Evaluation of the potential of extract of seaweed Eucheuma denticulatum as an alternative to antibiotic growth promoter in broiler chickens.

The seaweeds are in focus for their immunity and gut health-stimulating potentials in humans and farm animals, but their potential as a gut health-promoting agent and performance booster to replace antibiotic growth promoters (AGP) in broiler chicken-feed remains to be evaluated. In vivo feeding experiments were conducted on commercial broiler chickens (1-42 days post-hatch) to evaluate dried aqueous exact of red seaweed Eucheuma denticulatum (referred to as PBD 5). Each of the three test diets (basal diet with three dosing regimens of PBD5, 0.25 g kg -1 for 0-6 weeks, 0.25 g kg -1 for 0-4 weeks or 1.0 g kg -1 for 0-2 weeks), along with an AGP supplemented diet (Virginiamycin (V), 20 ppm in basal diet), and a control diet was fed to 13 pen replicates of five chicks in each. PBD5 at 1.0 g kg -1 diet for 0-2 weeks improved (P < 0.05) cumulative feed efficiency (4.65 % improvement at 28 d, and 3.74 % at 35 d) than the control and comparable to the V group and the trend in improvement persisted up to 42 d. The group fed with PBD5 @ 1.0 g kg -1 for 0-2 weeks had significantly (P < 0.05) higher serum IgG level, glutathione peroxidase levels, fat digestibility, and expression of occludin and avian beta-defensin 4 gene in the gut and a trend of increased expression of growth hormone receptor gene in the liver as compared to the control with no significant effect on body weight, phytohemagglutinin response or haemagglutination inhibition titer. At d 25 of age, fecal E. coli count was significantly (P < 0.01) lower in the seaweed extract groups and the V group as compared to the control. It can be concluded that dried aqueous extract of E. denticulatum at 1 g kg -1 diet for 0-2 weeks can be used as an alternative to antibiotic growth promoter in broiler chickens to improve feed efficiency and reduce gut pathogen load, and the improved performance was associated with increased expression of gut immunity and growth hormone receptor genes.

An Immobilized Form of a Blend of Essential Oils Improves the Density of Beneficial Bacteria, in Addition to Suppressing Pathogens in the Gut and Also Improves the Performance of Chicken Breeding.

Antimicrobial growth promoters (AGP) are used in chicken production to suppress pathogens in the gut and improve performance, but such products tend to suppress beneficial bacteria while favoring the development and spread of antimicrobial resistance. A green alternative to AGP with the ability to suppress pathogens, but with an additional ability to spare beneficial gut bacteria and improve breeding performance is urgently required. We investigated the effect of supplementation of a blend of select essential oils (cinnamon oil, carvacrol, and thyme oil, henceforth referred to as EO; at two doses: 200 g/t and 400 g/t feed) exhibiting an ability to spare Lactobacillus while exhibiting strong E. coli inhibition ability under in vitro tests and immobilized in a sunflower oil and calcium alginate matrix, to broiler chickens and compared the effects with those of a probiotic yeast (Y), an AGP virginiamycin (V), and a negative control (C). qPCR analysis of metagenomic DNA from the gut content of experimental chickens indicated a significantly (p < 0.05) lower density of E. coli in the EO groups as compared to other groups. Amplicon sequence data of the gut microbiome indicated that all the additives had specific significant effects (DESeq2) on the gut microbiome, such as enrichment of uncultured Clostridia in the V and Y groups and uncultured Ruminococcaceae in the EO groups, as compared to the control. LEfSe analysis of the sequence data indicated a high abundance of beneficial bacteria Ruminococcaceae in the EO groups, Faecalibacterium in the Y group, and Blautia in the V group. Supplementation of the immobilized EO at the dose rate of 400 g/ton feed improved body weight gain (by 64 g/bird), feed efficiency (by 5 points), and cellular immunity (skin thickness response to phytoheamagglutinin lectin from Phaseolus vulgaris by 58%) significantly (p < 0.05), whereas neither yeast nor virginiamycin showed a significant effect on performance parameters. Expression of genes associated with gut barrier and immunity function such as CLAUDIN1, IL6, IFNG, TLR2A, and NOD1 were significantly higher in the EO groups. This study showed that the encapsulated EO mixture can improve the density of beneficial microbes in the gut significantly, with concomitant suppression of potential pathogens such as E.coli and improved performance and immunity, and hence, has a high potential to be used as an effective alternative to AGP in poultry.

Effects of Dietary Antimicrobial Growth Promoters on Performance Parameters and Abundance and Diversity of Broiler Chicken Gut Microbiome and Selection of Antibiotic Resistance Genes.

Antimicrobial growth promoters (AGPs) are commonly used in broiler production. There is a huge societal concern around their use and their contribution to the proliferation of antimicrobial resistance (AMR) in food-producing animals and dissemination to humans or the environment. However, there is a paucity of comprehensive experimental data on their impact on poultry production and the AMR resistome. Here, we investigated the effect of five antimicrobial growth promoters (virginiamycin, chlortetracycline, bacitracin methyl disalicylate, lincomycin, and tylosin) used in the commercial broiler production in the Indian subcontinent and in the different parts of the world for three consecutive production cycles on performance variables and also the impact on gut bacteria, bacteriophage, and resistome profile using culture-independent approaches. There was no significant effect of AGPs on the cumulative growth or feed efficiency parameters at the end of the production cycles and cumulative mortality rates were also similar across groups. Many antibiotic resistance genes (ARGs) were ubiquitous in the chicken gut irrespective of AGP supplementation. In total, 62 ARGs from 15 antimicrobial classes were detected. Supplementation of AGPs influenced the selection of several classes of ARGs; however, this was not correlated necessarily with genes relevant to the AGP drug class; some AGPs favored the selection of ARGs related to antimicrobials not structurally related to the AGP. AGPs did not impact the gut bacterial community structure, including alpha or beta diversity significantly, with only 16-20 operational taxonomic units (OTUs) of bacteria being altered significantly. However, several AGPs significantly reduced the population density of some of the potential pathogenic genera of bacteria, such as Escherichia coli. Chlortetracycline increased the abundance of Escherichia phage, whereas other AGPs did not influence the abundance of bacteriophage significantly. Considering the evidence that AGPs used in poultry production can select for resistance to more than one class of antimicrobial resistance, and the fact that their effect on performance is not significant, their use needs to be reduced and there is a need to monitor the spread of ARGs in broiler chicken farms.

Supplementation of sulfur and folic acid improves performance of broiler chicken fed suboptimal concentrations of dietary methionine.

BACKGROUND: Supplementation of sulfur (S) and folic acid (FA) are known to spare the dietary requirement of methionine (Met) in chicken. An experiment was conducted to determine the effects of feeding graded concentrations of FA (0, 2, 4, 6, 8 mg kg-1 ) and two concentrations of S (0 and 2 g kg-1 ) in 5 × 2 factorial design. Diet without FA and S was considered as negative control (NC). Another diet with the recommended concentration of Met was considered as the positive control (PC). Each diet was offered ad libitum from day 1 to 42 to 10 pens of 20 male chicks each. RESULTS: Broilers fed the NC diet had lower body weight gain (BWG), high feed conversion ratio, lower ready-to-cook yield, higher lipid peroxidation and reduced concentrations of protein and albumin in serum. BWG improved with FA at day 21 and showed a trend of improvement at day 42. Similarly, BWG improved with S supplementation at day 42. The FA and S interaction indicated a significant reduction in lipid peroxidation with S supplementation at the majority of FA levels. Serum protein fractions increased with increased dietary FA content and increased further with S supplementation. CONCLUSION: Based on the results, it is concluded that the combination of FA (4 mg kg-1 ) and S (2 g kg-1 ) improved BWG. Similarly, the feed efficiency with 6 mg FA alone or with S was similar to the PC group. The improvement was probably due to the increase in protein accretion and reduction in lipid peroxidation with FA and S supplementation. © 2022 Society of Chemical Industry.

Effect of methyl donors supplementation on performance, immune responses and anti-oxidant variables in broiler chicken fed diet without supplemental methionine.

OBJECTIVE: Methionine (Met) is involved in methyl group transfer besides protein synthesis. As the availability is limited and cost is high for synthetic Met, reductions in its inclusion in broiler diet may be possible by supplementing the low Met diets with methyl donors (MD) like betaine (Bet), folic acid (FA), vitamin B12 (B12), and biotin (Bio). An experiment was conducted to study the effects of supplementing the MD on performance (average daily gain [ADG], daily feed intake, feed efficiency [FE]), anti-oxidant variables, immune responses and serum protein concentration in broilers fed sub-optimal concentrations of dietary Met. METHODS: Maize-soybean meal diet was used as control (CD). Different MD like Bet (0.2%), B12 (0.1 mg), FA (4 mg), or Bio (1.5 mg/kg) were supplemented to basal diet (BD) having no supplemental Met. The BD without MD was kept for comparison. Each diet was fed ad libitum to 10 replicates of 25 chicks in each from 1 to 42 d of age. RESULTS: At the end of experiment, the ADG in MD group was higher than BD and lower than CD. The FE improved with FA or Bet compared to the BD. Breast meat weight was higher in Bet compared to the BD, while it was intermediate between BD and CD in other groups. The lipid peroxidation reduced with Bio, B12, or Bet, while the glutathione peroxidase activity improved with Bio or B12 compared to the BD. Lymphocyte proliferation improved with Bet compared to the BD. The serum protein concentrations increased with FA, Bio, or Bet compared to those fed BD. CONCLUSION: It can be concluded that the ADG can be improved partially with supplementation of MD while the FE improved with FA or Bet. Some MD also reduced the stress indices and improved immune responses compared to the BD fed broilers.

Gut Microbial Composition Differs Extensively among Indian Native Chicken Breeds Originated in Different Geographical Locations and a Commercial Broiler Line, but Breed-Specific, as Well as Across-Breed Core Microbiomes, Are Found.

Gut microbiota plays an important role in the health and performance of the host. Characterizations of gut microbiota, core microbiomes, and microbial networks in different chicken breeds are expected to provide clues for pathogen exclusion, improving performance or feed efficiency. Here, we characterized the gut microbiota of "finishing" chickens (at the end of production life) of indigenous Indian Nicobari, Ghagus, and Aseel breeds, originating from the Nicobari island, coastal India, and the Indian mainland, respectively, as well as a global commercial broiler line, VenCobb 400, using 16S rDNA amplicon sequencing. We found that diversity, as well as richness of microbiota, was higher in indigenous breeds than in the broiler line. Beta diversity analysis indicated the highest overlap between Ghagus and Nicobari breeds and a very low overlap between the broiler line and all indigenous breeds. Linear discriminant analysis effect size (LEfSe) revealed 82 breed- or line-specific phylotype operational taxonomic unit (OTU) level biomarkers. We confirm the presence of breed specific and across-breed core microbiomes. Additionally, we show the existence of breed specific complex microbial networks in all groups. This study provides the first (and comprehensive) insight into the gut microbiota of three indigenous breeds and one commercial broiler line of chickens reared without antimicrobials, and underscores the need to study microbial diversity in other indigenous breeds.

EEG based interpretation of human brain activity during yoga and meditation using machine learning: A systematic review.

OBJECTIVES: The present investigation is to study the impact of yoga and meditation on Brain waves concerning physical and mental health. There are mainly three stages (steps) in the brain wave classification:(i) preprocessing, ii) feature extraction, and iii) classification. This work provides a review of interpretation methods of Brain signals (Electroencephalogram (EEG)) EEG during yoga and meditation. Past research has revealed significant mental and physical advantages with yoga and meditation. METHODS: The research topic reviewed focused on the machine learning strategies applied for the interpretation of brain waves. In addressing the research questions highlighted earlier in the general introduction, we conducted a systematic search of articles from targeted scientific and journal online databases that included PubMed, Web of Science, IEEE Xplore Digital Library (IEEE), and Arxiv databases based on their relevance to the research questions and domain topic. The survey topic is relatively nascent, and therefore, the scope of the search period was limited to the 20-year timeline that was deemed representative of the research topic under investigation. The literature search was based on the keywords "EEG", "yoga*" and "meditation*". The key phrases were concatenated using Boolean expressions and applied to search through the selected online databases yielding a total of 120 articles. The online databases were selected based on the relevancy of content with the research title, research questions, and the domain application. The literature review search, process, and classification were carefully conducted guided by two defined measures; 1.) Inclusion criteria; and 2.) Exclusion criteria. These measures define the criteria for searching and extracting relevant articles relating to the research title and domain of interest. RESULTS: Our literature search and review indicate a broad spectrum of neural mechanics under a variety of meditation styles have been investigated. A detailed analysis of various mental states using Zen, CHAN, mindfulness, TM, Rajayoga, Kundalini, Yoga, and other meditation styles have been described by means of EEG bands. Classification of mental states using KNN, SVM, Random forest, Fuzzy logic, neural networks, Convolutional Neural Networks has been described. Superior research is still required to classify the EEG signatures corresponding to different mental states. CONCLUSIONS: Yoga practice may be an effective adjunctive treatment for a clinical and aging population. Advanced research can examine the effects of specific branches of yoga on a designated clinical grouping. Yoga and meditation increased overall healthy brain activity.

Effect of Dietary Supplementation of Organic Trace Minerals at Reduced Concentrations on Performance, Bone Mineralization, and Antioxidant Variables in Broiler Chicken Reared in Two Different Seasons in a Tropical Region.

Two experiments were conducted to study the performance, antioxidant activity, and bone mineral variables in broilers fed organic trace minerals (OTM) at lowered concentrations in the diet. In experiment 1, a total of 1500 day-old broiler male chicks were randomly distributed into six groups with 10 replicates of 25 chicks each and housed in floor pens (1.90 × 1.22 m). One group was fed the maize-soybean meal-based control diet, supplemented with inorganic trace mineral (ITM) premix containing Mn, Zn, Fe, Cu, Se, and Cr at 50, 45, 40, 7.5, 0.30, and 0.25 mg/kg, respectively. The remaining groups of chicks were fed the diets, where the ITM premix was replaced with OTM mix so as to provide the respective minerals at graded levels in the diet (100, 75, 50, 40, and 30% of the control diet). Similarly, in experiment II, a total of 1350 day-old male broiler chicks were divided at random into six groups with 9 replicates of 25 chicks each and fed the maize-soybean meal-based control diet having ITM at levels similar to those of experiment I or the test diets, where the ITM was replaced with OTM so as to provide the minerals at 100, 80, 60, 40, and 20% of the ITM-based control diet. Each diet was fed ad libitum from 0 to 42 days of age. The body weight gain, feed intake, and feed efficiency were not affected by supplementing OTM at 30 and 20% in experiments I and II, respectively. Slaughter variables, activities of glutathione peroxidase and super oxide dismutase, and lipid peroxidation and ferric reducing activity in serum were not affected by supplementing OTM at the lowest level of 20% in the diet. Similarly, bone-breaking strength, ash, and Mn contents were not affected by supplementing OTM at 20% of the mineral concentration in control diet containing ITM. Deposition of Ca, P, Cu, and Fe in tibia ash increased with increased concentration of OTM in the diet. However, Zn content in tibia ash reduced with reduction in OTM level in the diet. Based on the results, it is concluded that trace mineral supplementation in organic form even at 20% of the concentration recommended for ITM may be sufficient in maize-soybean meal-based diet to support the optimum growth, bone ash, bone strength, and serum antioxidant status in commercial broilers.

Dietary supplementation of extracts of red sea weed (Kappaphycus alvarezii) improves growth, intestinal morphology, expression of intestinal genes and immune responses in broiler chickens.

BACKGROUND: Effects of supplementation of dried alkaline (referred to as MVP1) and aqueous (referred to as PBD1) extracts of Kappaphycus alvarezii, were evaluated in broiler (Vencobb 400) chickens (1-35 days post-hatch). In experiment I, each of the seven diets (basal diet with three levels (0.5, 1.5 or 5.0 g kg-1 diet) of MVP1 or PBD1 and a negative control was fed to 12 pen replicates containing five birds in each. In experiment II, each of three diets [a negative control, and PBD1 at two levels (1.0 or 1.5 g kg-1 diet)] was fed to 16 pen replicates of five chicks in each. RESULTS: Concentrations of total phenolics, phycobillins and free radical scavenging activity were higher (P < 0.01) whereas carrageenan was lower in PBD1 than in MVP1. In the experiment I, PBD1 at 1.5 g kg-1 diet improved (P < 0.05) body weight (BW) (7.11% higher). In the experiment II, both the treatments improved (P < 0.01) BW (9.18% and 8.47%, respectively) compared to the control. The group fed with PBD1@ 1.0 g kg-1 had higher (P < 0.05) haemagglutination inhibition titre, expression of intestinal claudin 2, TLR2A, NOD1, avian beta defensin 4, interleukin 2 and interleukin 6 genes than control. Treatments did not influence feed efficiency or levels of most of the antioxidant enzymes. Villus width and crypt depth were significantly higher in the group fed with 1.5 g kg-1 of PBD1. CONCLUSION: Supplementing dried aqueous extract of K. alvarezii at 1 g kg-1 diet may be an effective strategy to increase growth and immunity in broiler chickens. © 2020 Society of Chemical Industry.

Effect of incremental levels of dietary total sulfur amino acids on performance of white leghorn laying hens fed diets containing guar (Cyamopsis tetragonoloba) meal.

BACKGROUND: The present study evaluated the potential of increasing dietary levels of the most limiting amino acids such as total sulfur amino acids (TSAA) with respect to mitigating the adverse effects of the inclusion of guar meal (GM) in the diet of WL layers on egg production, feed efficiency and egg weight. In total, 4928 WL (Lohmann L) hens (32 week of age) were randomly allotted to eight treatments with seven replicates of 88 birds. The dietary treatments were a set of four maize-soybean meal based diets containing four graded concentrations of TSAA (5.85, 6.18, 6.51 and 6.84 g kg-1 ) and another set of four diets containing 100 g kg-1 guar meal (GM) with similar concentrations of TSAA. RESULTS: The inclusion of 100 g kg-1 GM in the diet caused a 20%, 23%, 35.5% and 6.7% reduction in egg production, daily egg mass, feed efficiency and egg weight, respectively, when the diet contained 5.85 g kg-1 TSAA. Increasing TSAA levels in GM groups reduced the adverse effects on egg production and feed efficiency 50%, with no effect on egg weight. The TSAA requirement of WL birds fed 10% GM was estimated as 6.28-7.39 g kg-1 diet at different ages. CONCLUSION: Increasing dietary TSAA to 7.39, 6.28, 6.53 and 6.37 g kg-1 or dTSAA (digestible TSAA) to 6.53, 5.32, 5.80 and 5.63 g kg-1 during 33-36, 37-40, 41-44 and 45-48 weeks of age, respectively, may be an effective and practical strategy for reducing the adverse effects of inclusion of 100 g kg-1 GM in the diet of WL layers. © 2018 Society of Chemical Industry.

A dithieno[3,2-b:2',3'-d]pyrrole based, NIR absorbing, solution processable, small molecule donor for efficient bulk heterojunction solar cells.

A novel, NIR absorbing organic small molecular donor material denoted as ICT3 with an A-D-D-D-A architecture having dithieno[3,2-b:2',3'-d]pyrrole (DTP) and butylrhodanine as donor and acceptor moieties, respectively, is synthesized and its thermal, photophysical, electrochemical and photovoltaic properties are explored. ICT3 has excellent stability over a broad range of temperatures with a decomposition temperature (Td corresponds to 5% weight loss) of 372 °C, soluble in most common organic solvents (solubility up to 30 mg mL-1) and suitable for solution processing during device fabrication. ICT3 has broad (520-820 nm) and intense visible region absorption (molar excitation coefficient is 1.69 × 105 mol-1 cm-1) and has suitable HOMO and LUMO energy levels with the [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) acceptor for efficient exciton dissociation and charge transfer. Bulk heterojunction solar cells (BHJSCs) with an indium tin oxide (ITO)/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS)/ICT3:PC71BM/poly(9,9-bis(3'-(N,N-dimethylamino)propyl)fluorene-2,7-diyl)-alt-(9,9-dioctylfluorene-2,7-diyl) (PFN)/aluminium (Al) structure are fabricated and the BHJSCs with the active layer as cast from chloroform solution displayed a power conversion efficiency (PCE) of 3.04% (JSC = 8.22 mA cm-2, VOC = 0.86 V and FF = 0.43). Annealing the active layer significantly improved the PCE of these BHJSCs. While thermal annealing of the active layer improved the PCE of the BHJSCs to 4.94%, thermal followed by solvent vapour annealing enhanced the PCE to 6.53%. X-ray diffraction and atomic force microscopy analyses are carried out on the active layer and these results revealed that annealing treatment improves the crystallinity and nanoscale morphology of the active layer, enriches the device exciton generation and dissociation efficiency, charge transport and collection efficiency and reduces carrier recombination. The observed higher PCE (6.53%) of the BHJSCs having ICT3 with a DTP donor moiety broadens the scope to develop new, efficient DTP based small molecular donor materials for BHJSCs.

Effect of supplementing different concentrations of organic trace minerals on performance, antioxidant activity, and bone mineralization in Vanaraja chickens developed for free range farming.

An experiment was conducted to determine the performance, antioxidation activity, and bone mineral content in Vanaraja chickens fed diet supplemented with organic trace minerals (oTM) at reduced levels. A total of 360 day-old chicks were selected and distributed randomly into 60 battery brooder pens. A maize-soybean meal-based control diet was supplemented with inorganic trace minerals (iTM), i.e., Mn, Zn, Fe, and Cu at 50, 45, 40, and 7.5 mg/kg, respectively, and varying concentration of oTM, i.e., Zn, Mn, Cu, Fe, I, Se, and Cr at 45, 50, 7.5, 40, 2, 0.30, and 0.25 mg/kg (diet II); 33.75, 37.50, 5.63, 30.0, 1.50, 0.23, and 0.19 mg/kg (diet III); 22.5, 25.0, 3.75, 20.0, 1.0, 0.15, and 0.13 mg/kg (diet IV); 18.0, 20.0, 3.0, 16.0, 0.80, 0.12, and 0.10 mg/kg (diet V); and 13.5, 15.0, 2.25, 12.0, 0.60, 0.09, and 0.08 mg/kg (diet VI), respectively. Each diet was allotted randomly to ten replicates and fed ad libitum from 1 to 42 days of age. The body weight at 14, 28, and 42 days was not affected by reducing the supplementation of oTM concentration in the diets. Similarly, feed intake at 14 days of age was not affected but reduced significantly (P < 0.05) in the group fed diet IV (50% oTM) compared to that in the other groups. The higher feed conversion ratio and increased concentration of Ca, P, and trace minerals in tibia were evident in the group fed oTM-supplemented diets compared to the diet containing iTM. Activities of glutathione peroxidase and ferric reducing ability in plasma did not differ in the groups fed on lower concentration of oTM compared to those fed on diet I (control diet). Therefore, it is concluded that the dietary supplementation of trace minerals can be reduced greatly when supplemented as organic form without affecting growth and antioxidant status in Vanaraja chickens.

Effect of supplementing organic selenium on performance, carcass traits, oxidative parameters and immune responses in commercial broiler chickens.

An experiment was conducted to determine the effect of supplementing various concentrations (0, 100, 200, 300, or 400 µg/kg diet) of organic Se on growth performance, carcass traits, oxidative stress, and immune responses in commercial broiler chickens reared in open-sided poultry house under tropical climatic conditions. Each diet was fed ad libitum to eight replicates consisting of six birds in each pen from 1 to 42 d of age. Body weight gain and feed efficiency, and relative weight of liver, abdominal fat and ready to cook yields were not affected (p>0.05) by organic Se supplementation to broiler diets. Lipid peroxidation in plasma decreased, while activities of glutathione peroxidase and glutathione reductase in plasma increased (p<0.01) linearly with Se concentration in diet. The ratios between heterophyls and lymphocytes and relative weight of lymphoid organs (bursa, spleen, and thymus), and antibody production to Newcastle disease vaccination were not affected (p>0.05) by Se supplementation to broiler diets. However, the cell-mediated immunity (lymphocyte proliferation ratio) increased (p<0.01) linearly with dietary Se concentration. The results of the present study indicate that the supplementation of Se did not influence body weight and feed efficiency. However, supplementation of Se increased antioxidant status and lymphocyte proliferation in broiler chickens.

Effect of nutrient density on production performance, egg quality and humoral immune response of brown laying (Dahlem Red) hens in the tropics.

A study was conducted to evaluate the effect of various concentrations of metabolizable energy (ME) with graded incremental levels of crude protein (CP) and essential amino acids (lysine and methionine) on production performance, egg quality and humoral immune response of Dahlem Red laying hens. Four experimental diets based on maize-soybean meal-deoiled rice bran were prepared. Diet 1 was fed as a control diet containing 2,600 kcal ME/kg, 15% CP, 0.75% Lys and 0.36% Met, and in the other three diets (D2, D3 and D4), concentrations of the above nutrients were increased by 2.5%, 5.0% and 7.5%, respectively. The levels of Ca (3.5%) and available P (0.32) were constant in all the diets. Each diet was offered ad libitum from 28 to 40 weeks of age to eight replicates containing six birds in each replicate. The egg production, egg weight and egg mass (in grams of egg per hen per day) were not affected by increasing the nutrient density up to 7.5% (2,795 kcal ME/kg diet) compared to the control group (2,600 ME/kg diet). However, feed consumption and feed efficiency (in grams of egg per gram of feed) were influenced by the variation in the nutrient density of diets. As the nutrient density increased by 5% (2,730 ME/kg diet), birds consumed significantly (P < 0.001) less feed. The birds in the 7.5% higher density group produced significantly (P < 0.05) higher egg mass per unit feed consumption compared to the control diet. Increasing nutrient density up to 7.5% had no effect on relative weight of albumen, yolk or shell. The Haugh unit, yolk colour and shell thickness were also not affected due to variation in the nutrient density. The humoral immune response measured at 34 and 40 weeks was progressively improved by increasing the nutrient density up to 5%. Increasing the nutrient density beyond 5% in the diet had no further influence on the humoral immune response. Based on the results of the present study, it can be concluded that Dahlem Red laying hens required 2,795 kcal/kg ME, 16% CP, 0.8% lysine and 0.4% methionine for eliciting optimum performance and immune response during 28 to 40 weeks of age.