RNA-sequencing analysis of shell gland shows differences in gene expression profile at two time-points of eggshell formation in laying chickens.

BACKGROUND: Eggshell formation takes place in the shell gland of the oviduct of laying hens. The eggshell is rich in calcium and various glycoproteins synthesised in the shell gland. Although studies have identified genes involved in eggshell formation, little is known about the regulation of genes in the shell gland particularly in a temporal manner. The current study investigated the global gene expression profile of the shell gland of laying hens at different time-points of eggshell formation using RNA-Sequencing (RNA-Seq) analysis. RESULTS: Gene expression profiles of the shell gland tissue at 5 and 15 h time-points were clearly distinct from each other. Out of the 14,334 genes assessed for differential expression in the shell gland tissue, 278 genes were significantly down-regulated (log2 fold change > 1.5; FDR < 0.05) and 413 genes were significantly up-regulated at 15 h relative to the 5 h time-point of eggshell formation. The down-regulated genes annotated to Gene Ontology (GO) terms showed anion transport, synaptic vesicle localisation, organic anion transport, secretion and signal release as the five most enriched terms. The up-regulated gene annotation showed regulation of phospholipase activities, alanine transport, transmembrane receptor protein tyrosine kinase signalling pathway, regulation of blood vessels diameter and 3, 5-cyclic nucleotide phosphodiesterase activity as the five most enriched GO terms. The putative functions of genes identified ranged from calcium binding to receptor activity. Validation of RNA-Seq results through qPCR showed a positive correlation. CONCLUSIONS: The down-regulated genes at 15 h relative to the 5 h time-point were most likely involved in the transport of molecules and synthesis activities, initiating the formation of the eggshell. The up-regulated genes were most likely involved in calcium transportation, as well as synthesis and secretory activities of ions and molecules, reflecting the peak stage of eggshell formation. The findings in the current study improve our understanding of eggshell formation at the molecular level and provide a foundation for further studies of mRNA and possibly microRNA regulation involved in eggshell formation in the shell gland of laying hens.

Upregulation of genes encoding digestive enzymes and nutrient transporters in the digestive system of broiler chickens by dietary supplementation of fiber and inclusion of coarse particle size corn.

BACKGROUND: Measures to improve bird performance have been sought due to the imminent phase out of in-feed antibiotics in poultry and continued demand for higher poultry feeding efficiency. Increasing grain particle size and dietary fibre may improve gizzard function, digestive efficiency and nutrient absorption. This study was conducted to evaluate the effect increased particle size of corn and inclusion of sugarcane bagasse (SB) on mRNA expression of genes encoding digestive enzymes and nutrient transporters in broilers. RESULTS: A total of 336 day-old Ross 308 males were assigned in a 2 × 2 factorial arrangement of treatments with corn particle size - coarse 3576 µm or fine 1113 µm geometric mean diameter, and SB - 0 or 2% inclusion. Feed conversion ratio (FCR), weight gain and feed intake were measured from d 0-10 and d 10-24. The relative gizzard weight and mRNA expression of genes encoding digestive enzymes and intestinal nutrient transporters were measured on d 24. During d 10-24, a particle size × SB interaction was observed for FCR (P < 0.01), where birds fed coarsely ground corn (CC) with 2% SB had lower FCR than those fed CC without SB. A particle size × SB interaction was observed for both expression of pepsinogen A and C (P < 0.01) which were negatively correlated with FCR on d 24. Addition of 2% SB upregulated pepsinogen A and C only in CC fed birds. Further, 2% SB also upregulated pancreatic amylase (AMY2A) and intestinal cationic amino acid transporter-1 (CAT1). Inclusion of dietary CC upregulated duodenal amino peptidase N (APN), jejunal alanine, serine, cysteine and threonine transporter-1 (ASCT1), and ileal peptide transporter-2 (PepT2). CONCLUSION: These results suggest that both SB and coarse particle size modulate expression of genes encoding important digestive enzymes and nutrient transporters and thus are directly related to bird performance. These findings provide insights into the combination effects of dietary fiber and particle size in the future management of broiler feeding.

Immune responses following experimental infection with Ascaridia galli and necrotic enteritis in broiler chickens.

Broilers commonly suffer from necrotic enteritis (NE). Other gastrointestinal infectious diseases affect poultry, including nematode infections which are considered a re-emerging disease in barn and free-range systems. The aim of this study was to characterize the immune response of broilers after artificial infection with NE and contrast these with responses to the nematode Ascaridia galli and determine whether immune parameters measured during the course of infection can be used to distinguish infected from uninfected birds. A total of 96 one-day-old male Ross 308 broiler chickens were used in this study. At 10 days of age, broilers were randomly assigned to one of the following treatment groups: control birds (n = 32), A. galli infected birds (n = 32), or NE infected birds (n = 32) and inoculated with the appropriate infective agents. The immune response of birds was monitored through evaluation of haematology parameters, acute phase protein production, and intraepithelial intestinal lymphocyte population changes at 11, 16, 20, and 32 days of age. T-helper cells (CD4+CD8-) increased significantly over time, and were significantly higher in A. galli and NE compared to day 10 controls. In conclusion, α-1 glycoprotein levels can distinguish birds with NE from other birds, including those infected with A. galli; also T-helper cell numbers can distinguish both NE and A. galli from uninfected birds and thirdly, 10 days post infection is the best time point to evaluate the bird's immune response for A. galli infections.

Dynamics of intestinal metabolites and morphology in response to necrotic enteritis challenge in broiler chickens.

Despite the relatively small contribution to metabolizable energy that volatile fatty acids (VFAs) provide in chickens, these organic acids have been reported to play beneficial roles in the gastrointestinal tract (GIT) of birds, for example, inhibition of the growth of some pathogenic bacteria. However, information regarding the dynamics of these metabolites in the GIT of chickens is still scarce, especially under disease conditions such as necrotic enteritis (NE). Here, we investigated the dynamics of VFAs and lactic acid, and intestinal morphology in response to NE predisposing factors, that is, excessive dietary fishmeal and Eimeria inoculation, and causative agent Clostridium perfringens producing NetB toxin. The experiment was designed in a 2 × 2 × 2 factorial arrangement of treatments with or without: fishmeal feeding, Eimeria inoculation and C. perfringens challenge. The results showed that these factors significantly influenced composition and concentration of VFAs and lactic acids, pH and histomorphometry in one way or another. These changes may be important for the onset of NE or only the synergetic responses to micro environmental stress. Eimeria appeared to be more important than fishmeal in predisposing birds to NE, thus the application of Eimeria in NE challenge provides more consistent success in inducing the disease. The metabolic responses to various adverse factors such as excessive dietary fishmeal and Eimeria infection are complex. Thus, intensive efforts are required to better understand NE so as to achieve the control of the disease in the absence of antibiotics.

Type 1 Metallothionein (ZjMT) Is Responsible for Heavy Metal Tolerance in Ziziphus jujuba.

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that are able to make cells to uptake heavy metals from the environment. Molecular and functional characterization of this gene family improves understanding of the mechanisms underlying heavy metal tolerance in higher organisms. In this study, a cDNA clone, encoding 74-a.a. metallothionein type 1 protein (ZjMT), was isolated from the cDNA library of Ziziphus jujuba. At the N- and C-terminals of the deduced amino acid sequence of ZjMT, six cysteine residues were arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC structure, respectively, indicating that ZjMT is a type 1 MT. Quantitative PCR analysis of plants subjected to cadmium stress showed enhanced expression of ZjMT gene in Z. jujuba within 24 h upon Cd exposure. Escherichia coli cells expressing ZjMT exhibited enhanced metal tolerance and higher accumulation of metal ions compared with control cells. The results indicate that ZjMT contributes to the detoxification of metal ions and provides marked tolerance against metal stresses. Therefore, ZjMT may be a potential candidate for tolerance enhancement in vulnerable plants to heavy metal stress and E. coli cells containing the ZjMT gene may be applied to adsorb heavy metals in polluted wastewater.

A Multifactorial Analysis of the Extent to Which Eimeria and Fishmeal Predispose Broiler Chickens to Necrotic Enteritis.

Necrotic enteritis (NE) is an important infectious disease in chickens. Predisposing factors play critical roles both in disease outbreaks in the field and in models for experimental induction of disease. Systematic manipulation and study of predisposing factors help to optimize methods for the experimental reproduction of disease. The nature of such factors may play a confounding role in challenge models and, therefore, warrant investigation to determine their importance in industry-relevant NE reproduction models. In the present study, we examined the roles of dietary fishmeal inclusion, Eimeria inoculation (E), and Clostridium perfringens challenge (C) on broiler growth performance and induction of NE infection. The results showed that E, preceding C, greatly increased the severity of NE induced in broiler chickens, but fishmeal addition played only a marginal role in the challenge model. Bird performance was significantly affected by all three factors during the 35-day experimental period. Fishmeal increased body weight, but statistically significant effects of fishmeal were not observed on feed conversion ratio (FCR) and feed intake. Both Eimeria and C. perfringens significantly reduced body weight gain and feed intake. E but not C led to significantly poorer FCR. These findings indicate that dietary fishmeal may be removed from the model to allow the performance results of challenged chicks to be equivalent to the performance of chicks in the field. In conclusion, the present study demonstrates that an NE challenge model without fishmeal is valid and removes bird performance bias in the model introduced by feeding high fishmeal diets, refining the model to facilitate the yield of more commercially relevant results.

Diversity of Thrips Species and Vectors of Tomato Spotted Wilt Virus in Tomato Production Systems in Kenya.

Thrips have been recognized as primary vectors of tomato spotted wilt virus (TSWV) with Frankliniella occidentalis (Pergande) reported as the most important and efficient vector, while other species such as Thrips tabaci Lindeman also include populations that can vector the virus. A study was undertaken to establish the diversity of thrips and presence of vectors for TSWV in four major tomato production areas in Kenya. The cytochrome oxidase 1 (CO1) gene was used to generate sequences from thrips samples collected from tomatoes and weeds, and phylogenetic analysis done to establish the variation within potential vector populations. Ceratothripoides brunneus Bagnall was the predominant species of thrips in all areas. F. occidentalis and T. tabaci were abundant in Nakuru, Kirinyaga, and Loitokitok but not detected at Bungoma. Other vectors of tospoviruses identified in low numbers were Frankliniella schultzei (Trybom) and Scirtothrips dorsalis Hood. Variation was observed in T. tabaci, F. occidentalis, and F. schultzei. Kenyan specimens of T. tabaci from tomato belonged to the arrhenotokous group, while those of F. occidentalis clustered with the Western flower thrips G group. The detection of RNA of TSWV in both of these species of thrips supported the role they play as vectors. The study has demonstrated the high diversity of thrips species in tomato production and the occurrence of important vectors of TSWV and other tospoviruses.

Dietary acylated starch improves performance and gut health in necrotic enteritis challenged broilers.

Resistant starch has been reported to act as a protective agent against pathogenic organisms in the gut and to encourage the proliferation of beneficial organisms. This study examined the efficacy of acetylated high amylose maize starch (SA) and butyralated high-amylose maize starch (SB) in reducing the severity of necrotic enteritis (NE) in broilers under experimental challenge. A total of 720 one-day-old male Ross 308 chicks were assigned to 48 floor pens with a 2 × 4 factorial arrangement of treatments. Factors were a) challenge: no or yes; and b) feed additive: control, antibiotics (AB), SA, or SB. Birds were challenged with Eimeria and C. perfringens according to a previously reported protocol. On d 24 and 35, challenged birds had lower (P < 0.001) livability (LV), weight gain (WG), and feed intake (FI) compared to unchallenged birds. Challenged birds fed SA and SB had higher FI and WG at d 24 and 35 (P < 0.05) compared to birds fed the control diet, while being significantly lower than those fed AB. Unchallenged birds fed SA or SB had higher FI at d 24 and 35 compared to those fed the control diet (P < 0.05). Birds fed SB had increased (P < 0.001) jejunal villus height/crypt depth (VH:CD) ratios at d 15, increased ileal (P < 0.001) and caecal (P < 0.001) butyrate levels at d 15 and 24, and decreased (P < 0.01) caecal pH at d 15. Birds fed SA had increased (P < 0.001) ileal acetate content at d 24 and decreased (P < 0.01) caecal pH at d 15. These results demonstrated that dietary acylated starch improved WG in birds challenged with necrotic enteritis. Depending on the acid used, starch acylation also offers a degree of specificity in short chain fatty acid (SCFA) delivery to the lower intestinal tract which improves gut health.

Use of yeast cell wall extract as a tool to reduce the impact of necrotic enteritis in broilers.

The use of a yeast cell wall extract derived from Saccharomyces cerevisiae (Actigen(®)) has been proposed as an alternative to in-feed antibiotics. This experiment was conducted to investigate the efficacy of yeast cell extract as an alternative to zinc bacitracin or salinomycin using a necrotic enteritis challenge model. A feeding study was conducted using 480-day-old male Ross 308 chicks assigned to 48 floor pens. A 2 × 4 factorial arrangement of treatments was employed. The factors were: challenge (- or +) and feed additive (control, zinc bacitracin at 100/50 mg/kg, yeast cell wall extract at 400/800/200 mg/kg, or salinomycin at 60 mg/kg in starter, grower, and finisher, respectively). Diets based on wheat, sorghum, soybean meal, meat and bone meal, and canola meal were formulated according to the Ross 308 nutrient specifications. Birds were challenged using a previously established protocol (attenuated Eimeria spp oocysts) on d 9 and 10(8) to 10(9) Clostridium perfringens (type A strain EHE-NE18) on d 14 and 15). Challenged and unchallenged birds were partitioned to avoid cross contamination. Challenged birds had lower weight gain, feed intake and livability compared to unchallenged birds on d 24 and d 35 (P < 0.05). Birds given zinc bacitracin, yeast cell wall extract, or salinomycin had improved weight gain and livability when compared to control birds given no additives. Challenge × additive interactions were observed for feed intake and weight gain on d 24 and d 35 (P < 0.01). The additives all had a greater positive impact on feed intake, weight gain, and livability in challenged than unchallenged birds. All challenged birds showed higher necrotic enteritis lesion scores in the small intestine sections when compared to unchallenged birds (P < 0.01). Birds fed yeast cell wall extract exhibited increased villus height, decreased crypt depth, and increased villus:crypt ratio when challenged. Yeast cell wall extract, zinc bacitracin, and salinomycin were effective in preventing performance decline from necrotic enteritis in the current study. This study indicates that yeast cell wall extract has promise as a tool for controlling necrotic enteritis.

Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis genes in the jejunum of broilers is modulated by fishmeal inclusion in the diet. Furthermore, we show for the first time suppression of CD36 mRNA levels in the intestine of broilers challenged with Eimeria or C. perfringens.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

The effect of sex and dietary crude protein level on nutrient transporter gene expression and cecal microbiota populations in broiler chickens.

It is well known that male and female broilers differ in their growth performance and that many physiological factors contribute to this difference. The aim of this experiment is to investigate if there are differences between male and female broilers in cecal microbiota and nutrient transporter gene expression and if these differences play a role in the growth performance of broilers. The possible effect of protein level and its interaction with sex on microbiota and expression of the nutrient transporters were also investigated. Samples were collected from male and female birds fed either standard crude protein (SCP) or reduced crude protein diets (RCP) at the age of d 35. The experiment was designed as a 2 × 2 factorial arrangement of treatments consisting of 448 Cobb 500 broilers assigned to 32-floor pens with 4 treatments, 8 replicates, and 14 birds per pen for performance measurements. The factors were sex (male or female) and dietary crude protein (CP) level (standard or reduced). Body weight gain (BWG), feed intake and feed conversion ratio were recorded for each pen. Sex had a significant effect on BWG and FCR (P < 0.001) where males had a significantly higher BWG and better FCR compared to females. There was a significant interaction between sex and protein level on feed intake (FI) (P < 0.05), where male birds had a higher FI compared to female birds only when the birds were fed SCP but not RCP diets. There was a significant interaction between CP level and sex on the expression of CAT2 (P = 0.02) and PEPT2 (P = 0.026) where the genes were significantly upregulated in females but only when the RCP diet was fed. The RCP diet upregulated the expression of BoAT (P = 0.03) as a main effect. Female birds had significantly higher expression of the PepT-2 gene compared to the males. The alpha diversity of the cecal microbiota showed differences among the treatments. The Shannon diversity index was statistically higher (P = 0.036) for males fed the SCP diet and the Chao1 index for evenness was statistically higher (P = 0.027) in females fed the SCP diet. There was also a difference in the relative abundance of the 15 most common genera found in the cecal content of the broilers in this experiment and lastly, the differential composition of microbiota between the different treatments was also significantly different. This study suggests that chickens are able to compensate for a reduction in AA substrates when fed a low CP diet through the upregulation of certain AA transporters, females may adapt to low CP diets better by such upregulation compared to males, and lastly, sex has an effect on the cecal microbial population and these differences contribute towards the performance differences between male and female broilers.

Re-evaluation of recent research on metabolic utilization of energy in poultry: Recommendations for a net energy system for broilers.

Different energy systems have been proposed for energy evaluation of feeds for domestic animals. The oldest and most commonly used systems take into account the fecal energy loss to obtain digestible energy (DE), and fecal, urinary and fermentation gases energy losses to calculate metabolizable energy (ME). In the case of ruminants and pigs, the net energy (NE) system, which takes into account the heat increment associated with the metabolic utilization of ME, has progressively replaced the DE and ME systems over the last 50 years. For poultry, apparent ME (AME) is used exclusively and NE is not yet used widely. The present paper considers some important methodological points for measuring NE in poultry feeds and summarizes the available knowledge on NE systems for poultry. NE prediction equations based on a common analysis of three recent studies representing a total of 50 complete and balanced diets fed to broilers are proposed; these equations including the AME content and easily available chemical indicators have been validated on another set of 30 diets. The equations are applicable to both ingredients and complete diets. They rely primarily on an accurate and reliable AME value which then represents the first limiting predictor of NE value. Our analysis indicates that NE would be a better predictor of broiler performance than AME and that the hierarchy between feeds is dependent on the energy system with a higher energy value for fat and a lower energy value for protein in an NE system. Practical considerations for implementing such an NE system from the commonly used AME or AMEn (AME adjusted for zero nitrogen balance) systems are presented. In conclusion, there is sufficient information to allow the implementation of the NE concept in order to improve the accuracy of feed formulation in poultry.

Readthrough events in plants reveal plasticity of stop codons.

Stop codon readthrough (SCR) has important biological implications but remains largely uncharacterized. Here, we identify 1,009 SCR events in plants using a proteogenomic strategy. Plant SCR candidates tend to have shorter transcript lengths and fewer exons and splice variants than non-SCR transcripts. Mass spectrometry evidence shows that stop codons involved in SCR events can be recoded as 20 standard amino acids, some of which are also supported by suppressor tRNA analysis. We also observe multiple functional signals in 34 maize extended proteins and characterize the structural and subcellular localization changes in the extended protein of basic transcription factor 3. Furthermore, the SCR events exhibit non-conserved signature, and the extensions likely undergo protein-coding selection. Overall, our study not only characterizes that SCR events are commonly present in plants but also identifies the recoding plasticity of stop codons, which provides important insights into the flexibility of genetic decoding.

Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna.

BACKGROUND: Reaumuria trigyna is an endangered small shrub endemic to desert regions in Inner Mongolia. This dicotyledonous recretohalophyte has unique morphological characteristics that allow it to tolerate the stress imposed by semi-desert saline soil. However, it is impossible to explore the mechanisms underlying this tolerance without detailed genomic information. Fortunately, newly developed high-throughput sequencing technologies are powerful tools for de novo sequencing to gain such information for this species. RESULTS: Two sequencing libraries prepared from control (C21) and NaCl-treated samples (T43) were sequenced using short reads sequencing technology (Illumina) to investigate changes in the R. trigyna transcriptome in response to salt stress. Among 65340 unigenes, 35495 (52.27%) were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways with a cut-off E-value of 10-5. These included 44 Gene Ontology (GO) terms, 119 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 25 Clusters of Orthologous Groups families. By comparing the transcriptomes from control and NaCl-treated plants, 5032 genes showed significantly differences in transcript abundance under salt stress (false discovery rate ≤ 0.001 and |log2Ratio| ≥ 1). These genes were significantly enriched in 29 KEGG pathways and 26 GO terms. The transcription profiles indicated that genes related to ion transport and the reactive oxygen species scavenging system were relevant to the morphological and physiological characteristics of this species. The expression patterns of 30 randomly selected genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-seq. CONCLUSIONS: The present study identified potential genes involved in salt tolerance of R. trigyna. The globally sequenced genes covered a considerable proportion of the R. trigyna transcriptome. These data represent a genetic resource for the discovery of genes related to salt tolerance in this species, and may be a useful source of reference sequences for closely related taxa. These results can also further our understanding of salt tolerance in other halophytes surviving under sodic stress.

Total amino acid level affects the results of standardized ileal digestibility assays for feed ingredients for swine.

It has been demonstrated that the fiber content of oilseed meals and total amino acids (AA) in distillers' grains products affects standardized ileal digestibility (SID) values from swine assays and that total protein concentration affects the AA digestibility in assays using chickens. This analysis was conducted to test the hypothesis that the total AA content of test samples affects the SID assay results. Databases containing total AA profiles and SID values of 20 feedstuffs commonly fed to pigs from 2 sources, AMINODat 5.0 (Evonik Industries, 2015) and the Nutrient Requirements of Swine, 12th edition (NRC, 2012) were used to compare AA concentration effects on standardized ileal digestibility coefficients. Databases were compared with AA and SID available in both data sets. The total AA values were similar for the 2 databases with an R 2 of 0.979 (P < 0.001). The linear relationship between digestibility coefficients from the 2 databases was highly significant, an R 2 of 0.810 (P < 0.001). Both databases had increased SID values with increasing AA contents within and across feed ingredients. The SID = f(AA concentration) relationship was confirmed with an individual paper. Since SID, as typically measured, is a function of both digestion and absorption, both processes following Michaelis-Menten kinetics, SID = f(AA concentration) may simply be a natural phenomenon. Other reasons for the relationship were explored. Methods of estimating endogenous AA losses and misapplication statistical procedures may contribute to variation in results and at least partially explain why SID = f(AA concentration).

Influence of sex and rearing method on performance and flock uniformity in broilers-implications for research settings.

Male and female broiler chickens differ in their growth performance, carcass part weights and nutrient requirements. The potential reasons for these differences have been explored by looking at differences in nutrient digestibility, nutrient transporter gene expression as well as gut microbiota populations between male and female birds. Studies have shown that male broilers have higher crude protein requirements compared to female broilers. The expression of monosaccharide and amino acid transporters show conflicting results as expression depends on the interactions between sex and bird age and breed as well as which tissue is sampled. Differences in microbiota populations between the genders were reported which may contribute towards performance differences, however research in this area is limited. The differences observed between the sexes contribute to increased variation in nutrition trials, and the potential to rear birds as equally mixed-sex becomes an option to reduce the variation introduced by the sex effect. Difference in rearing options obviously would only be feasible provided a quick, practical and cost-effective method of sexing birds is available, a topic that is also discussed in this review.

Buffered formic acid and a monoglyceride blend improve performance and modulate gut bacteria and immunity gene expression in broilers under necrotic enteritis challenge.

Due to the removal of antibiotics from animal feed, alternatives have been sought to control necrotic enteritis (NE) in broilers. The current study investigated the effects of buffered formic acid (Amasil NA) and monoglycerides of short- and medium-chain fatty acids (Balangut LS P) on the performance and gut health of broilers challenged with NE. A total of 816 as-hatched 1-d-old chicks (Cobb 500) were randomly assigned to 6 treatments with 8 replicates. Treatments were: T1) nonchallenged control; T2) NE challenged control; T3) Amasil NA (challenge plus Amasil NA, 0.3% throughout all phases); T4) Balangut LS P (challenge plus Balangut LS P, 0.5%, 0.3%, and 0.2% in the starter, grower and finisher phases, respectively; T5) Combined (challenge plus combination of T3 and T4); T6) Antibiotic (challenge plus Zn bacitracin, 0.05 % throughout all phases). Birds were orally gavaged with live Eimeria vaccine species (d 9) and with Clostridium perfringens (d 14 and 15). On d 16, birds were sampled to evaluate gut permeability, microbiota, and mRNA abundance in the jejunum. The data were analyzed in JMP software using one-way ANOVA with Tukey's test to separate means, and Kruskal-Wallis test was used for non-normally-distributed parameters. Results showed that Balangut LS P decreased (P<0.05) feed conversion ratio compared to nonchallenged ones at the end of the study. Balangut LS P reduced (P < 0.05) the level of cecal Bacteriods compared to nonchallenged group, whereas Amasil NA shifted the levels of ileal Bifidobacteria, Enterobacteriaceae, and Lactobacillus towards nonchallenged control (P > 0.05). NE challenge upregulated (P < 0.001) the expression of IL-21R, zeta chain of T cell receptor (ZAP70), and dual specificity phosphatase 4 (DUSP4) compared to nonchallenged birds, whereas Balangut LS P showed an intermediate (P > 0.05) expression pattern of these genes towards nonchallenged and antibiotic groups. In conclusion, combination of Balangut LS P and Amasil NA has the potential to be used as an additive to improve the performance and gut health of broiler chickens, especially under challenging conditions such as NE infections.

Xylanase and beta-glucanase improve performance parameters and footpad dermatitis and modulate intestinal microbiota in broilers under an Eimeria challenge.

Coccidiosis is an enteric disease of poultry worldwide that compromises gut health and growth performance. The current research investigated the effects of 2 doses of a multienzyme preparation on broilers' performance, gut health, and footpad dermatitis (FPD) under an Eimeria challenge. A total of 512 mixed-sex day-old chicks (Cobb 500) were randomly allocated to 4 treatments of 8 replicates. Treatments were: 1) nonchallenged control (NC); 2) NC + Eimeria challenge (CC); 3) CC + recommended level of xylanase and glucanase (XG, 100 g/t feed [on top]); 4) CC + double XG (2XG, 200 g/t feed). Eimeria spp. vaccine strains were gavaged on d 9 to induce coccidiosis in chickens. Performance parameters were evaluated during starter, grower, and finisher phases, and 4 birds per pen were euthanized on d 16 for sampling, FPD was scored on d 35, and litter moisture was analyzed on d 17 and 35. The data were analyzed using 1-way ANOVA with Tukey's test to separate means, and Kruskal-Wallis test was used for non-normally distributed parameters. The results showed that the Eimeria challenge was successful based on reduced weight gain and feed intake during grower phase, and higher FITC-d concentration, lesion score (female), and oocyst counts (d 14) in CC group compared to N.C. group, while XG and 2XG increased (P < 0.001) weight gain and improved FCR compared to CC and NC groups during finisher phase. The addition of X.G. and 2XG decreased litter moisture (P = 0.003) and FPD (P < 0.001) in challenged broilers compared to the N.C. group (d 35). Supplementing XG and 2XG reestablished the population of Lactobacillus in the cecum of challenged birds to an intermediate level between the NC and CC groups (P > 0.05). The inclusion of XG tended to increase the expression of Junctional adhesion molecule 2 (JAM2), which was not different from CC and NC groups (P > 0.05). In conclusion, the combination of xylanase and glucanase (Natugrain TS) improved the performance and modulated jejunal microbiota of broilers under mild Eimeria challenge.

Inclusion of up to 20% Black Soldier Fly larvae meal in broiler chicken diet has a minor effect on caecal microbiota.

Background: The Black Soldier Fly larvae (BSFL) are a source of nutrients and bioactive compounds in broiler diets. Some components of the BSFL may serve as a prebiotic or may impact the intestinal microbiota of the broilers by other modes of action, which in turn can affect the health and performance of broilers. Here, we investigate the impact of up to 20% BSFL in broiler diets on the diversity and composition of the broiler's microbiota. Methods: Four hundred broilers were fed five iso-nutritious experimental diets with increasing levels of BSFL meal reaching 0%, 5%, 10%, 15%, 20% in the finisher diets. Eight caecal content samples coming from each of the eight replicates per treatment were collected at two time points (day 21 and day 42) for DNA extraction and sequencing of the V3-V4 regions using Illumina MiSeq 2 × 300 bp pair-end sequencing with 341f and 805r primers. Analysis of variance and Spearman's correlation were performed, while QIIME2, DADA2, and Calypso were used for data analysis. Results: When broilers were 21 days of age, the abundance of two groups of sequence variants representing Enterococcus and unclassified Christensenellaceae was significantly lower (p-value = 0.048 and p-value = 0.025, respectively) in the 20% BSFL group compared to the 0% BSFL group. There was no relevant alteration in the microbiota diversity at that stage. On day 42, the Spearman correlation analysis demonstrated that the sequence variants representing the genus Coprococcus showed a negative relationship with the BSFL inclusion levels (p-value = 0.043). The sequence variants representing the genus Roseburia and Dehalobacterium demonstrated a positive relationship with the BSFL dietary inclusion (p-value = 0.0069 and p-value = 0.0034, respectively). There was a reduction in the dissimilarity index (ANOSIM) caused by the 20% BSFL dietary inclusion. Conclusion: The addition of up to 20% BSFL in broiler diets did not affect the overall caeca microbiota diversity or composition at day 21. On day 42, there was a reduction in the beta diversity caused by the 20% BSFL dietary inclusion. The abundance of the bacterial group Roseburia was increased by the BSFL dietary inclusion, and it may be beneficial to broiler immunity and performance.