Characterization of the mechanical properties of high-moisture meat analogues using low-intensity ultrasound: Linking mechanical properties to textural and nutritional quality attributes.

Plant-based meat analogues offer possible alternatives to meat consumption. However, many challenges remain to produce a palatable meat analogue as well as to understand the roles of different processing steps and ingredients on both the texture and nutritional properties of the final product. The goal of this paper is to help with addressing these challenges by using a low-intensity ultrasonic transmission technique, both online and 24 h after production, to investigate high-moisture meat analogues made from a blend of soy and wheat proteins. To understand the ultrasonic data in the context of traditional characterization methods, physical properties (meat analogue thickness, density, peak cutting force) and protein nutritional quality attributes of the meat analogues were also characterized separately. The ultrasonic velocity was found to decrease with the feed moisture content and to be strongly correlated (r = 0.97) with peak cutting force. This strong correlation extends over a wide range of moisture contents from 58% to 70%, with the velocity decreasing from about 1730 m/s to 1660 m/s over this range. The protein quality was high for all moistures, with the highest amino acid score and in vitro protein digestibility being observed for the highest moisture content treatment. The accuracy of the ultrasonic measurements was enhanced by the development of an innovative non-contact method, suitable for materials exhibiting low ultrasonic attenuation, to measure the meat analogue thickness ultrasonically and in a sanitary fashion - an advance that is potentially useful for online monitoring of production problems (e.g., extruder barrel-fill and cooling-die temperature issues). This study demonstrates, for the first time, the feasibility of using ultrasonic transmission techniques to measure both velocity and sample thickness simultaneously and provide information in real time during production that is well correlated with some textural and nutritional attributes of meat analogues.

Effects of phytase supplementation on production performance, egg and bone quality, plasma biochemistry and mineral excretion of layers fed varying levels of phosphorus.

Excessive fecal excretion of phosphorus (P) has increasingly become an environmental issue due to oversupply of P in layer rations, and thus it is imperative to minimize safety margins for P to ensure the sustainability of the egg industry. In this study, a 12-week feeding trial (22 to 34 weeks of age) was conducted to evaluate the effects of phytase supplementation on production performance, plasma biochemistry, egg and bone quality and P excretion of laying hens fed various levels of non-phytate P (NPP). Forty-eight Lohmann white laying hens were randomly allocated to one of six corn-soybean meal-oat-based diets: diets containing 2.0, 2.5 or 3.0 g/kg NPP without phytase, and diets containing 1.0, 1.5 or 2.0 g/kg NPP with phytase (1 000 U/kg diet) where phytase inclusion was expected to provide 1.0 g/kg NPP to laying hens, thus making the phytase-unsupplemented treatment served as a control for the phytase-supplemented treatment accordingly. Productive performance was recorded during the experimental period. Blood and egg samples were collected, and digestibility studies were conducted at weeks 6 and 12 of the experiment. Bone mineralization was evaluated at the end of the experiment. Egg weight and egg production, feed consumption, BW and feed conversion ratio of laying hens fed lower NPP diets supplemented with phytase were comparable to those of hens fed high NPP phytase-unsupplemented controls. Eggshell thickness, specific gravity, Haugh units, tibia bone mineral density, tibia ash percent, plasma P and other biochemical parameters were not significantly different among dietary treatments. Total P intake, excretion and retention were affected by diet (P < 0.001), but its deposition in eggs was not significantly different. Contrast analysis further showed that total P excretion of phytase present vs phytase absent was averagely reduced by 40.4 mg/hen per day (P < 0.01). Moreover, total P excretion was linearly (P < 0.01) reduced with lowering dietary NPP, and this relationship was similar regardless of whether phytase was supplemented or not. The results from this study indicated that NPP levels in laying hen diets could be reduced to 1.0 g/kg (excluding the portion of NPP released by phytase) with the inclusion of phytase, without negative effects on production performance and health of the hens, thereby diminishing P excretion into environment.

Effect of dietary n-3 polyunsaturated fatty acids on the composition of cecal microbiome of Lohmann hens.

Supplementation of n-3 fatty acids to poultry diets is widely acknowledged for its role in enhancing poultry products, however, little is known about the compositional responses of gut microbial communities to type and dosage of these supplements.  Here, we compared the effects of n-3 polyunsaturated fatty acids (PUFA), supplied as alpha-linolenic acid (ALA) or docosahexaenoic acid (DHA), on the composition of bacterial communities in ceca of laying hens.  Corn-soybean basal diets were supplemented with either flaxseed oil (FO, ALA-rich) or marine algal biomass (MA, DHA-rich), and each supplied 0.20 and 0.60% of total n-3 PUFA in the diet.  Lohmann LSL-Classic laying hens (n = 10/treatment) were randomly allocated to one of the 4 diets.  After 8 weeks of feeding, blood, liver and cecal digesta samples were obtained for plasma glucose, fatty acids, and short chain fatty acids analyses, respectively.  The gut bacterial communities were characterized using genomic DNA extracted from cecal contents, whereby the V3-V4 hypervariable region of the 16S rRNA gene was sequenced using the Illumina Miseq® platform.  Firmicutes and Bacteroidetes were the predominant phyla in both the FO- and MA-fed groups.  The relative abundance of Tenericutes, often associated with immunomodulation, was relatively higher (P<0.0001) in the FO than MA group.  Although the relative abundance of Bacteroides was greater for the FO- than the MA-fed group, this genus was negatively correlated (P<0.05) with total n-3 PUFA in the liver at higher dosages of both FO- and MA-fed hens.  Higher dose of FO (0.60%) and both dosages of MA (0.20 and 0.60%) substantially enriched several members of Firmicutes (e.g., Faecalibacterium, Clostridium and Ruminococcus) which are known to produce butyrate.  Moreover, co-occurrence network analysis revealed that, in the FO 0.60- and MA 0.20-fed hens, Ruminococcaceae was the most influential taxon accounting for about 31% of the network complexity.  These findings demonstrate that supplementation of different type and level of n-3 PUFA in hens' diets could enrich microbial communities with potential role in lipid metabolism and health.

Lipidomic characterization of omega-3 polyunsaturated fatty acids in phosphatidylcholine and phosphatidylethanolamine species of egg yolk lipid derived from hens fed flaxseed oil and marine algal biomass.

Membrane phospholipids, including phosphatidylcholine (PC) and phosphatidylethanolamine (PE), consist of distinct fatty acids occupying the sn-1 and sn-2 positions, reflecting the highly regulated nature of lipid biosynthesis. However, little is known about the influence of dietary lipids on the positional nature of fatty acids in tissues, including the enrichment of omega-3 polyunsaturated fatty acid (PUFA) in chicken egg yolk phospholipids. This study was undertaken to characterize the PC and PE species in egg lipids derived from Lohmann hens (n=10/treatment) randomly allocated to either a control (no supplementation), a flaxseed oil (FO) or a marine algal oil (MA) diet. Each of the FO or MA diets supplied three levels of total omega-3 PUFA (0.20, 0.40 and 0.60% of diet) that were provided for 6 weeks. A combination of multiplexed mass spectrometry (MS) experiments are used to determine total, isobaric, and position molecules for PC and PE in egg yolk. The distribution of phospholipids in the yolk was predominantly PC over PE (~72 vs. 23%, respectively) across treatments. The longer chain PUFA existed in the sn-2 position in the PC and PE. Although docosahexaenoic acid (22:6) formed isomers with fatty acids 16:0, 18:0 and 18:1; it was preferentially enriched in the egg in combination with 16:0 with both the FO and MA-fed groups in both lipid pools. All 22:6-containing isomers were enriched by ~2-fold more (P < 0.0001) with MA than FO, however, all isomers exhibited a plateau with the FO-fed group. In addition, the MS analyses of PCs revealed several isobaric species containing eicosapentaenoic acid (EPA, 20:5), however, in the PE, EPA formed only one isomer (i.e. in combination with 16:0). These results may assist to elucidate potential aspects regulating the limited enrichment of omega-3 PUFA, particularly EPA and docosahexaenoic acid (22:6) in chicken eggs.

Effects of additional dosage of vitamin D3, vitamin D2, and 25-hydroxyvitamin D3 on calcium and phosphorus utilization, egg quality and bone mineralization in laying hens.

Vitamin D is essential for the metabolism of calcium (Ca) and phosphorus (P) in birds. The objective of the study was to evaluate the effect of different isoforms of dietary vitamin D on Ca and P utilization, egg quality, and bone mineralization of laying hens. A total of 42 Lohmann white laying hens at 57 wk of age were randomly assigned to 7 dietary treatments for 6 wk. Dietary treatments were: 3,000 IU/kg Vit D3 as control, and control with additional 3,000 IU/kg 25-hydroxyvitamin D3 (T1), 9,000 IU/kg 25-hydroxyvitamin D3 (T2), 3,000 IU/kg vitamin D3 (T3), 9,000 IU/kg vitamin D3 (T4), 3,000 IU/kg of vitamin D2 (T5), or 9,000 IU/kg of vitamin D2 (T6). Egg production and egg quality were measured weekly. Fecal samples were collected at weeks 2 and 6 to measure Ca and P utilization. After 6 wk, the left tibia and femurs were collected to measure bone mineral density (BMD) and bone mineral content (BMC). A 1-way ANOVA with Tukey HSD means separation test was used for statistical analysis. There were no significant differences in egg production, egg quality, BMD, or BMC of tibia and femurs among the treatments (P > 0.05). T6 significantly reduced feed intake (P < 0.05). The apparent total tract digestibility (ATTD) of Ca was higher (P < 0.012) in treatments supplemented with additional vitamin D, irrespective of forms. The ATTD of P was higher (P < 0.0001) in T5 compared to the other treatments at both time points. The utilization of Ca and P by laying hens can be improved through the addition of different isoforms of vitamin D in diets. However, additional vitamin D supplementation to laying hens, regardless of forms, had no effect on either bone mineralization or measures of egg quality.

Assessment of the minimal available phosphorus needs of laying hens: Implications for phosphorus management strategies.

The oversupply of dietary phosphorus (P) leads to increased feed costs and discharge of excessive P to the environment, thus directly impacting the sustainability of egg production practices. The present study was conducted to better define the minimal available P needs of laying hens. Fifty-six Lohmann white laying hens were individually caged and fed one of 7 diets with graded levels of available P (0.15, 0.20, 0.25, 0.30, 0.35, 0.40, or 0.45%) for 12 weeks. Records were maintained for body weight, feed intake, and egg production during the experimental period. Blood and egg samples were collected and digestibility studies conducted at wk 6 and 12 of the experiment. At the end of the experiment, tibia characteristics and expression of the P transporters in the small intestine and kidney were determined. Lowering dietary available P from 0.45 to 0.15% generally reduced plasma P concentrations (P < 0.01), but hen productive performance, plasma calcium, parathyroid hormone and other constituents, tibia bone mineral density and content, tibia ash percentage, and mRNA abundance of the small intestine, and kidney type II sodium/phosphate cotransporter were not different among treatments. Specific gravity and eggshell thickness tended to increase with reducing dietary P (P < 0.05). Total P intake, excretion, and retention net amount decreased (P < 0.01) with reducing dietary P, but its retention rate (of intake) remained unchanged. Furthermore, changing dietary P did not affect calcium retention rate and net deposition of total P and calcium in eggs. These data indicate that reducing dietary available P up to 0.15% is adequate to maintain health and performance of layers. As such, this minimal available P estimate should serve as a benchmark for the assessment of P contents of commercial laying hen rations, with the goal of enhancing the sustainability of egg production.

Assessment of the minimal available phosphorus needs of pullets during the pre-laying period.

Dietary phosphorus (P) oversupply is costly to the poultry industry and represents a point source of excessive P discharge to the environment via manure application to soils. The current study was conducted to investigate the effects of dietary available P (AP) level on growth performance, plasma biochemistry, bone characteristics, and P flow in order to assess minimal AP needs of pullets during the pre-lay period. A total of 192 Lohmann pullet chicks were randomly allocated to one of 4 treatments with 8 replicate cages of 6 birds per cage. The pullets of Low (L; the lowest AP regimen) were fed diets in a sequence of 0.200, 0.175, and 0.150% AP for the age period of 0 to 4 wk, 4 to 8 wk, and 8 to 16 wk, respectively. The AP level was increased by an increment of 0.1% in each phase in Low-Medium (LM), Medium-High (MH) and High (H), thus making H (control) consisting of 0.500, 0.475, and 0.450% AP. Overall, there were no main effects of treatment on body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR). However, compared with the H treatment, BWG or FI of the L treatment was lower during the first 8 wk post-hatch, but became comparable or higher over the last 8 wk (P < 0.05); FCR of L birds was greater over wk 0 to 4 but became smaller over wk 8 to 16 (P < 0.05). Plasma P was lower in L than H treatments at wk 8 but turned comparable at wk 16 (P < 0.001). Bone characteristics were not different for pullets fed various AP regimens. Total P intake and excretion were reduced with lowering dietary AP (P < 0.001), but its retention percent was not affected. The results indicated that the lowest AP regimen (0.200-0.175-0.150%) in the current study was adequate to support healthy growth and development of pre-lay pullets, while reducing P excretion in manure.

Impact of dietary precursor ALA versus preformed DHA on fatty acid profiles of eggs, liver and adipose tissue and expression of genes associated with hepatic lipid metabolism in laying hens.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA), including alpha-linolenic acid (ALA) and preformed longer chain PUFA (LCPUFA, particularly docosahexaenoic acid, DHA) differ in their egg LCPUFA enrichment efficiency. However, mechanisms leading to these differences are unclear. To this end, omega-3 PUFA contents in different lipid classes, including triacylglycerol (TAG) and total phospholipid (PL) in yolk, liver and adipose, as well as the expression of key hepatic enzymes in lipid metabolism were evaluated in laying hens in response to changes in dietary supply. Seventy Lohmann hens (n=10/treatment) consumed either a control diet (0.03% total omega-3 PUFA), or the control with supplementation (0.20%, 0.40% and 0.60% total omega-3 PUFA) from either flaxseed oil or algal product, as sources of ALA (precursor) or DHA (preformed), respectively. The study was arranged in a completely randomized design, and data were analyzed using the Proc Mixed procedure of SAS. ALA accumulated as a function of intake (P<0.0001) in total and lipid classes of yolk, liver and adipose (TAG only) for ALA- and DHA-fed hens. Unlike flaxseed oil, preformed-DHA contributed to greater (P<0.0001) accumulation of LCPUFA in yolk total PL and TAG pool, as well as adipose TAG. This may relate to elevated (P<0.0001) expression of acyl-CoA synthetase (ACSL1). No difference in hepatic EPA level in total lipids was noted between both treatment groups; EPAliver=2.1493x-0.0064; R2=0.70, P<0.0001 (x=dietary omega-3 PUFA). The latter result may highlight the role of hepatic EPA in the regulation of LCPUFA metabolism in laying hens.

Performance and tissue fatty acid profile of broiler chickens and laying hens fed hemp oil and HempOmegaTM.

This study was conducted to determine the effects of hemp oil (HO) and HempOmega (HΩ), an equivalent product to HO, on performance and tissue fatty acid profile of layers and broiler chickens in two separate experiments. In the first experiment, forty 19-wk old Lohmann white laying hens were randomized to 1 of 5 dietary treatments, either a control diet or a control diet supplemented with 4 or 8% hemp oil provided by HO or HΩ, for a period of 6 wk (n = 8/diet). In experiment 2, 150-day-old mixed-sex (75 male; 75 female) Ross 308 chicks were randomly allocated into 5 dietary treatments, a control diet or a control diet supplemented with either 3 or 6% hemp oil provided by HO or HΩ, each with six replicates of 5 chicks for a 21-d feeding period. Performance of layers and broilers was not affected by dietary treatments. Animals provided with either HO or HΩ diets had greater total n-3 polyunsaturated fatty acids (PUFAs), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) in egg yolks, thighs, and breasts compared to the control diet (P < 0.01), and monounsaturated fatty acids (MUFAs) content of egg yolks and thighs decreased (P < 0.05). The levels of total n-6 PUFAs, linoleic acid (LA), or arachidonic acid (ARA) of the egg yolk and meat were generally not affected by dietary supplementation with HO or HΩ, but gamma-linolenic acid (GLA) was notably increased (P < 0.01). The current data show that inclusion of hemp oil up to 8% in layer diets and 6% in broiler diets provided by HO or HΩ does not negatively affect overall performance of birds and results in the enrichment of n-3 PUFAs and GLA in eggs and meat.

Effect of flaxseed oil and microalgae DHA on the production performance, fatty acids and total lipids of egg yolk and plasma in laying hens.

The incorporation of omega-3 polyunsaturated fatty acids (PUFA) in the egg is dependent on both the transfer efficiency of preformed dietary omega-3 fatty acids to the eggs as well as endogenous PUFA metabolism and deposition. Employing an experimental design consisting of 70 Lohmann LSL-Classic hens (n=10/treatment) in a 6-week feeding trial, we examined the impact of graded levels of either flaxseed oil (alpha-linolenic acid, ALA) or algal DHA (preformed docosahexaenoic acid, DHA), each supplying 0.20%, 0.40% and 0.60% total omega-3s. The control diet was practically low in omega-3s. Study parameters included monitoring the changes of fatty acid contents in yolk, measures of hen performance, eggshell quality, total lipids and fatty acid contents of plasma. Data were analysed as a complete randomized design using Proc Mixed procedure of SAS. No significant differences were observed between treatments with respect to hen performance, eggshell quality and cholesterol content in plasma and egg yolk. Individual and total omega-3 PUFA in the yolk and plasma increased (P<0.0001) linearly as a function of total omega-3 PUFA intake. At the highest inclusion levels, DHA-fed hens incorporated 3-fold more DHA in eggs compared with ALA-fed hens (179±5.55 vs. 66.7±2.25mg/yolk, respectively). In both treatment groups, maximal enrichment of total n-3 PUFA was observed by week-2, declined by week-4 and leveled thereafter. In addition, accumulation of DHA in egg yolk showed linear (P<0.0001) and quadratic (P<0.05) effects for flaxseed oil (R2=0.89) and algal DHA (R2=0.95). The current data, based on defined level of total omega-3s in the background diet, provides evidence to suggest that exogenous as well as endogenous synthesis of DHA may be subject to a similar basis of regulation, and serve to highlight potential regulatory aspects explaining the limitations in the deposition of endogenously produced omega-3 LCPUFA.

Increasing Levels of Dietary Hempseed Products Leads to Differential Responses in the Fatty Acid Profiles of Egg Yolk, Liver and Plasma of Laying Hens.

The limited efficiency with which dietary alpha-linolenic acid (ALA) is converted by hens into docosahexaenoic acid (DHA) for egg deposition is not clearly understood. In this study, dietary ALA levels were increased via the inclusion of hempseed (HS) and hempseed oil (HO) in hen diets, with the goal of assessing the effects on the fatty acid (FA) profiles of total lipids and lipid classes in yolk, liver and plasma. Forty-eight hens were individually caged and fed one of six diets containing either HS:10, 20 or 30, HO:4.5 or 9.0 (%, diet) or a control (containing corn oil), providing a range (0.1-1.28 %, diet) of ALA. Fatty acid methyl esters of total lipids and lipid classes, including phosphatidyl choline (PtdCho) and ethanolamine (PtdEtn) in yolk, plasma and liver were then determined. Levels of n-3 FAs in both total lipids and lipid classes increased in all tissues. ALA and eicosapentaenoic acid (EPA) increased linearly, while docosapentaenoic acid and DHA increased quadratically. The FA profiles of yolk closely reflected levels in both plasma and liver. While ALA was highly concentrated in the triacylglycerol, it was low but equally distributed between PtdCho and PtdEtn in all tissues; however, the net accumulation was lower (P < 0.0001) in liver compared to yolk and plasma. Levels of EPA and ALA in yolk-PtdEtn were linearly (P < 0.0001; R (2) = 0.93) associated, and reflected those in liver-PtdEtn (P < 0.0001; R (2) = 0.90). In the liver, a strong inverse correlation (P < 0.0001; r = -0.94) between PL-DHA and ALA-to-EPA ratio in PtdEtn supports theories of low substrate availability, possibly limiting the conversion of ALA into DHA for egg enrichment.

Hempseed Products Fed to Hens Effectively Increased n-3 Polyunsaturated Fatty Acids in Total Lipids, Triacylglycerol and Phospholipid of Egg Yolk.

Hempseed products represent potential alternative feed ingredients for poultry. However, their usage is not currently approved due to a lack of data to support their safety and efficacy. In this regard, the current study was conducted to assess the impact of dietary concentration of hempseed (HS) products and duration of their feeding to hens on the polyunsaturated fatty acid (PUFA) composition of egg yolk lipids. In the current study, 48 Lohmann LSL-Classic hens were individually housed in metabolism cages, in a completely randomized design, and provided one of six diets (wheat-barley-soybean-based) containing either HS (10, 20 and 30 %), hempseed oil (HO; 4.5 and 9.0 %) or no hempseed product (control) over 12 weeks. Increasing alpha-linolenic acid (ALA) intake via increasing dietary hempseed product inclusion, significantly (p < 0.0001) increased the n-3 PUFA contents of yolk total lipid. The values of ALA increased by 12-fold (152 ± 3.56 and 156 ± 2.42 mg/yolk) and docosahexaenoic acid (DHA) by twofold to threefold (41.3 ± 1.57 and 43.6 ± 1.61 mg/yolk) over the control, for the highest levels of HS and HO inclusion, respectively. Increasing levels of hemp products in laying hen diets proved effective in manipulating the fatty acid profile of the total lipid, triacylglycerol (TAG) and total phospholipid (PL) fractions of yolks, enhancing the n-3 fatty acids and reducing the n-6/n-3 ratio. The latter benefit was achieved within 4 weeks of feeding hens either HS- or HO-containing diets.

Performance, egg quality, and blood plasma chemistry of laying hens fed hempseed and hempseed oil.

The aim of this study was to compare the performance of hens (feed intake, rate of lay, egg weight, and BW gain), egg quality and blood biochemistry (enzymes, electrolytes, proteins, and other plasma constituents) of laying hens fed diets containing hemp products. Forty-eight Lohmann LSL-Classic (white-egg layers; 19 wk of age) were individually caged and fed 1 of 6 wheat-barley-soybean-based diets for a period of 12 wk. The diets consisted of hempseed (HS; 10, 20, or 30%), hempseed oil (HO; 4.5 or 9.0%), or a control diet (corn oil-based). All diets were formulated to contain similar levels of crude fat (11%), energy (2,800 kcal/kg), and CP (17%). Data were analyzed as a completely randomized design using the repeated measure analysis of the PROC MIXED procedure of SAS. The results indicated that the inclusion of up to 30 and 9.0% HS and HO, respectively, to diets of laying hens had no significant effects on hen performance, egg quality, or plasma level of metabolites (proteins, glucose, uric acid, and cholesterol) and electrolytes (Na, K, Cl, P, and Ca). Overall plasma enzyme concentrations, particularly gamma-glutamyl transferase, were significantly (P < 0.01) lowest at the 10 and 20% levels of HS inclusion, or at the 4.5% HO level of inclusion of the hempseed products compared with the higher levels or control fed hens. Similar effects were also observed for plasma aspartate aminotransferase levels but with the HS enriched diets only (P < 0.05), particularly being lowest at the inclusion levels of 10 and 20% HS compared with the control. The results may imply a possible protective effect of HS- and HO-containing diets, particularly at 10% HS, 20% HS, and 4.5% HO levels, on liver damage/injury. In summary, both HO and HS appear to be well tolerated by laying hens as judged by markers of plasma clinical chemistry supporting the safety and efficacy of hemp products for use in laying hen rations.

Performance, serum biochemical responses, and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide.

The present study was conducted to investigate the effects of dietary folic acid (FA) supplementation on performance, serum biochemical indices, and mRNA abundance of intestinal folate transporters in young and older laying hens after acute lipopolysaccharide (LPS) challenge. Two experiments were conducted separately involving 48 Shaver White young laying hens (24 wk of age) in experiment 1 and 48 Shaver White older laying hens (58 wk of age) in experiment 2. Birds were fed 2 diets in a complete randomized design. The diets were wheat-soybean meal based, with or without supplemental 4 mg of FA/kg of diet. Birds were fed for 8 wk, during which time feed consumption and egg production were monitored. At the end of each feeding experiment, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either Escherichia coli LPS or sterile saline. Four hours after injection, blood and intestinal samples were collected for further analysis. Compared with the control, dietary FA supplementation increased egg weight and egg mass and decreased serum glucose levels in the young laying hens, and reduced serum uric acid in the older laying hens (P < 0.05). Relative to saline injection, plasma homocysteine, serum calcium, and phosphorus levels were found to be lower in both young and older laying hens after LPS challenge (P < 0.05). Other serum biochemical variables and the mRNA expression of 2 folate transport genes in the small and large intestine were differentially affected by LPS challenge, and some of those responses varied with the age of the birds. Additionally, interactions between diet and LPS challenge were specifically found in the older laying hens. In summary, in addition to improving production performance, there were effects of dietary FA supplementation and its interaction with LPS challenge on biochemical constituents, and age played a role in the development of responses to diet and bacterial LPS infections.

Dietary and ontogenic regulation of fatty acid desaturase and elongase expression in broiler chickens.

Effects of diet and ontogeny on the expression of fatty acid desaturases and elongases were examined in broiler chickens. In Study 1, 120 day-old male chicks received one of six diets with LA:ALA ranging from 46:4 to 16:34, for 33 days. Total n-6 PUFA decreased, and n-3 PUFA increased in response to a decrease in the dietary LA:ALA. FADS1, FADS2, ELOVL2 and ELOVL5 mRNAs were highest (P<0.05) in birds fed lower LA:ALA diets. In Study 2, 60 day-old male chicks were fed a basal diet, and liver samples were collected on day of hatch, and on days 2, 7, 14, 21 and 35 post-hatch. Total n-6 and n-3 PUFA increased (P<0.01) from days 7 to days 21. FADS1, FADS2 and ELOVL2 mRNAs generally increased (P<0.01) with age. These findings provide evidence for the dietary and developmental regulation of PUFA metabolism in broiler chickens.

Post-hatching ontogeny of intestinal proton-coupled folate transporter and reduced folate carrier in broiler chickens.

Folate transporters, including the reduced folate carrier and the proton-coupled folate transporter, encoded by Slc19a1 and Slc46a1 genes respectively, play important roles in the transport of folate across biological membranes given the hydrophilic nature of folates. Although a number of studies have demonstrated that these two transporters are regulated ontogenetically in mammals, little data are available on their developmental patterns of expression in poultry. The objective of this study was to investigate the expression patterns of Slc19a1 and Slc46a1 in jejunal and cecal tissue of broiler chickens during post-hatching development. Post-hatch male chicks (Ross × Ross) had free access to water and a soybean/wheat-based diet. Jejunal, cecal and blood samples were collected on day-of-hatch but before feeding (D0), and on D2, D7, D14, D21 and D35 post-hatch (n = 8 at each time point), respectively. Plasma folate concentrations were low on the day of hatch and increased with maturation; by contrast, plasma homocysteine, a marker of folate status, was highest (P < 0.05) in the day-of-hatch birds and decreased thereafter. Increasing age reduced mRNA abundance of Slc19a1 (P < 0.05) in the jejunum and cecum. Abundance of Slc46a1 mRNA (P < 0.05) gradually decreased in the cecum with increasing age and that of Slc46a1 in the jejunum initially decreased and then increased to level similar to that of day-of-hatch. The study provides some initial data on ontogenetic regulation of Slc19a1 and Slc46a1 in the jejunum and cecum of the chicken and lays the ground work for future nutritional studies. Moreover, the expression of Slc19a1 and Slc46a1 transcripts in the cecum provides evidence of the potential for cecally derived folate to contribute to the folate status of the host.

Response of older laying hens to an Escherichia coli lipopolysaccharide challenge when fed diets with or without supplemental folic acid.

Folic acid plays a key role in nucleic acids and protein synthesis, and has been associated with anti-inflammatory effects in LPS-induced infections. To this end, a study was conducted to investigate the effects of dietary folic acid (FA) supplementation in older laying hens (58 to 66 wk of age) challenged with Escherichia coli lipopolysaccharide (LPS). A total of 24 Shaver White laying hens at 58 wk were fed 2 diets. The diets were wheat-soybean-based, with either 0 or 4 mg of supplemental FA per kg of diet. After 8 wk of feeding and at 66 wk, the hens were injected intravenously with 8 mg of LPS or saline per kg of BW. Four hours after injection, blood was collected and hens were euthanized to obtain spleen and cecal tonsils. The T cell subsets in the blood and the spleen (CD4+ and CD8+), total IgG, and biochemical constituents (total protein, albumin, globulin, and fibrinogen) were not influenced (P > 0.05) by dietary FA supplementation. However, LPS injection decreased (P < 0.05) biochemical constituents, CD4+, and CD8+ cells in the blood, whereas CD4+:CD8+ ratio and total IgG increased (P < 0.05), and fibrinogen was not influenced. Gene expression in the spleen and cecal tonsils was not influenced by dietary FA supplementation except a diet × challenge interaction for interleukin (IL)-8 in the spleen; IL-8 decreased in FA-fed hens that were treated with LPS. Also, FA supplementation decreased the expression of IL-8 in cecal tonsils. Relative to saline-injected hens, expression of IL-1ß, interferon-γ, and IL-10 increased in the LPS-injected hens in the spleen and cecal tonsils, IL-8 increased in LPS-injected hens only in the cecal tonsils, whereas Toll-like receptor 4, IL-4, IL-17, and IL-18 increased in the LPS-injected hens only in the spleen; however, LPS decreased expression of IL-13 in the cecal tonsils. In conclusion, FA did not affect inflammatory responses in older laying hens; more studies are required to investigate possible protective effects of FA in laying hens.

Immunomodulation in young laying hens by dietary folic acid and acute immune responses after challenge with Escherichia coli lipopolysaccharide.

We investigated the effects of dietary folic acid (FA) supplementation on immunological parameters in young laying hens under acute conditions of lipopolysaccharide (LPS) challenge. Twenty-four Shaver White laying hens at 24 wk were fed 2 diets in a completely randomized design. The diets were wheat-soybean based, with either 0 or 4 mg of supplemental FA per kilogram of diet. At 32 wk of age, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either LPS or saline. Four hours after injection, blood was collected and the hens were euthanized to obtain spleen and cecal tonsils. Heterophil:lymphocyte ratio, CD3+, CD4+, CD8+ T cells, and CD4+:CD8+ cells in the blood and spleen were not affected by dietary FA. Relative to saline-injected hens, LPS-injected hens had fewer (P < 0.05) CD3+, CD4+, CD8+, and CD4+:CD8+ cells in the blood, and no difference was found in the spleen. Total protein, albumin, and globulin were found to be higher (P < 0.05) in FA-supplemented hens compared with the control. However, total protein, albumin, and globulins decreased (P < 0.05) in the LPS-injected hens compared with the saline control. Expression of interleukin (IL)-1ß in cecal tonsils decreased (P < 0.05) in FA-supplemented hens, but no dietary influence was found on the expression of other genes in both the spleen and cecal tonsils. Lipopolysaccharide upregulated (P < 0.05) expression of IL-10 and interferon (IFN)-γ in the spleen, and IL-1ß, IL-10, and IFN-γ in the cecal tonsils, whereas the expression of Toll-like receptor (TLR)-4 and IL-8 was not influenced by LPS in the spleen and cecal tonsils. There was a diet × challenge interaction for total IgG, and cytokines IL-1ß and IL-18 in the spleen as well as IL-18 in the cecal tonsils. In conclusion, there were few interactions of dietary FA and LPS; however, FA increased biochemical constituents, enhanced generation of total IgG, as well as exhibiting pleoitropic effects in inflammatory responses.

Local and systemic innate immunity in broiler chickens supplemented with yeast-derived carbohydrates.

A study was conducted to assess the effect of yeast-derived carbohydrates (YDC) on performance and innate immune responses of broiler chickens. In total, 1,080 one-day-old birds were randomly assigned to one of 3 dietary treatments (n = 360): a standard broiler diet containing monensin (control), control + bacitracin methylene disalycylate (BMD), and YDC treatment (control + YDC at 0.02%, 0.01%, and 0.005% for starter, grower, and finisher, respectively). Weekly BW, feed intake (FI), and feed conversion ratio (FCR) were recorded. Immune organ weights, gut morphology, gene expression, heterophil:lymphocyte (H:L), and serum IgG were determined at d 42. No significant difference in FCR, FI, and mortality was observed among treatments. However, BW gain in starter phase was higher in control and YDC treatments compared with BMD treatment. Ileal villi height, crypt depth, and their ratio were not significantly different among treatments, whereas villi width was lower in control and YDC treatments compared with BMD treatment. The number of goblet cells per unit area in the ileum was lower in BMD treatment compared with control and YDC treatments. Expression of TLR2b and IL-6 in the ileum and cecal tonsils was not significantly different among treatments (P > 0.05). Expression of TLR4 was downregulated in YDC treatment compared with control in the ileum. Expression of IL-12p35 and IFN-γ were downregulated in the YDC treatment only in the cecal tonsils. Compared with the control, the expression of IL-10 in both the ileum and the cecal tonsils was downregulated in YDC treatment. Serum IgG and H:L ratio were lower and higher, respectively, in the YDC treatment compared with control and BMD treatments. In conclusion, dietary inclusion of YDC affected intestinal cytokines anti-inflammatory profile on a gut location associated immune pathways manner, suggesting different immune pathways that require further studies in this field.

Innate immune response to yeast-derived carbohydrates in broiler chickens fed organic diets and challenged with Clostridium perfringens.

Necrotic enteritis (NE) caused by Clostridium perfringens is a reemerging disease of economic importance in areas of the world where antibiotic growth promoters have been banned. The effect of mannan-oligosaccharide (MOS) supplementation in organic diets of broilers challenged with C. perfringens on performance, gut morphology, and innate immunity was investigated. Three hundred Ross-308 broilers were fed antibiotic-free certified organic starter and grower diets. On d 14, birds were orally challenged with 1 mL of C. perfringens culture at 3 × 10(10) cfu/bird. Treatments consisted of a control no-challenge (CO; 0 g/kg of MOS in the basal diet), control challenge (COC, 0 g/kg of MOS in the basal diet), and MOS challenge (2 g/kg of MOS in the basal diet). Challenge of birds resulted in decreased feed intake and BW gain (P = 0.048 and P = 0.026, respectively). Even though supplementation of diet with MOS improved feed intake (P = 0.985), BW gain and G:F were not improved compared with those of the CO group (P = 0.026 and P = <0.001, respectively). There was no significant difference among treatments in jejunal and ileal villus height, crypt depth, and goblet cells/mm(2) (P > 0.05). Quantitative real-time PCR showed that, in the ileum, the MOS diet resulted in an upregulation of toll-like receptor (TLR)2b, TLR4, interleukin (IL)-12p35, and interferon (IFN)-γ compared with CO (P = 0.003, P = 0.018, and P = 0.024, respectively). In the cecal tonsil, challenging birds with C. perfringens resulted in an upregulation of TLR2b compared with CO (P = 0.036), and MOS resulted in an upregulation of TLR4 (P = 0.018). In conclusion, feeding a MOS-supplemented diet to C. perfringens-challenged broiler chickens did not improve performance and gut morphology-associated responses. However, MOS was capable of altering TLR and cytokine profiles, where dual TLR2 and TLR4 pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN-γ, implying that MOS supplementation in C. perfringens-challenged chickens supports a proinflammatory effect via T-helper cell-1 associated pathways.