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Context Dietary supplementation with phytobiotics may form part of an alternative strategy to the use of antibiotics as growth promoters in the poultry industry. Essential oils of rosemary (Rosmarinus officinalis L.) may have antimicrobial and antioxidant properties. Aims The aims were to evaluate in vitro characteristics of non-encapsulated and encapsulated rosemary essential oils, and to investigate their effects on growth performance, intestinal morphology and microbial population in broiler chickens. Methods Essential oil main components were determined, and the minimum inhibitory concentration and 2,2-diphenyl-1-picrylhydrazyl radical assay methods were applied for determination of antimicrobial and antioxidant activities, respectively. In an in vivo study of performance, 1-day-old male chicks (n = 420) were randomly assigned to seven dietary treatments with five replicates of 12 birds in a completely randomised experimental design. Treatments were control (basal) diet, and basal diet supplemented with chitosan (encapsulating agent) at 150 mg/kg, encapsulated and non-encapsulated rosemary essential oils at both 150 and 300 mg/kg, and the antibiotic flavophospholipol at 650 mg/kg. Key results Non-encapsulated essential oil had stronger antioxidant activity than the encapsulated form (IC50 values 0.969 and 1.879 mg/mL, respectively). Non-encapsulated essential oil had greater antimicrobial effect against Escherichia coli, whereas the encapsulated form was more effective against Staphylococcus aureus. During the whole growing period of the in vivo study, supplementation with different levels of non-encapsulated or encapsulated essential oils significantly (P

Summary: The effects of nanoencapsulated cumin essential oil (EO) feeding broilers on growth performance, mucin 2 gene expression, blood parameters, and immune responses to phytohemagglutinin (PHA) and 2, 4-dinitrochlorobenzene (DNCB) were evaluated. In a completely randomized design, 1050 male day-old Ross 308 broilers were distributed into 7 dietary treatments with 5 replicates of 30 birds per replication. Dietary treatments included 1) control basal (no additives), 2) control antibiotic growth promoter (flavophospholipol at 650 mg/kg), control basal plus a solution of 3) 150 mg/kg of chitosan nanoparticles, free-form cumin EO at 4) 100 or 5) 200 mg/kg, and nanoencapsulated cumin EO at 6) 100 and 7) 200 mg/kg. The use of 200 mg/kg nanoencapsulated cumin EO significantly (P < 0.01) increased body weight gain and mucin 2 gene expression and improved feed conversion ratio compared with other treatment. The highest feed intake was significantly (P < 0.01) in the control treatment during day 15 to 18 and day 29 to 42. In birds fed with nanoencapsulated cumin EO, serum total triglyceride and low-density lipoprotein cholesterol levels were also significantly lower than birds in the control group. Sheep red blood cell antibody titer (35 and 42 d), immunoglobulin G (42 d), heterophilus (H), lymphocyte (L), and H/L ratio, PHA, and DNCB (42 d) were affected by the nanoencapsulation process of cumin EO (P

This survey aimed to evaluate the effects of dietary supplementation of different forms of savory essential oil (SEO) on growth performance, intestinal morphology and microbial population in broiler chickens. A total of 360 one-day-old male broilers were randomly allocated to 6 dietary treatment groups with 5 replicates per treatment and 12 birds per pen. The experiment consisted of a 2×3 factorial arrangement including two different forms [encapsulated SEO (ESEO) and nonencapsulated SEO (NSEO)] in three levels (0, 150, and 300 mg/kg diet) of SEO. Growth performance, jejunal morphology and intestine microbial population were examined. Our results revealed that feed intake was not influenced by the dietary treatments in different experimental periods. As well as, the experimental diets did not influence body weight gain (BWG) and feed conversion ratio (FCR) during the starter period. However, at the grower, finisher, and also whole rearing periods, broilers which received 150 mg/kg SEO had significantly higher BWG and lower FCR compared to the birds fed the control diet. The final body weight (FBW) was also higher in chickens fed with diet supplemented with 150 mg/kg SEO in comparison to the others. The results also revealed that 150 mg/kg SEO, significantly increased the concentration of Lactobacillus and decreased the intensity of coliforms in the ileal digesta in comparison to the control diet. Furthermore, villus height was significantly lower in birds fed the control diet than in the birds that consumed different levels of SEO. Eventually, the findings of this experiment revealed that dietary supplementation of SEO, especially at 150 mg/kg level, was effective in raising the populations of beneficial microorganisms in the gastrointestinal tract as well as improving intestinal morphology and growth performance of broilers.

The prepared protein and protein-ligand complexes (protease-SDS and protease-tween complexes) were used in MD simulation, performed using GROMACS v 4.6.5 with CHARMM36 all-atom force field. The CGenFF server (https://cgenff.umaryland.edu/) provides topologies and parameters of ligands compatible with the CHARMM36 all-atoms force field. Protein and protein-ligand complexes were soaked in a cubic box of water molecules, and the charges on the protein were neutralized by the addition of Na+ and Cl- ions. The energy of the system was minimised using the steepest descent algorithm to eliminate bad contact and clashes. The NVT and NPT ensembles were used during the equilibration to achieve the desired temperature (373.15 K for protease and 300 K for protease-ligand complex) and pressure (1 bar) for 100 ps and restraint forces of 1000 kJ/mol. Finally, 40 ns MD run were performed in triplicate after releasing all restraints. All bonds were constrained by the LINCS algorithm.

After the cultivation period, cells were collected by centrifugation at 3500 × g for 30 min at 4 °C and resuspended in 4 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole and 0.05% Tween 20 at a pH of 7). The mixture was sonicated with 4 × 40 sec pulses followed by 20 sec rest between cycles at 4 °C. The crude extracts were then centrifuged at 9000 × g for 30 min at 4 °C, and the resulting supernatant was heat-treated in a hot water bath at 90 °C for 15 min. Insoluble material was separated by centrifugation at 13000 × g for 10 min, and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide. The purified elution fractions were dialysed overnight in a 50 mM potassium-phosphate buffer (pH=7) at 4 °C.

The nucleotide sequence of Coh01147, previously obtained from Cohnella sp. A01 (Genbank Accession No: QEV88423) was translated to the amino acids using Expasy translate tool (http://web.expasy.org/translate). The resulting protein sequencewas used for further in silico analysis of the Cohnella sp. A01 derived protease 1147 protein. The biochemical properties of the protein, including molecular weight, isoelectric point, instability index, aliphatic coefficient, and grand average of hydropathy (GRAVY), were calculated using Expasy ProtParam tool (http://web.expasy.org/protparam). The presence of putative signal peptide and the location of its cleavage site was predicted using SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP). pFam (http://pfam.xfam.org/search) and MEROPS (http://MEROPS.sanger.ac.uk) databases were used for sequence alignment and identifying the peptidase family. The phylogenetic relationships of the protein sequence were analysed using ClustalW in MEGA 7 programs. Multiple alignments of the resulting protein family of protease 1147 (from MEROPS) was carried out using COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi), and the conserved areas were determined using DNAMAN software (www.lynnon.com). BLOSUM62 substitution matrix and a gap penalty = 12 were utilised in the search.

Background: The resistance of the bacteria and fungi to the innumerous antimicrobial agents is a major challenge in the treatment of the infections demands to the necessity for searching and finding new sources of substances with antimicrobial properties. The incorporation of the essential oils (EOs) in chitosan film forming solution may enhance antimicrobial properties. However, its use as the feeding additive in the poultry nutrition needs to clarify the product's activity against both pathogen and the useful microbes in the gastrointestinal tract. Objectives: In the present study, we carried out an in vitro investigation and evaluated the antimicrobial activity of chitosan film forming solution incorporated with essential oils (CFs+EOs) against microbial strains including Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Lactobacillus rahmnosus, Aspergillus niger and Alternaria alternate. Material and Methods: In three replicates, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of different treatments including: 1- essential oils (EOs), 2- chitosan film solution (CFs), and 3-chitosan film solution enriched with EOs (CFs+EOs) were determined against above mentioned microbes. Results: The results indicated that the chitosan solution enriched with essential oils (CFs+EOs) is capable of inhibiting the bacterial and fungal growth even at the lowest concentrations. The MIC and MBC for all the antimicrobial agents against Escherichia coli and Staphylococcus aureus were very low compared to the concentrations needed to inhibit the growth of useful bacteria, Lactobacillus rahmnosu and Enterococcus faecium. The antifungal activity of chitosan was enhanced as the concentration of EOs increased in the film solution. Conclusion: Chitosan-EOs complexes are the promising candidate for novel contact antimicrobial agents that can be used in animal feeds.

The addition of essential oil (EO) as chitosan encapsulated can increase the efficiency of these oils in broiler feeding. Therefore, the objective of the current research was to explore the antibacterial and antioxidant potential of garlic essential oil (GEO) (free vs. nanoencapsulated) and their effects on performance, gene expression of mucin2, microbial, and morphology of intestine in broilers. A total of 900 1-day-old male broilers (Ross 308) were assigned to six dietary treatments (0, 100, and 200 mg/kg free GEO and 0 (contain of chitosan), 100 and 200 mg/kg nanoencapsulated GEO) with a 2 × 3 factorial arrangement based on completely randomized design. Garlic essential oil encapsulation with chitosan significantly enhanced antibacterial and antioxidant parameters. At 100 mg/kg nanoencapsulated GEO had significant (P

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

1. The use of chitosan nanoparticles (CNPs) for the entrapment of active components has gained considerable interest in food and pharmaceutical industries due to its mucous adhesiveness, non-toxicity, biocompatibility and biodegradability.2. This study aimed to investigate the usefulness of feeding encapsulated thyme essential oil (E-TEO) on performance, blood parameters, immunity and ileal microflora in broilers.3. CNPs were prepared by using ionotropic gelation and structurally characterised by dynamic light scattering; scanning electron microscope and Fourier transform infrared spectroscopy. The encapsulation efficiency percentage of TEO in CNPs and its in vitro release were determined using ultraviolet visible spectroscopy and dialysis method, respectively.4. An in vivo experiment was conducted with five treatments of five replicates with ten chicks per pen. Dietary treatments consisted of an unsupplemented control diet, or supplemented with chlortetracycline (80 and 50 mg/kg during the starte...

The effects of a probiotic (protexin), a medicinal plant (basil essential oil) and an antibiotic growth promoter (avilamycin) as broiler feed additives on performance, carcass characteristics and some blood parameters of broilers were studied. A total of 600 Arian broilers were divided into six treatments, with four replicates of 25 birds. Treatments were a plant essential oil in three levels (200, 400 and 600 ppm), the probiotic (150 ppm), the antibiotic (150 ppm) and a control group with no additives. Birds in different treatments received the same diets during the experimental period. Growth performance, blood biochemical parameters and carcass traits were measured. There were no effects of dietary treatment on body weight gain, feed intake and feed conversion ratio of the broilers. Internal organ weights and carcass characteristics were not influenced by treatments; however, 200 ppm basil essential oil supplementation decreased abdominal fat (P < 0.05). Biochemical parameters including serum low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol were not statistically influenced. However, addition of 400 ppm basil essential oil into diet increased serum triglyceride as compared to the control (P < 0.05). Results of this experiment demonstrated that the herbal natural feed additives such as basil may be used as alternatives to an antibiotic growth promoter without any adverse effects on broiler production.

This experiment was conducted to investigate and compare the efficacy of different feed additives on performance, tibial dyschondroplasia (TD) incidence and tibia characteristics of male broilers fed low-calcium diets. A completely randomized design, with six treatments and five replicates of five chicks per each was used. Experimental treatments were: (i) Basal diet containing recommended level of calcium (0.9%) as control treatment (Ctrl), (ii) low-calcium (0.67%) diet without any additive (LC), (iii) low-calcium diet + probiotic (2 g/kg diet), (iv) low-calcium diet + prebiotic (2 g/kg diet), (v) low-calcium diet + synbiotic [mix of probiotic and prebiotic (each 2 g/kg diet)], (vi) low-calcium diet + organic acid (1.5 g/kg diet). Birds were reared in an open-sided house system under natural tropical condition until 21 days of age. Feeding with low-calcium diet negatively influenced broiler performance (body weight, body weight gain and feed conversion ratio) and tibia characteristics, whereas dietary inclusion of all feed additives had beneficial effects on above-mentioned parameters and helped the birds to overcome problems related to low-calcium diets. Different treatments had no effect on TD incidence.

Summary High environmental temperature has detrimental effects on the gastrointestinal tract of poultry. An experiment was conducted to determine the effect of acute heat stress on endogenous amino acid (EAA) flow in broiler chickens. A total of 90, day-old broiler chicks were housed in battery cages in an environmentally controlled chamber. Chicks were fed a nitrogen-free diet on day 42 following either no heat exposure (no-heat) or 2 weeks exposure to 35 ± 1 °C for 3 h from days 28 to 42 (2-week heat) or 1 week exposure to 35 ± 1 °C for 3 h from days 35 to 42 (1 week heat). The most abundant amino acid in the ileal flow was glutamic acid, followed by aspartic acid, serine and threonine in non-heat stressed group. The EAA flow in 1-week heat and 2-week heat birds were significantly (p

1. An experiment was conducted to determine the effects of supposedly unpleasant physical treatment on broiler performance, small intestinal development and ameliorating role of probiotics. 2. The following treatments were applied from day one: (1) chicks exposed to normal human contact fed basal diet (control); (2) chicks were exposed to unpleasant physical treatment and fed basal diet (UPT-BD); and (3) chicks were exposed to unpleasant physical treatment and fed basal diet supplemented with Lactobacillus (UPT-BDL). Chicks were exposed to UPT from days 1 to 21. Different segments of gastrointestinal tract were sampled at 14, 28, 35 and 42 d of age. 3. Broilers of UPT-BD had lower feed consumption compared with control group at 7 d of age. Overall, UPT-BDL birds showed higher body weight gain (BWG) and better feed conversion ratio (FCR) over the course of the experiment. 4. Birds of UPT-BD had lower concentrations of lactic, propionic and butyric acids in the caecum as compared with other groups at 14 d of age. Acetic acid concentration was profoundly decreased in both UPT groups compared to the control. 5. Duodenal villus height of UPT-BD broilers showed a slight reduction compared to the control and UPT-BDL birds at 14 d of age. Afterwards until day 42, UPT-BDL birds showed the highest villus height among treatments in different parts of the small intestine. 6. The results suggested that, even though UPT did not have significant inhibitory effects on the development of the small intestine and broiler performance, it negatively affected bacterial metabolic end products in the caecum, which could be ameliorated by the addition of Lactobacillus.

The objective of the present study was to isolate, identify and characterize new LAB strains with high probiotic potentials from Iranian (Isfahan) indigenous chickens. From 90 isolated LABs, 11 isolates had high growth rate under different stress conditions, including acid (pH 2.5), bile (0.5% oxgall), salt (6-15%) and temperatures 15 and 45°C, and their aggregation time was less than 120 min. Based on the molecular identification using 16S rDNA sequencing and phylogenetic analysis, the isolates belonged to two Lactobacillus salivarius and L. reuteri species. The isolates showed different tolerance to 16 clinically and veterinary relevant antibiotics, and most of them were resistant to or semi-tolerant of 7-15 different studied antibiotics. The Es11, Es12, Es3 and Es13 strains with resistance to or semi-tolerance of 15, 14 and 13 different antibiotics, respectively, were the most tolerant strains. The selected isolates showed a wide range of antimicrobial activity against 7 different pathogenic strains. All the isolates exhibited antagonistic activity against E. coli, Enterococcus hirae, Salmonella enterica and Staphylococcus aureus. The isolates Es6 and Es11 with high antagonistic activity and resistance against 6 of the studied pathogens were the most powerful antagonistic isolates. The values and types of adhesion to the Caco-2 cell cultures were significantly different (0-40 bacteria/Caco-2 cell), and the maximum adhesion was observed for the isolates Es6 and Es13 with 35 and 40 bacteria adhesion/cell, respectively. Finally, based on all the experiments, 7 strains, including Es1, Es6, Es7, Es11, Es12 and Es13, were selected for the further in vivo assays and possible use in the poultry industry.

The effects of probiotic (Protexin®, Protexin Veterinary, Somerset, UK), medicinal plant (basil essential oil) and an antibiotic growth promoter (avilamycin) as broiler feed additives on ileal bacteria of broilers were studied. A total of 600 Arian broilers were divided into 6 treatments, with 4 replicates of 25 birds. Treatments were a plant essential oil in 3 level (200, 400 and 600 ppm), the probiotic (150 ppm), an antibiotic (150 ppm) and a control group with no additives. Birds in different treatments received the same diets during the experimental period. The results showed that probiotic treatment significantly decreased total bacteria counts (P0.05). The lowest and highest lactic acid bacteria in ileum were obtained by the control group and in birds receiving 400 ppm basil essential oil, respectively. Moreover, addition of 600 ppm of basil essential oil into diet decreased the number of E.coli colonies as compared to other treatments (P

The influence of stress due to rough handling (RH) on gastrointestinal tract (GIT) environmental pH, concentration of short chain fatty acids (SCFAs) and modulatory roles of two Lactobacillus strains was investigated in broiler chickens. Equal number of chicks was randomly assigned to one of the following treatment groups; (i): no handling + basal diet (control), (ii): RH and + basal diet (RH-BD), (iii): RH + basal diet supplemented with 107 CFU of each Lactobacillus strain per gram of feed (RH-BDL). Birds fed dietary Lactobacilli from day one until the end of the experiment and subjected to RH from day 1 to 21. Digesta from different GIT regions were collected at 14, 28, 35 and 42 days of age and SCFAs and pH were measured. Duodenal, ileal and cecal lactate concentrations together with cecal butyrate level were significantly (P < 0.05) decreased in RH-BD birds compared with the control and RH-BDL at 14 and 21 days of age. Acetate concentration was significantly (P < 0.05) reduced in the jejunum, ileum and cecum of both RH-BD and RH-BDL birds at 14 days of age. It can be concluded that, stressful condition over the course of the GIT microbial development, negatively affected microbial activity by reduction of the lactate, acetate (as the main substrate for butyric producing bacteria) and ultimately butyrate concentration along the GIT and particularly cecum. This adverse effect was effectively ameliorated by Lactobacillus supplementation.Key words: Gastrointestinal tract (GIT), SCFAs, Lactobacilli, stress.

Stressors may influence chicken susceptibility to pathogens such as Salmonella enterica. Feed withdrawal stress can cause changes in normal intestinal epithelial structure and may lead to increased attachment and colonization of Salmonella. This study aimed to investigate modulatory effects of epigenetic modification by feed restriction on S. enterica serovar Enteritidis colonization in broiler chickens subjected to feed withdrawal stress. Chicks were divided into four groups: ad libitum feeding; ad libitum feeding with 24-h feed withdrawal on day 42; 60% feed restriction on days 4, 5, and 6; and 60% feed restriction on days 4, 5, and 6 with 24-h feed withdrawal on day 42. Attachment of S. Enteritidis to ileal tissue was determined using an ex vivo ileal loop assay, and heat shock protein 70 (Hsp70) expression was evaluated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Feed withdrawal stress increased S. Enteritidis attachment to ileal tissue. However, following feed withdrawal the epigenetically modified chickens had significantly lower attachment of S. Enteritidis than their control counterparts. A similar trend with a very positive correlation was observed for Hsp70 expression. It appears that epigenetic modification can enhance resistance to S. Enteritidis colonization later in life in chickens under stress conditions. The underlying mechanism could be associated with the lower Hsp70 expression in the epigenetically modified chickens.

From a total of 656 broiler farms with the permission of exploitation in Guilan province, data was gathered from 20% of active farms including 85 units (Capacity of over 1663000 portions of broiler chickens) to evaluate the management of nutrition and nourish using questionnaire. The questionnaires were completed by the interview method. The methods of making feed, feed formulation, the shape of grain, the frequency of feeding, the type of dietary supplement and food additives, the use of experts, feed conversion ratio, causes of mortality, the age of mortality, drinking and feeding systems, and how to use fine nourishing (in the shape of supplement or concentrate) were considered in this study. The performance was calculated for each broiler farm. According to the production index, the farms were divided into three groups of weak (200+25), medium (250+25), and good (300+25) and their differences were compared. According to the results, among the managerial factors, the factors such as: feeding system, water quality, the conformity of ration in nutrition with the needs of commercial strains and drinking management, have the most portion in creating the three groups of good, medium and weak, among the broiler rearing farms. Thus, in the study of each group’s feeding system, the good group has the highest percentage of using automatic systems (86/60) and the weak group has the highest percentage of using manual systems. In comparing three groups regarding the drinking water quality, it was observed that good, medium and weak groups drink 92%, 61.84%, and 75% fresh water, respectively. Furthermore, in the weak group, most of the farms have used the water with tolerable hardness or saltiness. Finally, the conformity of strain’s requirement with diet was examined among groups. The results showed that 16.5% of units followed this conformity and the percentages of the good, medium, weak groups was 11.12%, 28%, 53.5%, respectively.