Transcriptome-based analysis of key signaling pathways affecting the formation of primordial germ cell in chickens

Bone morphogenetic protein 4(BMP4)can induce the formation of chicken primordial germ cells(PGCs)in vitro;however,its regulatory mechanism in poultry remains unknown.This study aimed to use RNA-seq to analyze PGCs in chicken embryos and iPGCs induced by BMP4 in vitro,clarify the internal regulatory factors of PGCs,analyze the mechanism of the formation of PGCs,and lay a theoretical foundation for the further optimization of PGCs induction systems.Embryonic stem cells(ESCs),PGCs and iPGCs induced by BMP4 in vitro were collected.The transcriptional maps of the three cell types were studied using RNA-seq.The results showed 6,142 genes differentially expressed between PGCs and iPGCs,of which 2,728 were upregulated in iPGCs and 3,414 were downregulated in iPGCs.Compared to that in ESCs,BMP4 was significantly upregulated in PGCs and iPGCs.KEGG results showed that both the TGF-βand Wnt signaling pathways were activated during the formation of PGCs in vitro and in vivo,and the activation was more significant during iPGCs induced by BMP4.The expression of Nodal,an inhibitory factor of TGF-βsignaling,was significantly decreased in PGCs and iPGCs,but was not expressed in iPGCs,which further supports our conclusion.Additionally,the Lysosome and PI3K-AKT signaling pathways were significantly enriched in PGCs and iPGCs,respectively.Further,transmission electron microscopy(TEM)results showed that the number of autolysosomes was significantly higher after the addition of BMP4,which is consistent with the KEGG results.Furthermore,the number of PGCs was significantly reduced after ATG14 was interfered in vivo and in vitro.In conclusion,this study screened out the key signaling pathways during the formation of PGCs,aiming to provide help for enriching the mechanism network regulating PGCs formation in chicken and laying a theoretical foundation for further improving the efficiency of inducing PGCs in vitro.

Retinol is involved in the intestinal regeneration and strengthens the intestinal barrier during refeeding in broiler chickens

Fasting is typically used before feeding metabolizable energy assessment in broilers.Previous studies have shown that fasting cause atrophy of the intestinal villus.Whether fasting affects intestinal permeability during refeeding by altering barrier function and nutrient absorption is of concern.Here,23-d-old broilers were randomly assigned to 5 treatments,fasted for 0,12,24,36,and 48 h,respectively,and then refed for 2 d,to study the impact of different duration of fasting on the intestinal regeneration and barrier function during refeeding.Results showed that the intestinal morphology in fasted birds was recovered in 2 d of refeeding at most.As fasting durations increased,enterocytes per intestinal villus were linearly and quadratically increased(both P<0.05),whereas goblet cells per intestinal villus was linearly decreased(both P<0.05).Besides,the mRNA level of lysozyme was linearly decreased as fasting durations increased during refeeding(both P<0.05),while quadratically increased mucin 2 was observed only after 1 d of refeeding(P<0.05).Linear increase effects were observed for claudin 2 and zonula occludens-1with increased fasting durations after 1 d of refeeding(all P<0.05),and linear and quadratical effects were observed for claudin 2 at 2 d of refeeding(both P<0.05).Besides,we found that intestinal permeability to creatinine,4 and 70 kD dextran were linearly and quadratically decreased with increased fasting durations at 6 h and 1 d of refeeding(all P<0.05).Furthermore,jejunum proteomic from birds refed for 6 h showed that birds fasted for 36 h showed increased antimicrobial peptides and upregulated retinol metabolism when compared to the nonfasted birds(P<0.05).Further study showed that retinyl ester catabolism was inhibited during fasting and enhanced during refeeding.Results of intestinal organoid culture showed that retinol benefits the cell proliferation and enterocyte differentiation.In conclusion,the intestinal permeability to small and large molecules was decreased during refeeding by strengthening the intestinal barrier function,and the activated retinol metabolism during refeeding is involved in the intestinal regeneration and strengthens the intestinal barrier.

A novel live attenuated vaccine candidate protects chickens against subtype B avian metapneumovirus

Avian metapneumovirus(aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses. Subtype B aMPV has recently become the dominant epidemic strain in China. We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate. The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation.The LN16-A strain did not revert to a virulent strain after five serial passages in chickens. The genomic sequence of LN16-A differed from that of the parent wild-type LN16(wtLN16) strain and had nine amino acid mutations. In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4^(+) T lymphocytes, and the Th1(IFN-γ) and Th2(IL-4 and IL-6)cytokines. We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation. Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

Significant genomic introgression from grey junglefowl(Gallus sonneratii)to domestic chickens(Gallus gallus domesticus)

Background Chicken is one of the most numerous and widely distributed species around the world,and many studies support the multiple ancestral origins of domestic chickens.The research regarding the yellow skin phenotype in domestic chickens(regulated by BCO2)likely originating from the grey junglefowl serves as crucial evidence for demonstrating the multiple origins of chickens.However,beyond the BCO2 gene region,much remains unknown about the introgression from the grey junglefowl into domestic chickens.Therefore,in this study,based on wholegenome data of 149 samples including 4 species of wild junglefowls and 13 local domestic chicken breeds,we explored the introgression events from the grey junglefowl to domestic chickens.Results We successfully detected introgression regions besides BCO2,including two associated with growth trait(IGFBP2 and TKT),one associated with angiogenesis(TIMP3)and two members of the heat shock protein family(HSPB2 and CRYAB).Our findings suggest that the introgression from the grey junglefowl may impact the growth performance of chickens.Furthermore,we revealed introgression events from grey junglefowl at the BCO2 region in multiple domestic chicken breeds,indicating a phenomenon where the yellow skin phenotype likely underwent strong selection and was retained.Additionally,our haplotype analysis shed light on BCO2 introgression event from different sources of grey junglefowl into domestic chickens,possibly suggesting multiple genetic flows between the grey junglefowl and domestic chickens.Conclusions In summary,our findings provide evidences of the grey junglefowl contributing to the genetic diversity of domestic chickens,laying the foundation for a deeper understanding of the genetic composition within domestic chickens,and offering new perspectives on the impact of introgression on domestic chickens.

Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains worldwide.To reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies.Crossed beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal side.Carbonic anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(P<0.05).However,both were low-expressed in short mandibular condyle side of affected birds(P<0.05).The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes.Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA.Our research suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow slowly.CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

Proteo-transcriptomic profiles reveal key regulatory pathways and functions of LDHA in the ovulation of domestic chickens(Gallus gallus)

Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.Results Critical biochemical events involved in ovulation in domestic chickens(Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle(F1) and postovulatory follicle(POF1) in continuous laying(CL) and intermittent laying(IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes(DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A(LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.Conclusions This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.

Major regulatory factors for reproductive performances of female chickens

The reproductive performance of female chickens is critical for determining the efficiency of production and productivity and thus profitability.Studies have shown that the reproductive performance of female chickens is mainly regulated by the feed,hormones,genes,and light conditions.Herein,we review the major factors regulating female chicken reproductive performance and assess the reproductive organs and their functions.In the current review,we highlight how the interconnections of hormones,candidate genes,and photo-stimulation regulate female chicken reproductive hormones and thus regulate the reproductive organ performance.In this regard,the roles of main hormones[gonadotropinreleasing hormone(GnRH)and genes(GnRH-Ⅰ)]in regulating sexual maturation and ovarian development and maintenance by influencing the survival and function of follicular granulosa cells were also reviewed.In addition,the current review also highlights how feeding female chickens with diets and artificial light-emitting diodes(LEDs)support the effective functioning of their reproductive capacity through the stimulation of sexual maturity at an appropriate age and regeneration of aged reproductive organs.

Dietary Macleaya cordata extract supplementation improves the growth performance and gut health of broiler chickens with necrotic enteritis

Background The poultry industry needs effective antibiotic alternatives to control outbreaks of necrotic enteritis(NE)caused by Clostridium perfringens.Methods The aim of this study was to investigate the effects of dietary supplementation with Macleaya cordata extract(MCE)on the immune function and gut microbiota of broilers with NE.A total of 2881-day-old broiler chicks were randomly assigned to a 2×2 factorial arrangement with two concentrations of dietary MCE supplementation(0 or 350 mg/kg of diet)and two disease challenge statuses(control or NE).Results The results revealed that NE significantly increased the feed conversion rate(FCR),mortality,intestinal lesion score,the levels of IL-1β,IL-17 and IFN-γ/IL-4 in serum and IL-17/IL-10 in the jejunal mucosa,m RNA levels of TLR2,IFN-γand p Ig R in the jejunum,and Clostridium perfringens concentrations in the cecum.NE significantly decreased the body weight(BW),body weight gain(BWG),jejunal villus height,V/C,m RNA level of AMPK-α1 in jejunum,IL-4 level in the jejunal mucosa and lactic acid bacteria abundance in the cecum.MCE significantly increased BW,BWG,jejunal villus height,V/C,m RNA levels of occludin,ZO-1 and AMPK-α1 in the jejunum,the levels of Ig A and Ig G in serum and IL-10 in the jejunal mucosa and m RNA levels of NF-κB,IL-10 and MHC-II in the jejunum.Additionally,MCE significantly decreased the FCR,mortality,intestinal lesion score,jejunal crypt depth,the levels of IFN-γand IL-17 in serum and IL-17/IL-10 in the jejunal mucosa,Clostridium perfringens concentrations in the cecum,and m RNA levels of IL-17/IL-10 in the jejunum.Moreover,NE significantly increased the abundance of bacteria that are associated with inflammation,obesity and depression(Alistipes,Barnesiella,Intestinimonas,RF39 and UCG-005)and significantly decreased the abundance of short-chain fatty acid(SCFA)-producing bacteria(Anaerotruncus,Butyricicoccus and Bacteroides)in the cecum.MCE significantly increased the abundance of SCFA-producing bacteria(Streptococcus,Ruminococcus_torques_group and Lachnospiraceae_NK4A136_group)and significantly reduced the abundance of bacteria that are associated with inflammation and obesity(Alistipes,Barnesiella and UCG-010)in the cecum.In the cecum of broilers with NE,the relative abundance of Barnesiella and Alistipes was higher and that of Lachnoclostridium and Shuttleworthia was lower.Interestingly,these trends were reversed by the addition of MCE to the diet.Spearman correlation analysis showed that Barnesiella and Alistipes were associated with enhanced intestinal inflammation and inhibited growth performance,whereas Lachnoclostridium and Shuttleworthia were associated with anti-inflammatory effects.Conclusions MCE ameliorated the loss of growth performance in broiler chickens with NE,probably by regulating the intestinal barrier,immune function,and gut microbiota.

Effects of Supplemental Glutamine and Lysine on Growth Performance of Broiler Chickens

The optimum levels of Lysine and Glutamine needed for growth performance and maintenance of the chicken broilers were evaluated in a randomized 3 × 4 factorial arrangement of dietary treatments. The battery cages measured 99 × 66 × 25 cm that can be sufficient for 5 birds. Day old Chicken broilers totaling 180 were assigned to dietary treatments comprising of 3 concentrations of Lysine (0.85, 1.14, and 1.42) each in combination with 4 concentrations of Glutamine (0, 1, 2, and 3). Each dietary treatment was replicated 3 times and each replication had 5 birds. The birds were given feed and water ad libitum with a 23-hour light regimen for a period of 4 weeks. Then, the experimental birds were evaluated for body weight gain, feed consumption, and feed conversion in order to determine their optimum requirement for dietary Lysine and Glutamine. Based on the findings of this study, the highest performance was observed in birds fed the diet supplemented with 1.42 lysine and 1% glutamine, but the highest improvement in feed conversion was observed in diet contain 1.14 and 1.42 with 1% and 3% glutamine, respectively. Birds fed 1.42 lysine and 1% glutamine had the highest total body weight gain and feed consumption. The lysine requirements in the diet for Chicken are between 1.14 and 1.42 with glutamine level of 1%.

Influencing Factors and Variation Trends of Muscle pH in Chickens after Slaughter

[Objective] This study aimed to investigate influencing factors and variation trends of muscle p H in chickens after slaughter. [Method] Beijing You chickens and Lohmann brown chickens were reared under the same conditions and slaughtered at the age of 8, 16 and 20 weeks, respectively. At different time（15 min, 1, 4, 8,12, 24 and 48 h） after slaughter, p H value of breast muscles and leg muscles was measured. [Result] The results showed that muscle position, post-slaughter time,gender and week age were important factors influencing p H value, but the breed had little effect. Within 1 h after slaughter, p H value of breast muscles and leg muscles declined rapidly, which reached the minimum level at 12 h after slaughter,and then increased slowly. In addition, p H value of leg muscles exhibited higher decline and increase rates than that of breast muscles to some extent. [Conclusion]This study provided basis for breeding of chickens with excellent meat quality.

Characterization of Genetic Polymorphism of Novel MHC B-LBⅡ Alleles in Chinese Indigenous Chickens

Genetic polymorphism of the major histocompatibility complex （MHC） B-LBⅡ gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB Ⅱ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB Ⅱsequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LBⅡ alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LBⅡ gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB Ⅱ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%±0.94% and 14.64%±2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the β1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB Ⅱ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

Effects of Astragalus Powder on Serum Lipid and Body Fat Content of Fast Large-scale Quality Chickens

[ Objective] To study the effects of astragalus powder on serum lipids and body fat contents of fast large-scale quality chickens, and thus provide a theoretical basis for its application in poultry production,[ Method ] All the 180 1-day-old healthy Liangfeng chickens were randomly divided into six groups. Group 1 was the control group, fed with basal diet; group 2, group 3 and group 4 were fed with basal diet added with 0.75%, 1.0% and 1.25% astragalus powder, respectively; group 5 and group 6 were separately fed with basal diet added with 1.0% and 1.25% astragalus pow- der, but the astragalus powder was used for 10 d and then was forbidden for 10 d in turn during the whole experiment. The content of triglyceride （TG）, total cholesterol （TCHO）, high-density lipoprotein-Cholesterol （ HDL-C）, and low-density lipoprotein-Cholesterol （LDL-C） were respectively determined when the chickens were 35-day-old and 63-day-old, while the percentage of abdominal fat （PAF） as well as intramuscular fat （IMF） and subcutaneous fat ply （SFP） was measured when the chickens were 63-day-old. [ Result] In 35-day-old chickens, the levels of TCHO were sig- nificantly or very significantly lower in group 3, group 4, and group 6 than in control group （ P 〈0.05 or 0.01 ） ; the levels of LDL-C significantly lower in group 3 and group 6 （ P〈0. 05） ; the level of HDL-C significantly higher in group 5 （P〈0.05）. In 63-day-old chickens, the levels of TCHO and LDL-C were significantly or very significantly lower in group 3, group 4, and group 5 than in control group （ P 〈 0.05 or 0.01 ） while the levels of HDL-C were significantly higher in these groups; the PAF was significantly lower in group 3 （ P 〈 0.05）. [ Condasion] Astragalus powder should re- duce the levels of TG, TCHO and LDL-C, promote HDL-C content, and also play a certain regulative role in deposition of abdominal fat.

Study on the Elimination Regularity of Ronidazole Residues in Chickens

[Objective] This study aimed to investigate the elimination regularity of ronidazole residues in chickens. [Method] Normal, healthy, 85-day-old Jinling broilers were selected as experimental chickens and fed with complete feed containing different concentrations of ronidazole. Ronidazole residues in chicken feather and edible tissues were detected by high performance liquid chromatography-tandem mass spectrometry （LC-MS/MS） method to compare the elimination regularity. [Result] Data analysis showed that the amount of ronidazole residues in chicken feather and edible tissues was positively correlated with the concentration of ronidazole administered. After drug withdrawal, there was a significant correlation between the elimination of ronidazole residues in chicken feather and edible tissues. Thus, the prediction model of ronidazole residues in chicken feather could be established. [Conclusion] This study could improve in vivo detection system of ronidazole residues and provide reference for monitoring and controlling drug residues in livestock and poultry oroduction.

Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.

Effects of Bacillus coagulans supplementation on the growth performance and gut health of broiler chickens with Clostridium perfringens-induced necrotic enteritis

Background: The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis(NE) due to Clostridium perfringens.Methods: This study was conducted to investigate the effects of feeding Bacil us coagulans on the growth performance and gut health of broiler chickens with C. perfringens-induced NE. Two hundred and forty 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two dietary B. coagulans levels(0 or 4 × 109 CFU/kg of diet) and two disease chal enge statuses(control or NE chal enged).Results: NE-induced reduction in body weight gain was relieved by the addition of B. coagulans into broiler diets compared with the NE-infected birds. NE infection damaged intestinal morphological structure, promoted intestinal C.perfringens growth and liver invasion, and enhanced anti-C. perfringens specific sI gA concentrations in the gut and specific IgG levels in serum compared with the uninfected birds. NE infection significantly(P < 0.05) decreased mucin-2(at 14 d post-infection(DPI), tol-like receptor 2(TLR2, at 7 and 14 DPI), TLR4(at 7 and 14 DPI), tumor necrosis factor super family15(TNFSF15, at 7 and 14 DPI), lysozyme(LYZ, at 14 DPI) and fowlicidin-2(at 7 and 14 DPI) mR NA levels, whereas it dramatical y(P = 0.001) increased IFN-γ mR NA levels at 7 DPI. However, chal enged birds fed diets supplemented with B.coagulans showed a significant(P < 0.01) decrease in gut lesion scores, decreased C. perfringens numbers in the cecum and liver, and an increase in fowlicidin-2 mR NA levels in compared with the uninfected birds. In addition, compared with the non-supplemented group, dietary inclusion of B. coagulans improved intestinal barrier structure, further increased specific sI gA levels and alkaline phosphatase(IAP) activity in the jejunum, enhanced the expression of jejunum lysozyme mR NA, and inhibited the growth, colonization, and invasion of C. perfringens; in contrast, it reduced serum-specific IgG concentrations and jejunum IFN-γ mR NA levels.Conclusion: These results indicated that dietary B. coagulans supplementation appeared to be effective in preventing the occurrence and reducing the severity of C. perfringens-induced NE in broiler chickens.

Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens

Background： Necrotic enteritis caused by Clostfidium perffingens infection leads to serious economic losses in the global poultry production. In the present study, we investigated the protective effects of essential oils （EO, which contained 25 % thymol and 25 % carvacrol as active components） supplementation on growth performance, gut lesions, intestinal morphology, and immune responses of the broiler chickens infected with C. perfringens. A total of 448 1-day-old male broiler chicks were allocated into eight treatment groups following a 4 x 2 factorial arrangement with four dietary EO dosages （0, 60, 120, or 240 mg/kg） and two infection status （with or without C. perfringens challenge from d 14 to 20）. Results： The challenge did not impair the growth performance of birds, but induced gut lesions and increased crypt depth in the ileum （P ≤ 0.05）. It also down-regulated the claudin-1 and occludin mRNA expression （P ≤0.05）, up-regulated the mRNA expression of interleukin-113 （P≤ 0.05）, tended to increase the toll-like receptor （TLR） 2 mRNA expression （P 〈 0.10） in the ileum, and enhanced the mucosal secretory IgA production （P 〈 0.05）. In the challenged birds, dietary EO supplementation linearly alleviated the gut lesions and improved the ratio of villus height to crypt depth （P ≤0.05）, and the supplementation of 120 and 240 mg/kg EO increased the serum antibody titers against Newcastle disease virus （P≤ 0.05）. Regardless of challenge, the EO supplementation showed a tendency to linearly elevate the feed conversion efficiency between 14 and 28 d of age as well as the occludin mRNA expression （P〈 0.10）, and linearly inhibited the mRNA expression of TLR2 and tumor necrotic factor-o in the ileum （P≤ 0.05）. Conclusions： The dietary supplementation of EO could alleviate the intestinal injury by improving intestinal integrity and modulating immune responses in the C. perffingens-challenged broiler chickens.

Nutritional value of a partially defatted and a highly defatted black soldier fly larvae (Hermetia illucens L.) meal for broiler chickens: apparent nutrient digestibility,apparent metabolizable energy and apparent ileal amino acid digestibility

Background: The study aimed to determine the apparent total tract digestibility coefficients(ATTDC) of nutrients, the apparent metabolizable energy(AME and AMEn) and the amino acid(AA) apparent ileal digestibility coefficients(AIDC)of a partially defatted(BSFp) and a highly defatted(BSFh) black soldier fly larvae meal. The experimental diets were: a basal diet and two diets prepared by substituting 250 g/kg(w/w) of the basal diet with BSFp or BSFh, respectively.Results: Significant differences were found between BSFp and BSFh meals for ATTDC of the nutrients: BSFp resulted more digestible than BSFh, except for ATTDC of CP which did not differed between meals, while a statistical trend was observed for ATTDC of DM and EE. The AME and AMEn values were significantly(P < 0.05) different between the two BSF meals, with higher levels for BSFp(16.25 and 14.87 MJ/kg DM, respectively). The AIDC of the AA in BSFp ranged from 0.44 to 0.92, while in BSFh they ranged from 0.45 to 0.99. No significant differences were observed for the AA digestibility(0.77 and 0.80 for BSFp and BSFh, respectively), except for glutamic acid, proline and serine that were more digestible in the BSFh meal(P < 0.05).Conclusions: Defatted BSF meals can be considered as an excellent source of AME and digestible AA for broilers with a better efficient nutrient digestion. These considerations suggested the effective utilization of defatted BSF larvae meal in poultry feed formulation.

Black soldier fly defatted meal as a dietary protein source for broiler chickens:Effects on growth performance,blood traits,gut morphology and histological features

Background: The present study has evaluated the effects of different inclusion levels of a partially defatted black soldier fly(Hermetia illucens L.; HI) larva meal on the growth performance, blood parameters and gut morphology of broiler chickens. A total of 256 male broiler chickens(Ross 308) were reared from d 1 to d 35 and assigned to4 dietary treatments(8 replicates/treatment and 8 birds/replicate). HI larva meal was included at increasing levels(0, 5%, 10% and 15%; HI0, HI5, HI10 and HI15, respectively) in isonitrogenous and isoenergetic diets formulated for 3 feeding phases: starter(1–10 d), growing(10–24 d) and finisher(24–35 d). Two birds per pen were slaughtered at d 35 and morphometric investigations and histopathological alterations were performed.Results: The live weight(LW) showed linear and quadratic responses to increasing HI larva meal(maximum for HI10 group). Average daily gain(ADG) showed a linear and quadratic responses to HI meal(maximum for HI10 group) during starter and growing periods. A linear decrease was observed for ADG during the finisher period. The daily feed intake(DFI) showed a linear and quadratic effect during the starter period(maximum for HI10 group).Linear and quadratic responses were observed for the feed conversion ratio(FCR) in the growing period and for the whole period of the experiment. The FCR showed a linear response in the finisher period(maximum for HI15).No significant effects were observed for the blood and serum parameters, except for the phosphorus concentration,which showed linear and quadratic responses as well as glutathione peroxidase(GPx) activity, the latter of which showed a linear response. The HI15 birds showed a lower villus height, a higher crypt depth and a lower villus height-to-crypt depth ratio than the other groups.Conclusions: Increasing levels of dietary HI meal inclusion in male broiler chickens may improve the LW and DFI during the starter period, but may also negatively affect the FCR and gut morphology, thus suggesting that low levels may be more suitable. However, no significant effects on the haematochemical parameters or histological findings were observed in relation to HI meal utilization.

Influence of starch sources and dietary protein levels on intestinal functionality and intestinal mucosal amino acids catabolism in broiler chickens

Background: There is growing interest in carbohydrate and protein nutrition to enhance the efficiency of animal production.Reduced-crude protein diets depress environmental pollution and feeding cost,but the challenge to their adoption is maintaining digestive function and growth performance of birds.The present study was conducted to evaluate the influence of different dietary starch sources and protein levels on intestinal functionality and mucosal amino acid catabolism.Methods: Six dietary treatments,based on maize and soybean meal,were offered to 360 AA+male chicks from 6 to 35 d post-hatch as a 3 × 2 factorial array.Either waxy rice or amylose was added to a conventional maize-soy diet to provide three sources of starch with different digestion rates and relatively high and low dietary protein levels.Growth performance,parameters of intestinal functionality and concentrations of free amino acid in the portal circulation were determined.Results: In the grower phase,starch source influenced(P < 0.02) weight gain as diets containing amylose supported significantly higher weight gains than waxy rice.Significant increase of ileal ATP concentrations and Na^+/K^+-ATPase activity were found in amylose treatment.Also,amylose decreased BrdU positive cell numbers and down-regulated m RNA expression for CASP-3.GOT activity in the ileum was higher(P < 0.01) in birds offered low protein diets and there was a trend(P = 0.057) for waxy rice as a starch source to increase ileal GOT activities.There was a significant influence on the concentration of seventeen amino acids in the portal circulation with tryptophan the one exception.Waxy rice as a starch source generated 13.6% and 22.4% numerically higher concentrations of non-essential amino acids than maize and amylose,respectively.Conclusions: Amino acid catabolism in the gut mucosa is subject to nutritional regulation.Given that amino acids can be spared from catabolism in the gut mucosa by supplementation of amylose,it follows their post-enteral availability would be improved and intestinal energy would be derived more efficiently from glucose.

In vitro antibacterial activity of thymol and carvacrol and their effects on broiler chickens challenged with Clostridium perfringens

Background： In the post-antibiotic era, essential oils （EO） are promising alternatives to growth-promoting antibiotics. The aim of the present study was to investigate the antibacterial activities of an EO product and its components thymol and carvacrol in vitro, and the efficacy of EO to control Clostridium perffingens challenge in broiler chickens. Results： The in vitro minimum inhibitory concentration assay showed strong antibacterial activity of the EO product, thymol, and carvacrol against pathogenic Escherichia col~, C. perffingens, and Salmonella strains, and weak activity towards beneficial Lactobacillus strains. Besides, an additive effect was observed between thymol and carvacrol. The in vivo study was carried out with 448 male broiler chicks following a 4 x 2 factorial arrangement to test the effects of EO supplementation （0, 60, 120, or 240 mg/kg EO in wheat-based diet）, pathogen challenge （with or without oral gavage of C. perfr/ngens from day 14 to day 20） and their interactions. Each treatment consisted of eight replicate pens （seven birds/pen）. The challenge led to macroscopic gut lesions, and resulted in a significant increase in ileal populations of C. perffingens and Eschefichia subgroup （P ≤ 0.05） on day 21. Dietary EO supplementation did not influence C. perfringens numbers, but linearly alleviated intestinal lesions on day 21 and 28 （P= 0.010 and 0.036, respectively）, and decreased Eschefichia populations in ileum with increased EO dosages （P = 0.027 and 0.071 for day 21 and 28, respectively）. For caecum, EO quadratically influenced Lactobacillus populations on day 21 （P = 0.002）, and linearly decreased the numbers of total bacteria and Eschefichio on day 28 （P = 0.026 and 0.060, respectively）. Mean thymol and carvacrol concentrations in the small intestine were 0.21 and 0.20 μg/g in intestinal digesta （wet weight）, respectively, for birds fed 60 mg/kg EO, and 0.80 and 0.71 μg/g, respectively, for birds fed 240 mg/kg EO. Conclusions： These results indicated that dietary EO supplementation could affect intestinal microbiota and alleviate intestinal lesions in broilers, which may contribute in controlling C. perffingens infection in broiler chickens.