 5, 15, 25, and 35 days of age, one bird per cage for each strain of chicken was randomly selected. Birds with the body weight closest to the average from each cage were selected, marked, and kept in their cage until being euthanized. Then, the selected bird was injected with a mixed dose of ketamine 10% and xylazine 20% intramus- cularly to put the bird into a deep sedation and anaesthe- sia. After dissection, intestinal tracts were removed from the carcasses immediately and luminal contents of the duodenum, jejunum, ileum and cecum were aseptically collected into a labeled sterile 15-mL tube. The entire process of collecting intestinal contents was performed on a thoroughly cleaned workbench and required less than 30 min. Samples were placed on ice and immedi- ately transported to the laboratory and stored in −80 °C freezer until analysis. DNA Extraction, 16S rDNA Gene Amplicon Production and High-Throughput Sequencing Total DNA was extracted from contents of each luminal content samples (duodenum, jejunum, ileum, and ce- cum) using a QIAamp DNA Stool Mini Kit (QIAGEN, CA, Hamburg, Germany) according to the manufactur- er’s instructions. The DNA concentration was evaluat- ed by measuring optical density using Nano-Drop 2000 (Thermo Electron Corporation, Waltham, MA, USA) at a wavelength of 260 and 280 nm. The integrity of the DNA extracts was assessed visually using 1.0% agarose gel (containing ethidium bromide) electerophoresis. The variable regions V3-V4 of the 16S rDNA gene were amplified and sequenced. The PCRs were per- formed in triplicate in a total volume of 20 μL contain- ing 5 μM of each primer, 10 ng of DNA template, 4 μL 1× FastPfu buffer, 2.5 mM dNTPs, and 0.4 μL of FastPfu polymerase (TransGen Biotech, Beijing, China). PCR conditions were as follows: Initial denaturation at 95 °C for 2 min; followed by 25 cycles of denaturation 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s and then, a final extension at 72 °C for 5 min. PCR products were separated on 2% agarose gels, and purified using the DNA gel extraction kit (Axygen Sci- entific Inc., Union City, CA, USA). Amplicons produced form different intestinal luminal content samples were sent to a commercial company (BGI Genomic Lab, Tai Po Industrial Zone, New Territories, Hong Kong, China) for sequencing on the Illumina MiSequencing platform. 49Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi Sequencing Analysis All the raw sequences obtained from Illumina Miseq were firstly filtered for quality control to get operation- al sequences. The quality control and analysis of the sequences were performed using the software Quanti- tative Insights into Microbial Ecology (QIIME, v1.8.0) (Caporaso et al., 2010). The paired-end reads from the DNA fragments were merged using FLASH (Magoc and Salzberg, 2011). Sequences data was treated by read trimming and identification of V3-V4 sequences and set of sequences with ≥97% identity were defined as an operational taxonomic unit (OTU). The UCLUST (Edgar, 2010), clustering method was used to cluster operational taxonomic units. The defined OTUs were assigned to different taxonomic levels (phylum, class, genus and families) at a cutoff of 97%. The clustered OTUs were also used to construct the rarefaction curves and calculate the Shannon and Simpson diversity indi- ces, abundance-based coverage estimators (ACE), Chao 1 richness, and coverage percentage by Good’s method. Bioinformatics and Statistical Analysis Bioinformatics and statistical analyses were performed using the QIIME and R package (v3.1.1). The alpha-di- versity indices (Chaol, ACE, Shannon diversity index, and Simpson index) were calculated to establish the relative abundance and diversity of the sequences. Beta diversity was determined using unweighted Unifrac distance metrics to evaluate the structure and distribu- tion of the microbial genetic communities among the samples. Differences in the Unifrac distances for pair- wise comparisons among groups were calculated us- ing Student’s t-test and the Monte Carlo permutation test with 1000 permutations. Metastats and R package (v3.1.1) (James et al., 2009) were used to compare and determine which taxonomic groups were significantly different between groups of samples based on intestinal segments and age period. The differences were consid- ered to be significant at p < 0.05. The obtained p-value was adjusted by a Benjamini–Hochberg false discovery rate correction (Function ‘p.adjust’ in the stats package of R (v3.1.1)). Results Bacterial Taxonomic Composition of Duodenum Bacteria were classified according to their respective Phylum and Class, found in the duodenum of broiler chickens at different age periods, are presented in Ta- ble 1. Seventeen bacterial florae at the class level were detected in the duodenum. Of the 30248 detected se- quences, the most abundant Class was Bacilli, at 93.4 % of the total sequences. Clostridia accounted for 2.9 % out of the total detected sequences. Actinobacteria and Proteobacteria sequences represent 1.01 % and 0.694 %, respectively of the total sequences. Across different age periods Bacilli were considered the dominant group with 98.9 % at Day 5, 94.6 % at Day 15, 89.9 % at Day 25 and 88.7 % at Day 35 of sequences. Clostridia sequences fluctuated from 0.062 % at Day 5, 1.78 % at Day 15, 4.61% at Day 25, and 6.00 % at Day 35. Chloroplast were detect- ed at very small percentage across all age periods except at Day 25 of age was detected at 3.39 %. Both Actinobac- teria and Proteobacteria group-related sequences were detected at smaller percentage across all age periods. Bacterial Taxonomic Composition of the Jejunum Bacteria were classified according to their respective Phylum and Class, found in the jejunum of broiler chick- ens at different ages are presented in Table 2. Seventeen bacterial florae at the Class level were detected in the jejunum. Of the 28678 reads, Bacilli were the most abun- dant, at 79.6 % of the total sequences. Clostridia account- ed for 7.61% of the total sequences. Actinobacteria and Proteobacteria sequences represented 4.46 % and 4.44%, respectively of the total sequences. Across different age periods Bacilli were the dominant group, representing 86.4 % at Day 5, 93.3 % at Day 15, 91.5 % at Day 25 and 40.1 % at Day 35 of sequences. Clostridia sequences var- ied from Day 5: 4.20%, Day 15: 4.33%, Day 25: 0.859% and Day 35: 23.60%. Actinobacteria sequences were Day 5: 2.33%, Day 15: 0.318%, Day 25: 0.109%, and Day 35: 17.4%, while sequences related to Gammaproteobacteria were Day 5: 0.015%, Day 15: 0.625%, Day 25: 4.94%, and Day 35: 12.0%. Bacterial Taxonomic Composition of the Ileum Bacteria were classified according to their respective Phylum and Class, found in the ileum of broiler chick- ens at different ages are presented in Table 3. Seventeen bacterial florae at the Class level were detected in the ile- um. Of the 31007 reads, Bacilli were the most abundant, at 96.1 % of the total sequences. Only few of Clostridia (0.239%) related to the total sequences were detected. Actinobacteria and Proteobacteria represented a very small percentage of 0.893% and 0.232%, respectively, of the total sequences. Across different age periods Bacilli were the dominant group, representing 91.1 % at Day 5, 99.0 % at Day 15, 99.6 % at Day 25 and 95.2 % at Day 35 of sequences. Clostridia sequences fluctuated from 0.565% at Day 5, 0.140% at Day 15, 0.107% at Day 25, and 0.050% at Day 35. Proteobacteria group-related sequences were detected at smaller percentages across all age periods. Bacterial Taxonomic Composition of the Cecum Bacteria were classified according to their respective Phylum and Class, found in the cecum of broiler chick- ens at different ages are presented in Table 4. Seventeen bacterial florae at the Class level were detected in the cecum. Of the 28035 reads, Clostridia were the most abundant, at 63.4 % of the total sequences. Bacilli were detected at 13.1% of the total sequences. Actinobacte- ria and Proteobacteria sequences represented 6.01% and 50 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis 1.75%, respectively of the total sequences. Across differ- ent age periods Clostridia were the dominant group, rep- resenting 40.8% at Day 5, 73.5% at Day 15, 75.0% at Day 25 to 62.6% at Day 35 of the sequences. Bacilli sequences fluctuated from 16.3% at Day 5, 21.0% at Day 15, 11.8% at Day 25, and 2.32% at Day 35. Actinobacteria sequences were Day 5: 22.0%, Day 15: 0.809%, Day 25: 1.18%, and Day 35: 0.0%. Proteobacteria group-related sequences were detected at smaller percentages across age periods. Table 1. Abundance of bacterial 16S rDNA sequences (n=30248) identified from the duodenum flora of 500 Cobb broiler chicken. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Actinobacteria Coriobacteriia Thermoleophilia 43 (0.531) 0 (0) 0 (0) 191 (2.32) 3 (0.036) 0 (0) 46 (0.531) 0 (0) 0 (0) 0 (0) 15 (0.285) 0 (0) Bacteroidetes Cyanobacteria Firmicutes Proteobacteria Tenericutes Thermi Total Bacteroidia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Deinococci Other 0 (0) 0 (0) 34 (0.420) 8005 (98.9) 5 (0.062) 0 (0) 5 (0.062) 1 (0.012) 0 (0) 3 (0.037) 0 (0) 0 (0) 1 (0.012) 0 (0) 8097 0 (0) 0 (0) 20 (0.243) 7777 (94.6) 146 (1.78) 2 (0.024) 36 (0.438) 27 (0.328) 0 (0) 11 (0.134) 5 (0.061) 3 (0.036) 3 (0.036) 0 (0) 8224 0 (0) 2 (0.023) 293 (3.39) 7785 (89.9) 399 (4.61) 0 (0) 25 (0.289) 6 (0.069) 0 (0) 84 (0.970) 6 (0.069) 0 (0) 11 (0.127) 0 (0) 8657 8 (0.152) 175 (3.32) 0 (0) 4675 (88.7) 316 (6.00) 42 (0.797) 0 (0) 0 (0) 1 (0.019) 0 (0) 0 (0) 32 (0.607) 0 (0) 6 (0.114) 5270 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day Table 2. Abundance of bacterial 16S rDNA sequences (n=28678) identified from the jejunum flora of Cob 500 broiler chickens. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Actinobacteria Coriobacteriia Thermoleophilia 154 (2.33) 0 (0) 0 (0) 27 (0.318) 0 (0) 0 (0) 8 (0.109) 0 (0) 0 (0) 1090 (17.4) 0 (0) 0 (0) Bacteroidetes Cyanobacteria Firmicutes Proteobacteria Tenericutes Thermi Total Bacteroidia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Deinococci Other 0 (0) 0 (0) 427 (6.45) 5718 (86.4) 278 (4.20) 0 (0) 38 (0.574) 5 (0.076) 0 (0) 0 (0) 1 (0.015) 0 (0) 0 (0) 0 (0) 8584 0 (0) 0 (0) 64 (0.755) 7906 (93.3) 367 (4.33) 13 (0.153) 38 (0.448) 10 (0.118) 0 (0) 0 (0) 53 (0.625) 0 (0) 0 (0) 0 (0) 7872 0 (0) 0 (0) 175 (2.39) 6705 (91.5) 63 (0.859) 1 (0.014) 16 (0.218) 0 (0) 0 (0) 0 (0) 362 (4.94) 0 (0) 0 (0) 0 (0) 7582 0 (0) 0 (0) 0 (0) 2504 (40.1) 1475 (23.6) 429 (6.87) 0 (0) 0 (0) 0 (0) 0 (0) 751 (12.0) 0 (0) 0 (0) 0 (0) 7217 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day 51Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi Differences of Bacterial Communities among Samples from Different Intestinal Segments The p-value distribution of 16S rDNA gene sequence li- braries used to compare the quantitative differences of microbial communities among samples from different intestinal segments of broiler chickens are presented in Table 5. Statistical comparisons of the libraries showed that the composition of the Duodenum-Jejunum, Duo- denum-Ileum, Cecum-Duodenum, Cecum-Ileum Ce- cum-Jejunum bacterial microbiota differed significantly (p < 0.05), implying that each region established its own bacterial community. The number of Actinobacteria, Alphaproteobacteria, Bacilli, Bacteroidia, Betaproteo- Table 3. Abundance of bacterial 16S rDNA sequences (n=31007) identified from the ileum flora of Cobb 500 broiler chicken. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Proteobacteria Tenericutes Thermi Total Actinobacteria Coriobacteriia Thermoleophilia Bacteroidia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Deinococci Other 220 (2.44) 0 (0) 0 (0) 3 (0.033) 0 (0) 466 (5.16) 8222 (91.1) 51 (0.565) 1 (0.011) 55 (0.610) 4 (0.044) 0 (0) 0 (0) 1 (0.011) 0 (0) 0 (0) 0 (0) 9023 56 (0.655) 0 (0) 0 (0) 2 (0.023) 0 (0) 7 (0.082) 8458 (99.0) 12 (0.140) 0 (0) 5 (0.059) 3 (0.035) 0 (0) 0 (0) 1 (0.012) 0 (0) 0 (0) 0 (0) 8544 1 (0.013) 0 (0) 0 (0) 0 (0) 0 (0) 17 (0.227) 7467 (99.6) 8 (0.107) 0 (0) 1 (0.013) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 7494 0 (0) 87 (1.46) 0 (0) 40 (0.673) 7 (0.118) 0 (0) 5659 (95.2) 3 (0.050) 137 (2.30) 0 (0) 0 (0) 0 (0) 0 (0) 2 (0.034) 11 (0.185) 0 (0) 0 (0) 5946 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day Table 4. Abundance of bacterial 16S rDNA sequences (n=28035) identified from the cecum flora of Cobb 500 broiler chicken. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Actinobacteria Coriobacteriia Thermoleophilia 1535 (22.0) 25 (0.359) 1 (0.014) 57 (0.809) 21 (0.298) 2 (0.028) 93 (1.18) 0 (0) 0 (0) 0 (0) 114 (1.85) 0 (0) Bacteroidetes Cyanobacteria Firmicutes Proteobacteria Tenericutes Thermi Total Bacteroidia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Deinococci Other 35 (0.502) 12 (0.172) 1103 (15.8) 1134 (16.3) 2839 (40.8) 24 (0.344) 165 (2.37) 78 (1.12) 0 (0) 0 (0) 11 (0.158) 0 (0) 5 (0.072) 0 (0) 6967 32 (0.454) 13 (0.185) 92 (1.31) 1480 (21.0) 5177 (73.5) 24 (0.341) 63 (0.895) 58 (0.824) 1 (0.014) 0 (0) 22 (0.312) 1 (0.014) 0 (0) 0 (0) 7043 2 (0.025) 0 (0) 855 (10.9) 925 (11.8) 5902 (75.0) 2 (0.025) 83 (1.05) 5 (0.064) 0 (0) 0 (0) 1 (0.013) 0 (0) 0 (0) 0 (0) 7868 2010 (32.65) 27 (0.439) 0 (0) 143 (2.32) 3851 (62.6) 6 (0.097) 0 (0) 0 (0) 5 (0.081) 0 (0) 0 (0) 1 (0.016) 0 (0) 0 (0) 6157 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day 52 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis bacteria, Clostridia and Erysipelotrichia differed sig- nificantly across different intestinal segments (p < 0.05). Bacilli were the dominant 16S rDNA sequences in the duodenum, jejunum, and ileum libraries, whereas Clos- tridia were the dominant 16S rDNA sequences in the cecum libraries. Table 5. P-value distribution of 16S rDNA gene sequence libraries compared the abundance differences of microbial commu- nities among samples from different segments for cobb 500 broiler chicken. P-Value Class Duodenum- Jejunum Duode- num-Ileum Jejunum- Ileum Cae- cum-Duo- denum Caecum- Jejunum Caecum- Ileum 4C0d-2 Actinobacteria Alphaproteobacteria Bacilli Bacteroidia Betaproteobacteria Chloroplast Clostridia Coriobacteriia Deltaproteobacteria Epsilonproteobacteria Erysipelotrichi Flavobacteriia Gammaproteobacteria Lentisphaeria Mollicutes Sphingobacteriia Verrucomicrobiae 0.270 0.921 0.575 0.901 0.026 0.191 0.327 0.512 0.828 0.501 0.479 0.726 0.233 0.699 0.124 0.328 0.483 0.581 0.797 0.724 0.673 0.051 0.153 0.203 0.826 0.542 0.778 0.289 0.108 0.427 0.124 0.614 0.447 0.682 0.745 0.630 1.000 0.879 0.737 0.543 0.514 0.322 0.684 0.603 0.236 0.405 1 0.485 0.507 0.029 0.303 0.000 0.011 0.018 0.218 0.002 0.058 0.192 0.270 0.003 0.069 0.096 0.146 0.403 0.074 0.415 0.015 0.025 0.000 0.007 0.042 0.142 0.002 0.071 0.183 0.600 0.004 0.501 0.096 0.107 0.103 0.463 0.052 0.108 0.000 0.029 0.005 0.083 0.002 0.070 0.285 0.766 0.003 0.362 0.098 1 0.117 Table 6. P-value distribution of 16S rDNA gene sequence libraries compared the abundance differences of microbial commu- nities among sample from different ages period for Cobb 500 broiler chickens. P-Value Class Day 5-Day 15 Day 5-Day 25 Day 5 - Day 35 Day 15-Day 25 Day 15- Day35 Day 25- Day 35 4C0d-2 Actinobacteria Alphaproteobacteria Bacilli Bacteroidia Betaproteobacteria Chloroplast Clostridia Coriobacteriia Deltaproteobacteria Epsilonproteobacteria Erysipelotrichi Flavobacteriia Gammaproteobacteria Lentisphaeria Mollicutes Sphingobacteriia Verrucomicrobiae 0.005 0.054 0.678 0.299 0.165 0.027 0.871 0.130 1 0.592 0.501 0.123 0.342 0.342 0.247 0.046 0.235 0.924 0.317 0.158 0.746 0.875 0.226 0.288 0.035 0.758 0.621 0.497 0.347 0.347 0.198 0.004 0.036 0.914 0.344 0.051 0.283 0.854 0.290 0.171 0.069 0.511 0.111 0.249 0.676 0.499 0.299 0.226 0.016 0.112 0.669 0.750 0.614 0.031 0.848 0.733 0.342 0.043 0.652 0.501 0.556 0.475 0.361 0.361 0.199 0.748 0.178 0.695 0.738 0.532 0.147 0.806 0.659 0.248 0.080 0.907 0.501 0.217 0.228 0.369 0.561 0.337 0.191 0.012 0.304 0.913 0.678 0.922 0.464 0.856 0.556 0.660 0.193 0.648 0.332 1 0.347 1.000 0.760 53Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi Differences of Microbial Communities among Samples The p-value distribution of 16S rDNA gene sequence li- braries comparing the quantitative differences of micro- bial communities among samples from broiler chickens at different age groups are presented in Table 6. Statisti- cal comparisons of the libraries revealed that there were no significant differences (p > 0.05) between the micro- bial compositions at different age groups: Day 5–15, Day 5–25, Day 5–35, Day 15–25, Day 15–35, and Day 25–35. The results of the statistical evaluation at certain age groups revealed that the percentage of bacterial micro- biota of Actinobacteria, Alphaproteobacteria, Chloro- plast and Epsilonproteobacteria varied significantly (p< 0.05). The average percentage of Actinobacteria detected at Day 5 (6.83%) of age was significantly higher (p < 0.05) than at Day 15 (1.03%), at Day 25 (0.458%) and at Day 35 (4.35%) of age. The average percentage of Alphapro- teobacteria was detected at significantly higher level (p < 0.05) at Day 35 (0.904%) of age than at Day 5 (0.458%) of age. The average percentage of Chloroplast detected at Day 15 (0.598%) was significantly lower (p< 0.05) than at Day 25 (4.23%) of age. The average percentage of Ep- silonproteobacteria at Day 25 (0.243%) was significant- ly higher (p < 0.05) from those of the other age groups. Taxonomic Composition Distribution of the Bacterial Community in Intestinal Segments Percentage of relative abundance of bacterial commu- nity of Cobb 500 broiler chickens determined from dif- Figure 3. Percentage of relative abundance of bacterial community of Cobb 500 broiler chickens determined from different intestinal segments at different age periods from 16S rDNA libraries. 54 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis ferent intestinal segments at different age periods from 16S rDNA libraries are presented in Figure 1. From Fig- ure 1, it can be seen that the diversity of the bacterial community in the intestinal segments of broiler chick- ens changed from one age period to the next. Species that exhibited an abundance less than 0.5% in all sam- Table 8. Abundance of bacterial 16S rDNA sequences (n=31255) identified from the jejunum flora of local Omani chickens. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Lentisphaerae Proteobacteria Tenericutes Thermi Total Actinobacteria Coriobacteriia Bacteroidia Flavobacteriia Sphingobacteriia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Lentisphaeria Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Verrucomicrobiae 200 (2.33) 4 (0.047) 1 (0.012) 2 (0.023) 0 (0) 0 (0) 1578 (18.4) 6484 (75.5) 215 (2.50) 3(0.035) 0 (0) 92 (1.07) 4 (0.047) 0 (0) 0 (0) 1 (0.012) 0 (0) 0 (0) 8584 2 (0.025) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 18 (0.229) 7616 (96.8) 225 (2.86) 9 (0.114) 0 (0) 1 (0.013) 0 0 (0) 0 (0) 1 (0.013) 0 (0) 0 (0) 7872 49 (0.646) 1 (0.013) 0 (0) 0 (0) 0 (0) 0 (0) 268 (3.53) 7092 (93.5) 101 (1.33) 0 (0) 0 (0) 29(0.382) 11 (0.145) 0 (0) 23 (0.303) 8 (0.106) 0 (0) 0 (0) 7582 109 (1.51) 1 (0.014) 0 (0) 0 (0) 0 (0) 1 (0.014) 178 (2.47) 6889 (95.5) 10 (0.139) 0 (0) 0 (0) 25 (0.346) 3 (0.042) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.014) 7217 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day Table 7. Abundance of bacterial 16S rDNA sequences (n=33443) identified from the duodenum flora of local Omani chickens. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Lentisphaerae Proteobacteria Tenericutes Thermi Total Actinobacteria Coriobacteriia Bacteroidia Flavobacteriia Sphingobacteriia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Lentisphaeria Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Verrucomicrobiae 213 (2.55) 6 (0.072) 2 (0.024) 0 (0) 4 (0.048) 0 (0) 502 (6.00) 6065(72.5) 1096 (13.1) 6 (0.072) 0 (0) 419 (5.01) 39 (0.466) 1 (0.012) 0 (0) 9 (0.108) 0 (0) 0 (0) 8362 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (0.037) 8023 (97.7) 150 (1.83) 10 (0.122) 0 (0) 4 (0.049) 2 (0.024) 0 (0) 0 (0) 8 (0.097) 15 (0.183) 0 (0) 8215 21 (0.235) 2 (0.022) 2 (0.022) 0 (0) 0 (0) 2 (0.022) 23 (0.257) 8667 (97.0) 121 (1.35) 2 (0.022) 0 (0) 11 (0.123) 11 (0.123) 1 (0.011) 71 (0.795) 1 (0.011) 0 (0) 0 (0) 8935 144 (1.82) 0 (0) 3 (0.038) 0 (0) 0 (0) 5 (0.063) 50 (0.630) 7618 (96.1) 29 (0.366) 1 (0.013) 0 (0) 15 (0.189) 12 (0.151) 0 (0) 15 (0.189) 0 (0) 39 (0.492) 0 (0) 7931 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day 55Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi ples were classified into “others”. The intestinal segments of duodenum, jejunum, and ileum had a higher relative abundance of Bacilli, and as the birds aged, the percent- age of Bacilli decreased, whereas the cecum had a higher relative abundance of Clostridia and as the birds aged, the percentage of Clostridia increased. Table 9. Abundance of bacterial 16S rDNA sequences (n=31337) identified from the ileum flora of local Omani chicken. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Lentisphaerae Proteobacteria Tenericutes Thermi Total Actinobacteria Coriobacteriia Bacteroidia Flavobacteriia Sphingobacteriia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Lentisphaeria Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Verrucomicrobiae 116 (1.29) 1 (0.011) 1 (0.011) 0 (0) 0 (0) 0 (0) 2170 (24.1) 6531 (72.5) 26 (0.289) 3 (0.033) 0 (0) 144 (1.60) 9 (0.100) 0 (0) 0 (0) 2 (0.022) 0 (0) 0 (0) 9003 2 (0.026) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 192 (2.49) 6814 (87.0) 471 (6.01) 1 (0.013) 0 (0) 3 (0.038) 2 (0.026) 0 (0) 0 (0) 349 (4.45) 0 (0) 0 (0) 7834 12 (0.155) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 16 (0.206) 7674 (98.9) 57 (0.734) 0 (0) 0 (0) 0 (0) 1 (0.013) 0 (0) 3 (0.039) 0 (0) 0 (0) 0 (0) 7763 147 (1.72) 2 (0.023) 1 (0.012) 0 (0) 1 (0.012) 33 (0.387) 768 (9.00) 7090 (83.1) 398 (4.66) 2 (0.023) 0 (0) 70 (0.820) 7 (0.082) 4 (0.047) 9 (0.105) 5 (0.059) 0 (0) 0 (0) 8537 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day Table 10. Abundance of bacterial 16S rDNA sequences (n=31337) identified from the ileum flora of local Omani chicken. Abundance of Sequence (No. of Sequence [%]) at Day: Phylum Class Day 5 Day 15 Day 25 Day 35 Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Lentisphaerae Proteobacteria Tenericutes Thermi Total Actinobacteria Coriobacteriia Bacteroidia Flavobacteriia Sphingobacteriia 4C0d-2 Chloroplast Bacilli Clostridia Erysipelotrichi Lentisphaeria Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Mollicutes Verrucomicrobiae 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 657 (9.33) 5417 (76.9) 222 (3.15) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 745 (10.58) 0 (0) 0 (0) 7041 0 (0) 31 (0.397) 1320 (16.9) 0 (0) 0 (0) 0 (0) 0 (0) 29 (0.371) 6361(81.5) 45 (0.576) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 22 (0.282) 0 (0) 0 (0) 7808 0 (0) 21 (0.336) 695 (11.1) 0 (0) 0 (0) 0 (0) 0 (0) 17 (0.272) 4953 (79.2) 143 (2.29) 1 (0.016) 0 (0) 0 (0) 31 (0.496) 7 (0.112) 354 (5.66) 0 (0) 30 (0.480) 6252 0 (0) 98 (1.36) 2187 (30.4) 0 (0) 0 (0) 119 (1.65) 0 (0) 471 (6.54) 4191 (58.2) 57 (0.792) 2 (0.028) 0 (0) 0 (0) 11 (0.153) 0 (0) 2 (0.028) 0 (0) 60 (0.834) 7198 Note: Values in the parentheses are abundance of Sequence (No. of Sequence [%]) at Day 56 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis Bacterial Taxonomic Composition of the Du- odenum of Local Omani Chickens across Age Periods Bacteria classified according to their respective Phylum and Class, found in the duodenum of local Omani chick- ens at different ages are presented in Table 7. Eighteen bacterial microbiota at the Class level were found in du- odenum. Of the 33443 reads, Bacilli were the most abun- dant, at 90.8% of the total sequences, while sequences related to Clostridia accounted for 4.17% of the total se- quences. Actinobacteria and Chloroplast represented a very small percentage of 1.13% and 1.72%, respectively of the total sequences. Across different age periods Ba- cilli were the dominant group, representing 72.5 % at Day 5, 97.7 % at Day 15 and 97.0 % at Day 25 and 96.1 % at Day 35 of the sequences. Clostridia sequences fluc- tuated from 13.1% at Day 5, 1.83% at Day 15, 1.35% at Day 25 and 0.366% at Day 35. Both Actinobacteria and Bacteroidia - related sequences were Day 5: 2.55%, Day 15: 0.0%, Day 25: 0.235% and Day 35: 1.82% and Day 5: 0.024%, Day 15: 0.0%, Day 25: 0.022%, and Day 35: 0.038%, respectively. Proteobacteria group-related sequences were detected at smaller percentages across age periods. Bacterial Taxonomic Composition of the Jeju- num of Local Omani Chickens Across Age Periods Bacteria classified according to their respective Phylum and Class, found in the jejunum of local Omani chick- ens at different ages are presented in Table 8. Eighteen bacterial microbiota at the Class level were found in jejunum. Of the 31255 reads, Bacilli were the most abundant, at 89.84% of the total sequences. Chloroplast represented 6.53% of the total sequences. Clostridia accounted for 1.76% of the total sequences. Actinobac- teria and Gammaproteobacteria represented a small percentage of 1.15 % and 0.032 %, respectively of the total sequences. and Chloroplast were 6.53%. Across different age periods Bacilli were the dominant group, representing 75.5% at Day 5, 96.8% at Day 15, 93.5% at Day 25 to 95.5% at Day 35 of the sequences. Clostrid- ia sequences fluctuated from 2.50% at Day 5, 2.86% at Day 15, 1.33% at Day 25, and 0.139% at Day 35. Chlo- roplast sequences were 18.4% at Day 5, 0.229% at Day 15, 3.53% at Day 25 to 2.47% at Day 35 of the sequenc- es. Actinobacteria sequences were Day 5: 2.33%, Day 15: 0.025%, Day 25: 0.646%, and Day 35: 1.51%, while Gammaproteobacteria sequences were Day 5: 0.012%, Day 15: 0.013%, Day 25: 0.106%, and Day 35:0.0%. Bacterial taxonomic composition of the ileum of Local Omani chickens across age periods Bacteria classified according to their respective Phylum and Class, found in the ileum of local Omani chickens at different ages are presented in Table 9. Eighteen bac- terial microbiota at the Class level were found in ileum. Of the 31337 reads, Bacilli were the most abundant, at 84.8% of the total sequences. Chloroplast accounted for 9.49% of the total sequences. Clostridia and Gammapro- teobacteria represented a small percentage of 2.87% and 1.07%, respectively of the total sequences. Only a few Actinobacteria (0.88%) related sequences were detected. Table 11. P-value distribution of 16S rDNA gene sequence libraries compared the abundance differences of microbial commu- nities among samples from different segments for cobb 500 broiler chicken. P-Value Class Duodenum- Jejunum Duode- num-Ileum Jejunum- Ileum Cae- cum-Duo- denum Caecum- Jejunum Caecum- Ileum 4C0d-2 Actinobacteria Alphaproteobacteria Bacilli Bacteroidia Betaproteobacteria Chloroplast Clostridia Coriobacteriia Deinococci Deltaproteobacteria Epsilonproteobacteria Erysipelotrichi Gammaproteobacteria Mollicutes Thermoleophilia Unclassified 0.881 0.374 0.467 0.485 0.831 0.918 0.037 0.753 0.863 0.564 1 0.497 0.681 0.228 0.194 1 0.514 0.352 0.885 0.900 0.390 0.048 0.068 0.820 0.283 0.646 1 0.556 0.080 0.606 0.094 0.500 0.680 0.119 0.635 0.767 0.320 0.318 0.765 0.901 0.394 0.851 0.538 0.619 0.505 0.495 0.208 0.670 0.006 0 0.314 0.038 0.019 0.002 0.045 0.268 0.239 0.347 0.075 0.566 0.138 0.512 0.347 0.212 0.823 0.039 0 0.318 0.218 0.009 0.002 0.046 0.174 0.141 0.263 0.070 0.464 0.087 1 0.370 0.203 0.611 0.055 0 0.299 0.027 0.018 0.001 0.010 0.324 0.162 0.324 0.068 0.768 0.126 0.324 57Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi Proteobacteria sequences represented 1.72% of the total sequences. Across different age periods Bacilli were the dominant group, representing 72.5% at Day 5, 87.0% at Day 15, 98.8% at Day 25 to 83.1% at Day 35 of the se- quences. Clostridia sequences fluctuated from 0.289% at Day 5, 6.01% at Day 15, 0.734% at Day 25, and 4.66% at Day 35. Chloroplast sequences were Day 5: 24.1%, Day 15: 2.49%, Day 25: 0.206%, and Day 35: 9.0%, while Actinobacteria sequences were Day 5: 1.29%, Day 15: 0.026%, Day 25: 0.155%, and Day 35: 1.72%. Proteobac- teria group-related sequences were detected at smaller percentages across all age periods. Bacterial Taxonomic Composition of the Cecum of Local Omani Chickens Across Age Periods Bacteria classified according to their respective Phylum and Class, found in the cecum of local Omani chickens at different ages are presented in Table 10. Eighteen bac- terial microbiota at the Class level were found in cecum. Of the 28299 reads, Clostridia were the most abundant, at 73.9% of the total sequences. Bacteroidia and Gam- maproteobacteria accounted for 14.9 % and 3.97 %, re- spectively of the total sequences. Bacilli and Erysipelotri- chi represented a small percentage of 4.15% and 1.65%, respectively of the total sequences. Across different age periods Clostridia were the dominant group, represent- ing 76.9% at Day 5, 81.5% at Day 15, 79.2% at Day 25 to 58.2% at Day 35 of the sequences. Bacilli sequences fluctuated from 9.33% at Day 5, 0.371% at Day 15, 0.272% at Day 25, and 6.54% at Day 35. Bacteroidia sequences were Day 5: 0.0%, Day 15: 16.9%, Day 25: 11.1%, and Day 35: 30.4%, while Erysipelotrichia sequences were Day 5: 3.15%, Day 15: 0.576%, Day 25: 2.29%, and Day 35: 0.792%. Differences of Microbial Communities among Samples from Different Intestinal Segments of Local Omani Chicken The p-value distribution of 16S rDNA gene sequence li- braries used to compare the quantitative differences of microbial communities among samples from different intestinal segments of local Omani chickens is present- ed in Table 11. Statistical comparisons of the libraries showed that the composition of the Duodenum-Jeju- num, Duodenum-Ileum, Cecum-Duodenum, Cecum-Il- eum Cecum-Jejunum bacterial microbiota differed significantly (p < 0.05), suggesting that each region es- tablished its own bacterial community. The number of Alphaproteobacteria, Bacilli, Betaproteobacteria, Chlo- roplast, Clostrdia and Coriobacteriia differed signifi- cantly across different intestinal segments (p < 0.05). Bacilli were the dominant 16S rDNA sequences in the duodenum, jejunum, and ileum libraries, whereas Clos- tridia were the dominant 16S rDNA sequences in the cecum libraries. Differences of Microbial Communities among Samples from Local Omani Chickens of Differ- ent Age Groups The p-value distribution of 16S rDNA gene sequence libraries comparing the quantitative differences of mi- crobial communities among samples from local Omani chickens at different age groups are presented in Table 12. Statistical comparisons of the libraries revealed that there were no significant differences (p > 0.05) between Table 12. P-value distribution of 16S rDNA gene sequence libraries compared the abundance differences of microbial com- munities among samples from different segments for cobb 500 broiler chicken. P-Value Class Day 5-15 Day 5-25 Day 5-35 Day 15-25 Day 15-35 Day 25-35 4C0d-2 Actinobacteria Alphaproteobacteria Bacilli Bacteroidia Betaproteobacteria Chloroplast Clostridia Coriobacteriia Deinococci Deltaproteobacteria Epsilonproteobacteria Erysipelotrichi Gammaproteobacteria Mollicutes Thermoleophilia Unclassified 0.315 0.343 0.736 0.925 0.347 0.746 0.669 0.839 0.185 0.068 1 0.159 0.574 0.052 0.259 0.347 0.442 0.875 0.850 0.933 0.511 0.337 0.812 0.929 0.598 0.119 0.494 0.264 0.681 0.483 0.624 0.376 0.688 0.481 0.057 0.599 0.328 0.128 0.094 0.868 0.144 0.681 0.485 0.163 0.934 0.215 0.335 0.111 0.386 0.277 0.271 0.620 0.911 0.213 0.575 0.742 0.779 0.277 0.634 0.066 0.277 0.021 0.872 0.121 0.248 0.287 0.092 0.230 0.285 0.129 0.388 0.188 0.554 0.112 0.244 0.121 0.057 0.494 0.065 0.555 0.344 0.080 0.143 0.860 0.717 0.373 0.201 0.661 0.123 0.536 0.107 58 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Evaluation of the Intestinal Bacterial Community of Local Omani and Cobb 500 Broiler Chickens Raised in an Open-Sided House Using 16S rDNA-Based Analysis the microbial compositions at different age groups: Day 5–15, Day 5–25, Day 5–35, Day 15–25, Day 15–35, and Day 25–35. The results of the statistical evaluation at certain age groups revealed that the percentage of bacte- rial microbiota of 4C0d-2 varied significantly (p< 0.05). The average percentage of 4C0d-2 was detected at sig- nificantly higher level (p < 0.05) at Day 35 (0.529%) of age than at Day 15 (0.0%) of age. Taxonomic Composition Distribution of the Bacterial Community in Intestinal Segments at the Class-Level of Local Omani Chickens From Figure 2, it can be seen that the diversity of the bacterial community of intestinal segments of local Omani chickens changed from one age period to the next. Species of that exhibited an abundance less than 0.5% in all samples were classified into “others”. The in- testinal segment of duodenum, jejunum, and ileum had a higher abundance of Bacilli, and as the birds aged, the percentage of Bacilli decreased, whereas the cecum had a higher abundance of Clostridia, and as the birds aged, the percentage of Clostridia increased. Discussion The aim of this study was to generate a phylogenetic diversity census of bacteria identified in the intestinal segments (duodenum, jejunum, ilium and cecum) of Local Omani and Cobb 500 broiler chickens raised in open-sided house from 0 to 35 day of age using 16S rD- NA-based analysis. However, little is known about the Figure 4. Percentage of relative abundance of bacterial community of local Omani chickens.and determined from different intestinal segments at different age periods from 16S rDNA libraries. 59Research Paper Al-Balushi , El Tahir, Asi, El-Zaiat, Al-Abri, Al-Kharousi, Al-Marzooqi intestinal bacterial community composition and succes- sion for birds especially those with various growth rates such as the indigenous chickens raised in naturally ven- tilated open-sided house system. The open-sided house is widely practiced by a majority of small and medium scale farmers in the developing countries. A greater understanding of the chicken gut function and micro- biology will enhance chicken’s health and productivity raised in naturally ventilated open sided house system. The data obtained in the study revealed the hetero- geneity of bacterial populations found in different intes- tinal segments as derived from molecular detection and bioinformatics analysis. As a result, the current study’s findings have been confined to the most quantitative- ly significant bacterium classes. Therefore, this study focused solely on Bacilli and Clostridia, the two most common groups in the Firmicutes phylum. The statis- tical analyses of microbial community libraries with- in each breed among samples from different intestinal segments at different age groups revealed no significant differences (p > 0.05) in the current investigation. Quite the contrary, the bacterial microbiota of each breed differed significantly (p<0.05) across distinct intestinal segments, suggesting that each region established its bacterial community with different relative abundances. It is anticipated that diverse bacteria will emerge in various intestine segments as each segment’s function and environmental circumstances differ from one anoth- er (Rehman et al., 2007; Wise and Siragusa, 2007). In the current study the most reflective differences in the mi- crobial population in the intestinal segments of the two breeds was detected between Day 5 to 25. One possible explanation is that at early age periods, immediately after hatching, is the most critical period in the life of the chick. During this early age period, there is the transition from yolk to oral nutrition associated with major physical and functional development of the digestive tract and organs (Ravindran, 2003) resulting in unstable environmen- tal conditions of the digestive tract’s microecosystem. Different studies observed similar results that the microbial community structure varies with age and the microbial community structure was impartially stable and is replaced by a stable bacterial community once the rate of the intestinal development lessened (Mackie et al., 1999; Xu et al., 2003; Amit-Romach et al., 2004). The unique microbial community at 3-5 days of age suggests that the early bacterial community is relatively transient and is replaced by a stable bacterial community later in life (Lumpkins et al., 2010; Glendinning et al., 2019). In the current study, our data showed that Bacilli was the most dominant Class in the duodenal flora of Cobb 500 at a younger age than Local Omani breed (Cobb 500: Day5 98.9 %; Local Omani: Day5 72.5 %). Clostridia was the second most abundant Class in the duodenum. Their levels were high in Local Omani than in Cobb 500 at an early age (Local Omani: Day5 13.1 %; Cobb 500: Day5 0.062 %, respectively). It is well documented that the chicken cecum and its mucosal tissue are dominated by Clostridia related species (Bjerrum et al., 2006; Gong et al., 2007; Lund et al., 2010). Clostridia are mainly involved in fermentation and can ferment a wide variety of sub- strates including monosaccharides and polysaccharides (Jones and Woods, 1986). The larger number of Clos- tridia in the duodenum of Local Omani chickens during early intestinal development possibly have no or limited function in nutrient absorption and subsequently the duodenum functions at a lessened level when compared to that of Cobb 500 chickens. According to Al-Marzooqi et al. (2020) the higher abundance of Clostridia that are associated with fermentation act in an inhibitory fashion for nutrient absorption in the intestine of the local Oma- ni chickens and the villus development is slowed down. The morphological analysis in a study by Al-Balushi (2021) showed that villus height to villus width ratio in Cobb 500 broilers was significantly higher compared to the Local Omani chickens (14.18 versus 9.74, respective- ly p < 0.01). It is assumed that an increased villus height is paralleled by an increased digestive and absorptive function of the intestine due to increased absorptive surface area, expression of brush border enzymes, and nutrient transport systems (Amat et al., 1996). Entero- cyte enzymatic activity and structure are two of the most important features of the intestinal mucosal phys- iology (Caspary, 1992). Al-Marzooqi et al., (2019) con- cluded that villus development has a profound effect on the growth performance of the chickens. Many studies proved that variations in bacterial population between certain broiler lines might be attributed to differences in villi height, which could result in a wider distance between the crypt and the lumen with increased villus height, creating a niche for specific bacteria (Suau et al., 1999; Salzman et al., 2002; Lumpkins et al., 2010). As a result, villi height might be referred as a contrib- uting element to the bacterial community’s habitat. Conclusion The dynamics of the gut microbial community or microbial balance are still far from fully understood. However, the future development of the Local Omani chicken breed for rapid growth production requires a further selection of lines that should take in con- sideration the intestine’s overall developmental rate. In addition, future studies will need to look into his- tological alterations related to intestinal function. It is essential to establish baseline values for pro- duction parameters of Local Omani chickens and characterize the overall performance of Local Oma- ni chickens. Future research should be focused by identifying gut bacteria that can be associated with improved/poor chicken growth performance. 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