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Bone issues such as osteoporosis are major concerns for the laying hen industry. A study was conducted to improve bone-health in pullets. A total of 448 one-day-old Hyline W36 pullets were randomly assigned to four treatments (8 rep; 14 birds/rep) until 17 weeks (wks). Dietary treatments were: 1) vitamin D3 at (2,760 IU/kg) (D), 2) vitamin D3 (2,760 IU/kg)+62.5 mg 25-(OH)D3/ton (H25D), 3) vitamin D3 (2,760 IU/kg) + 62.5 mg 25-(OH)D3/ton + high Ca&P (H25D + Ca/P), and 4) vitamin D3 (2,760 IU/kg) + high Ca&P (D + Ca/P). The high calcium (Ca) and phosphorus (P) diet was modified by increasing both high calcium and phosphorus by 30% (2:1) for the first 12 wks and then only increasing P for 12–17 wks to reduce the Ca to P ratio. At 17 wk, growth performance was measured, whole body composition was measured by dual energy x-ray absorptiometry (DEXA), and femur bones were scanned using Micro-computed tomography (Micro-CT) for bone 3D structure analyses. The data were subjected to a one-way ANOVA using the GLM procedure, with means deemed significant at p < 0.05. There was no significant outcome for growth performance or dual energy x-ray absorptiometry parameters. Micro-computed tomography results indicated that the H25D + Ca/P treatment had lower open pore volume space, open porosity, total volume of pore space, and total porosity in the cortical bone compared to the D + Ca/P. It also showed that a higher cortical bone volume/tissue volume (BV/TV) in the H25D + Ca/P than in the D + Ca/P. Furthermore, the H25D + Ca/P treatment had the lowest trabecular pattern factor and structure model index compared to the other treatments, which indicates its beneficial effects on trabecular structural development. Moreover, the H25D + Ca/P had a higher trabecular percentage compared to the D and 25D, which suggests the additional high calcium and phosphorus supplementation on top of 25D increased trabecular content in the cavity. In conclusion, the combination of 25D with higher levels of high calcium and phosphorus could improve cortical bone quality in pullets and showed a beneficial effect on trabecular bone 3D structural development. Thus, combination of a higher bio-active form of vitamin D3 and higher levels of high calcium and phosphorus could become a potential feeding strategy to improve bone structural integrity and health in pullets.

Organic acids and essential oils are commonly used in the poultry industry as antimicrobials and for their beneficial effects on gut health, growth performance, and meat quality. A common postharvest storage fungal colonist, Aspergillus flavus, contaminates corn, the primary component of poultry feed, with the highly detrimental mycotoxin, aflatoxin. Aflatoxin adversely affects poultry feed intake, feed conversion efficiency, weight gain, egg production, fertility, hatchability, and poultry meat yield. Both organic acids and essential oils have been reported to inhibit the growth of A. flavus. Thus, we evaluated if the inhibitory synergy between combined essential oils (cinnamon, lemongrass, and oregano) and organic acids (acetic, butyric, and propionic) prevents A. flavus growth. The study confirmed that these compounds inhibit the growth of A. flavus and that synergistic interactions do occur between some of them. Overall, cinnamon oil was shown to have the highest synergy with all the organic acids tested, requiring 1000 µL/L air of cinnamon oil and 888 mg/kg of butyric acid to fully suppress A. flavus growth on corn kernels. With the strong synergism demonstrated, combining certain essential oils and organic acids offers a potentially effective natural method for controlling postharvest aflatoxin contamination in poultry feed.

The objective of present study was to investigate the effects of alpha-lipoic acid (ALA) dietary supplementation on the behavior, physiological and oxidant stress indicators, and bone quality in broilers under high stocking density (HSD) stress. A total of one thousand eight hundred 22-day-old Arbor Acres male broiler chicks were randomly allocated to 18 pens (2.97 × 2.03 m) in 3 groups: 14 birds/m2 (NSD, normal stocking density) or 18 birds/m2 (HSD) or 18 birds/m2 plus 300 mg/kg ALA dietary supplement (HSD + ALA, high stocking density + alpha-lipoic acid). Each treatment had 6 replicates, and the experiment lasted 3 wk. The HSD group was significantly lower than the NSD group (P 0.05) in the standing and preening behavior, alkaline phosphatase activity, PTH concentration, H/L ratio, CAT, T-AOC, MDA, SOD, and GSH-Px. These results indicate that the increase of stocking density lowered the bone quality, increased the physiological and oxidative stress indicators, and modified the behavior of broilers, whereas ALA dietary supplementation could counteract the reduction in the performance and physiological responses of broilers under high-density environmental stress.

This study evaluated the effects of maternal fish oil supplementation rich in n-3 PUFA on the performance and bone health of offspring broilers at embryonic development stage and at market age. Ross 708 broiler breeder hens were fed standard diets containing either 2.3% soybean oil (SO) or fish oil (FO) for 28 days. Their fertilized eggs were collected and hatched. For a pre-hatch study, left tibia samples were collected at 18 days of incubation. For a post-hatch study, a total of 240 male chicks from each maternal treatment were randomly selected and assigned to 12 floor pens and provided with the same broiler diets. At 42 days of age, growth performance, body composition, bone microstructure, and expression of key bone marrow osteogenic and adipogenic genes were evaluated. One-way ANOVA was performed, and means were compared by student's t-test. Maternal use of FO in breeder hen diet increased bone mineral content (p < 0.01), bone tissue volume (p < 0.05), and bone surface area (p < 0.05), but decreased total porosity volume (p < 0.01) during the embryonic development period. The FO group showed higher body weight gain and feed intake at the finisher stage than the SO group. Body composition analyses by dual-energy X-ray absorptiometry showed that the FO group had higher fat percentage and higher fat mass at day 1, but higher lean mass and total body mass at market age. The decreased expression of key adipogenic genes in the FO group suggested that prenatal FO supplementation in breeder hen diet suppressed adipogenesis in offspring bone marrow. Furthermore, no major differences were observed in expression of osteogenesis marker genes, microstructure change in trabecular bone, or bone mineral density. However, a significant higher close pores/open pores ratio suggested an improvement on bone health of the FO group. Thus, this study indicates that maternal fish oil diet rich in n-3 PUFA could have a favorable impact on fat mass and skeletal integrity in broiler offspring.

ABSTRACT: β-mannan is a nonstarch polysaccharide found in hulled and dehulled soybeans that can survive drying-toasting phase of processing soybeans and have antinutritive effects in poultry. β-mannanase is an active enzyme (endohydrolase) that can hydrolyze β-mannan to reduce its antinutritional effects. Two experiments were conducted to evaluate the effects of β-mannanase supplementation in low energy/protein diets on egg production, egg quality, and apparent ileal digestibility of the dry matter (DM), crude protein (CP), and amino acids in 21-week-old Single Comb White Leghorn hens (Hy-Line W-36). A total of 192 hens (8 replicates of 6 hens per treatment) for a production study (Exp. 1) and a total of 64 hens (8 replicates of 2 hens per treatment) for a digestibility study (Exp. 2) were randomly allocated to 4 experimental treatments in a 2 × 2 factorial arrangement. Four dietary treatments were control (CS) based on corn and 44% CP soybean meal (ME: 2,850 kcal/kg CP: 18.5%) and CS-low energy/protein (CSL) (ME: 2,750 kcal/kg CP: 17.5%), with or without 0.05% β-mannanase enzyme. Hens were fed the experimental diets for 14 d for the digestibility study and 8 wk for the production study. Hen-day egg production (HDEP), weekly feed intake, FCR, and biweekly egg quality parameters were measured. Significant interaction on feed intake (P < 0.01) was observed between energy/protein and enzyme. At 3, 6 and 8 wk, the feed intake and FCR of CSL with enzyme were significantly lower than those of CSL without enzymes. The main effects indicated that birds fed diets without inclusion of β-mannanase had higher feed intake than those fed diets with enzymes at 4, 7, and 8 wk. The inclusion of β-mannanase significantly increased (P < 0.05) HDEP at 2, 3, 5, and 7 wk. However, there was no significant effect of nutrient density or enzyme supplementation on egg quality parameters. The digestibility study showed that the inclusion of β-mannanase significantly improved (P < 0.01) apparent ileal digestibility of lysine, histidine and tryptophan in the diet. The results of these experiments indicate that supplementation of β-mannanase could reduce the feed intake and FCR and improve HDEP and apparent ileal digestibility of key amino acids in corn/soy diets fed to laying hens.

This study aimed to determine the effect of the housing environment and laying hen strain on tibia and femur properties. A 3 × 2 factorial arrangement of 3 housing environments (conventional cages [CC], enriched colony cages [EC], and free range [FR]) and 2 laying hen strains (Hy-Line W-36 [W-36] and Hy-Line Brown [HB]) in a completely randomized design was conducted from 32 to 85 wk of age. Six left tibias were collected at 8 different time points (38, 45, 52, 59, 65, 72, 79, and 85 wk of age), whereas 6 left femurs were collected at 3 time points (38, 65, and 85 wk of age). Tibias were evaluated for tibia breaking strength (TBS) and ash percentage, whereas femurs were evaluated for bone mineral density (BMD), bone mineral content, bone volume as a fraction tissue volume, and porosity percentage from total, cortical, medullary, and trabecular bones. The higher TBS (P = 0.0005) and ash percentage (P = 0.045) was observed in hens raised in FR systems compared with those raised in the CC. Overall, TBS of W-36 hens was significantly greater than that of HB hens (P 0.05). An interaction between the housing environment and hen strain was observed for BMD (P = 0.04), wherein W-36 hens raised in the FR system had higher BMD than HB hens. Similarly, hens raised in FR systems had higher trabecular bone volume than those raised in CC (P = 0.022). Hen strain influenced total and cortical bone properties: BMD, bone volume as a fraction tissue volume, and porosity percentage, wherein W-36 hens had better properties than HB hens (P

1,25-dihydroxyvitamin D3 (1,25OHD) has been suggested to play an important role in osteogenic differentiation and mineralization. However, limited data have been reported in avian species. In the present study, the direct role of 1,25OHD on osteogenic differentiation and mineralization in chicken mesenchymal stem cells (cMSCs) derived from day-old broiler bones was investigated. cMSCs were treated with control media (C), osteogenesis media (OM), OM with 1, 5, 10, and 50 nM 1,25OHD, respectively. The messenger RNA (mRNA) samples were obtained at 24 and 48 h and 3 and 7 days to examine mRNA expression of key osteogenic genes [runt related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 2 chain (COL1A2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and alkaline phosphatase (ALP)]. Cells were stained at 7, 14, and 21 days using Von Kossa (mineralization), Alizarin Red (AR; mineralization), and Alkaline Phosphatase (early marker) staining methods. From the mRNA expression results, we found a time-dependent manner of 1,25OHD on osteoblast differentiation and mineralization. In general, it showed an inhibitory effect on differentiation and mineralization during the early stage (24 and 48 h), and a stimulatory effect during the late cell stage (3 and 7 days). The staining showed 1,25OHD had an inhibitory effect on ALP enzyme activities and mineralization in a dosage-dependent manner up to 14 days. However, at 21 days, there was no difference between the treatments. This study provides a novel understanding of the effects of 1,25OHD on osteogenic differentiation and mineralization of cMSCs depending on cell stage and maturity.

A study was conducted to understand the effects of 25-hydroxyvitamin D3 (25OHD) and 1,25-dihydroxyvitamin D3 (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D3 metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses (n=8). Gene expression for key osteogenesis markers (RUNX2: runt-related transcription factor 2; BMP2: bone morphogenetic protein 2; COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatese), adipogenesis markers (PPAR-γ: peroxisome proliferator-activated receptor gamma; FASN: fatty acid synthase; and FABP4: fatty acid binding protein 4), myogenesis markers (MYOG: myogenin; MYOD1: myogenic differentiation 1; and MYF5: myogenic factor 5), and the enzyme responsible for vitamin D3 inactivation (CYP24A1: cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker, ALP, was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers (FAPB4 and FASN). In contrast, the expression of myogenesis markers (MYOD1 and MYF5) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker, SPP1, was increased by 25D-H. MYOD1 and MYF5 were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D3 metabolite retention was assessed by measuring CYP24A1 expression. At 1h, there were no differences in CYP24A1 expression. At 3h, all treatments upregulated CYP24A1 expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher CYP24A1 expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.

Cobb broilers (COBB) have been heavily selected for their production performance in the past several decades, while the Athens Canadian Random Bred (ACRB) chickens, a meat-type breed, have been kept as a non-selected control strain. The purpose of this study was to compare these two lines of chickens at late embryonic development and identify the molecular markers and fatty acid profiles underlining their differences in growth performance due to selection. Fertilized eggs of the ACRB (n = 6) and COBB (n = 6) were used at 14 and 18 embryonic days. Genes involved in lipogenesis and myogenesis were measured using quantitative real-time reverse transcroption-polymerase chain reaction (RT-PCR), and fatty acid (FA) compositions of egg yolk, muscle, and liver were measured using gas chromatography. COBB had higher egg weight, embryo weight, and breast and fat ratio. The gene expression in the liver showed an interaction between age and breed on FASN expression, with the highest level in COBB at E18. ACRB had higher ApoB and MTTP expression, but lower SREBP-1 expression compared to COBB. No difference was found in myogenesis gene expression in the muscle between two breeds. For the FA composition, muscle was largely affected by both breed and age. Yolk and liver were affected mainly by breed and age, respectively. Constant interaction effects in docosahexaenoic acid (DHA), indicating the highest level in all the tested tissues of ACRB at E14 and the constant main effects with higher myristic, palmitic, and gondoic, but lower linolenic acid in the liver and yolk of COBB compared to the levels in those of ACRB. Finally, fat accumulation in the liver had no obvious difference between the breeds but was higher when embryo was older. In conclusion, broiler breed affects egg, embryo, and tissue weight, as well as FA composition in initial egg yolk and throughout the embryonic development. The highest docosahexaenoic percentage was observed in ACRB, indicating that genetic selection may result in fatty acid profile changes such as lower DHA content in chicken tissues and eggs.

An experiment was conducted to evaluate the effect of different levels of inorganic copper and zinc on growth performance, intestinal permeability, intestinal lesion scores, oocyst shedding, antioxidant properties and bone quality in broilers challenged with Eimeria spp. A total of 360 d-old male Cobb broiler chickens were housed in floor cages for 12 days at the Poultry Research Center. At 12 days of age, birds were placed in grower Petersime batteries and distributed in a completely randomized design with 10 birds per cage, six replicates per treatment, and six treatments. There were six corn-soybean meal-based dietary treatments: non-challenged control (NC), challenged control (CC), 100 ppm Cu (100 Cu), 150 ppm Cu (150 Cu), 80 ppm Zn (80 Zn), and 100 ppm Zn (100 Zn). Broilers received the treatment diets for 9 days (12–20d). Birds, except NC, were challenged with Eimeria maxima (50,000 oocysts/bird), Eimeria tenella (50,000 oocysts/bird), and Eimeria acervulina (250,000 oocysts/bird) on 14d. On 20d, the growth performance was recorded, and one bird/cage was used for analysis of intestinal permeability, antioxidant properties and bone quality. Lesion score was recorded at 20 days of age in eight birds/cage. The means were subjected to ANOVA and, when significant, compared by Duncan's test. Intestinal permeability was significantly improved when birds received the 100 Zn diet (P < 0.05). In addition, lesion scores on duodenum were reduced when broilers received diets 150 Cu as compared to CC diet (P < 0.05). However, growth performance was not positively influenced by inclusion of inorganic minerals as compared to the NC diet. Furthermore, activity of superoxide dismutase and bone quality were not affected, whereas glutathione status was improved with mineral supplementation in all groups. This study showed that Cu and Zn supplementation improves intestinal integrity during the Eimeria spp. infection, suggesting that Cu and Zn supplementation would be a potential strategy to reduce detrimental effects of Eimeria infection in broilers.

This study was designed to evaluate the effects of dietary supplemental DL-methionine (MET) on live performance and meat yield for broilers raised to a common weight. A total of 1552 one-day old Ross 708, sexed broilers were randomly distributed to 32 pens resulting in eight treatments (TRT) of four replicates with 44 male or 53 female/pen. A randomized complete block with a 2 × 4 (sex × 4 MET levels 0, 0.5, 1, and 2 g/kg) factorial arrangement of TRT was used. A common weight of 2400 g was approached by day 46 (1 and 2 g MET/kg feed) and day 48 (0 and 0.5 g MET/kg feed). Supplementation of MET at 1, and 2 g/kg had a lower (p < 0.01) feed conversion ratio (FCR) at day 46/48 than broilers fed 0.5 g MET/kg. Broilers without supplemental MET had the worst (p < 0.01) feed conversion and average daily gain (ADG) at day 46/48. Birds fed 0 g MET/kg of feed had lower (p < 0.05) whole eviscerated carcass without giblets (WOG), yield than birds fed 2 g MET/kg of feed. Additionally, birds fed 0 g MET/kg of feed had lower (p < 0.05) breast fillet and tender percent yields than birds fed supplemental MET. Elimination of MET from organic broiler diets resulted in reduced ADG, breast fillet yield and feed efficiency of meat yield of broilers raised to day 46/48. Reduction in MET supplementation below current levels reduced the efficiency of meat production of organic broilers raised to day 46/48.

Changes in medullary and cortical bone structure with age remain unclear. Twenty Hy-Line W36 hens, 25 or 52 weeks of age, were euthanized, and both tibiae were collected when an egg was present in the magnum. Serial cross sections of the tibiae were stained with Alcian blue. The bones were scanned using micro-computed tomography. Trabecular width (Tb.Wi) was significantly higher (p < 0.05) in 25-week-old hens, whereas medullary bone tissue volume (TV) was significantly higher (p < 0.01) in 52-week-old hens. 25-week-old hens had significantly higher (p < 0.01) bone volume fraction (BVF = calcified tissue / TV). Moreover, the cortical bone parameters were significantly higher (TV and bone mineral content (BMC) at p < 0.05, and bone volume (BV) and BVF at p < 0.01) in younger hens. Open porosity and total porosity, which indicate less density, were significantly higher (p < 0.01) in older hens. Older hens showed significantly higher (p < 0.01) tibial diaphysis TV than younger hens. Younger hens had significantly higher (p < 0.01) BV, BVF and bone mineral density (BMD) of the tibial diaphysis. These findings reveal that reductions in medullary bone quality might be associated with age-related low estrogen levels and stimulation of osteoclastic bone resorption by parathyroid hormone. Cortical bone quality decreased with enlargement of the Haversian canals and loss of volume, with a longer egg-laying period leading to osteoporosis.

Chicken mesenchymal stem cells (MSCs) can be used as an avian culture model to better understand osteogenic, adipogenic, and myogenic pathways and to identify unique bioactive nutrients and molecules which can promote or inhibit these pathways. MSCs could also be used as a model to study various developmental, physiological, and therapeutic processes in avian and other species. MSCs are multipotent stem cells that are capable of differentiation into bone, muscle, fat, and closely related lineages and express unique and specific cell surface markers. MSCs have been isolated from numerous sources including human, mouse, rabbit, and chicken with potential clinical and agricultural applications. MSCs from chicken compact bones have not been isolated and characterized yet. In this study, MSCs were isolated from compact bones of the femur and tibia of day-old male broiler chicks to investigate the biological characteristics of the isolated cells. Isolated cells took 8–10 days to expand, demonstrated a monolayer growth pattern and were plastic adherent. Putative MSCs were spindle-shaped with elongated ends and showed rapid proliferation. MSCs demonstrated osteoblastic, adipocytic, and myogenic differentiation when induced with specific differentiation media. Cell surface markers for MSCs such as CD90, CD105, CD73, CD44 were detected positive and CD31, CD34, and CD45 cells were detected negative by PCR assay. The results suggest that MSCs isolated from broiler compact bones (cBMSCs) possess similar biological characteristics as MSCs isolated from other chicken tissue sources.

Development of antibiotic resistance in foodborne pathogens, Salmonella spp. and Campylobacter, is a public health concern. Public demand to reduce the use of sub-therapeutic antibiotic growth promoters (AGP) in poultry feeding has resulted in greater adoption of antibiotic-free poultry production systems. There is a need to understand the effects of AGP removal from poultry feed on gut microbiota and its impact on prevalence of foodborne pathogens. The effect of antibiotic withdrawal from poultry feed on gut microbial community, host performance and immunity, and prevalence of Salmonella and Campylobacter was evaluated. Birds were raised on three phase diets (starter [d0-22], grower [d23-35] and finisher [d36-42]) with and without bacitracin dimethyl salicyclate (BMD). At early growth stage, bird performance was improved (P ≤ 0.05) with BMD treatment, whereas performance was better (P ≤ 0.05) in control group (no BMD in the feed) at the time of commercial processing. Acetate and butyrate production was affected (P ≤ 0.05) by age, whereas propionate production was affected (P ≤ 0.05) by both the treatment and age. The bacterial communities in the cecum were more diverse (P ≤ 0.001) and rich compared to the ileal communities, and they shifted in parallel to one another as the chicks matured. Differences in diversity and species richness were not observed (P > 0.05) between the BMD-fed and control groups. Comparing all ages, treatments and diets, the composition of cecal and ileal bacterial communities was different (P ≤ 0.001). Inclusion of BMD in the feed did not affect the bacterial phyla. However, predictable shift in the ileal and cecal bacterial population at lower taxonomic level was observed in control vs BMD-fed group. Cytokines gene expression (IL-10, IL-4, IFN-γ, beta-defensin, and TLR-4) was affected (P≤ 0.05) in the BMD-fed group at early stages of growth. The prevalence of foodborne pathogens, Campylobacter spp. and Salmonella spp. showed higher abundance in the ilea of BMD-fed chicks compared to control group. Overall, this study provided insight of the impact of AGP supplementation in the feed on gut microbial modulations, bird performance, host immunity and pathogen prevalence. This information can assist in designing alternative strategies to replace antibiotics in modern poultry production and for food safety.