Ovo Inoculation Research Articles

Adapted tissue assay for the assessment of ileal granulocyte degranulation following in ovo inoculation with select bacteria or coccidial challenge in chickens.

A previously described heterophil degranulation assay was adapted for use with ileal mucosal tissue via quantification of β-D-glucuronidase and assay end product 4-methylumbelliferone (4-MU). Three initial experiments evaluated the effect of in ovo inoculations of Citrobacter freundii (CF) or mixed lactic acid bacteria (LAB) on ileal granulocyte degranulation. Inoculations were administered on embryonic d18, body weights (BW) were recorded on day of hatch (DOH) and d10 to calculate body weight gain (BWG), and ileal mucosal scrapings were collected on DOH or d10 for the 4-MU assay. In all experiments, treatments were statistically analyzed relative to control groups. Treatments minimally affected BWG in all in ovo experiments (p > 0.05) relative to respective control groups. Similarly, ileal degranulation in in ovo treatments did not statistically differ (p > 0.05). Based on BWG, in ovo treatments may have induced low-level inflammation unable to elicit detectable changes via the 4-MU assay. Four subsequent experiments were conducted to evaluate effects of Eimeria maxima (EM) on ileal degranulation. Treatments included non-inoculated controls and low, medium, or high EM infection. Across all four experiments, final BW or BWG over the inoculation period were suppressed (p < 0.05) in EM groups relative to respective controls with the exception of EM-low (p = 0.094) and EM-medium (p = 0.096) in one trial. Ileal mucosal scrapings for the 4-MU assay were collected on day of peak lesions. Resulting values were reduced (p < 0.05) for EM treated birds in three experiments with the exception of EM-medium (p = 0.247). No differences were observed in one experiment (p = 0.351), which may have been attributed to a variation in strain of infecting Eimeria. Although refinement for low level inflammation is warranted, results indicate successful adaptation of the 4-MU assay for use with intestinal tissue during significant gastrointestinal inflammation.

Synergistic influence of probiotic and florfenicol on embryonic viability, performance, and multidrug-resistant Salmonella Enteritidis in broiler chickens

This study was conducted during the period of August 2021 to April 2022 and divided into two parts. The first part involved the isolation and characterization of Salmonella from 200 diseased broiler chickens collected from farms in Dakahlia Governorate, Egypt, with the detection of its antimicrobial susceptibility. The second experimental part involved in ovo inoculation of probiotics and florfenicol to evaluate their effects on hatchability, embryonic viability, growth performance traits and the control of multidrug-resistant Salmonella Enteritidis infections post hatching. The point prevalence of Salmonella in the internal organs of diseased chickens was 13% (26/200), including 6 serotypes: S. Enteritidis, S. Typhimurium, S. Santiago, S. Colindale, S. Takoradi and S. Daula. Multidrug resistance was found in 92% (24/26) of the isolated strains with a multiantibiotic resistance index of 0.33–0.88 and 24 antibiotic resistance patterns. The in ovo inoculation of probiotic with florfenicol showed significant improvement in the growth performance parameters compared with other groups and had the ability to prevent colonization of multidrug resistant S. Enteritidis in the majority of the experimental chicks, and the remaining chicks showed very low colonization, as detected by RT‒PCR. These findings suggested the application of in ovo inoculation techniques with both probiotics and florfenicol as a promising tool to control multidrug-resistant S. Enteritidis in poultry farms.

Evaluation of Newcastle disease virus LaSota strain attenuated by codon pair deoptimization of the HN and F genes for in ovo vaccination

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken’s hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.

In ovo Inoculation of Bacillus subtilis and Raffinose Affects Growth Performance, Cecal Microbiota, Volatile Fatty Acid, Ileal Morphology and Gene Expression, and Sustainability of Broiler Chickens (Gallus gallus).

Banning antibiotic growth promoters has negatively impacted poultry production and sustainability, which led to exploring efficient alternatives such as probiotics, probiotics, and synbiotics. Effect of in ovo injection of Bacillus subtilis, raffinose, and their synbiotics on growth performance, cecal microbial population and volatile fatty acid concentration, ileal histomorphology, and ileal gene expression was investigated in broilers (Gallus gallus) raised for 21 days. On 300 h of incubation, a total of 1,500 embryonated eggs were equally allotted into 10 groups. The first was non-injected (NC) and the remaining in ovo injected with sterile distilled water (PC), B. subtilis 4 × 105 and 4 × 106 CFU (BS1 and BS2), Raffinose 2 and 3 mg (R1 and R2), B. subtilis 4 × 105 CFU + raffinose 2 mg (BS1R1), B. subtilis 4 × 105 CFU + raffinose 3 mg (BS1R2), B. subtilis 4 × 106 CFU + raffinose 2 mg (BS2R1), and B. subtilis 4 × 106 CFU + raffinose 3 mg (BS2R2). At hatch, 60 chicks from each group were randomly chosen, divided into groups of 6 replicates (10 birds/replicate), and fed with a corn–soybean-based diet. In ovo inoculation of B. subtilis and raffinose alone or combinations significantly improved body weight, feed intake, and feed conversion ratio of 21-day-old broilers compared to NC. Cecal concentrations of butyric, pentanoic, propionic, and isobutyric acids were significantly elevated in R1, R2, BS2R1, and BS2R2, whereas isovaleric and acetic acids were significantly increased in R1 and BS2R1 compared to NC. Cecal microbial population was significantly altered in treated groups. Ileal villus height was increased (p < 0.001) in BS1, R2, and BS2R2 compared to NC. The mRNA expression of mucin-2 was upregulated (p < 0.05) in synbiotic groups except for BS1R1. Vascular endothelial growth factor (VEGF) expression was increased (p < 0.05) in BS2, R1, BS1R1, and BS1R2 compared to NC. SGLT-1 expression was upregulated (p < 0.05) in all treated birds except those of R1 group compared to NC. The mRNA expressions of interleukin (IL)-2 and toll-like receptor (TLR)-4 were downregulated (p < 0.05) in BS2 and R1 for IL-2 and BS1R1 and BS2R2 for TLR-4. It was concluded that in ovo B. subtilis, raffinose, and synbiotics positively affected growth performance, cecal microbiota, gut health, immune responses, and thus the sustainability of production in 21-day-old broilers.

In ovo inhibition of avian pox virus replication by mangosteen rind and red ginger ethanolic extracts.

Background and Aim:Avian pox is a contagious disease caused by the avian pox virus (APV). Mangostin and γ-mangostin in mangosteen rind (MR) and gingerol in red ginger (RG) exhibit antiviral activity. In this study, we evaluated the effect of MR and RG ethanolic extracts on APV based on pock lesions on the chorioallantoic membrane (CAM) of specific pathogen-free (SPF) embryonated chicken eggs (ECEs).Materials and Methods:Three APVs from chicken isolates (C1, C2, and C3), one APV from a pigeon isolate (P), 1.5% and 3% MR ethanolic extract, 5% and 10% RG ethanolic extract, and a combination of 1.5% MR and 5% RG at 0.1 mL/egg were inoculated in ovo (7th day incubation, chorioallantoic route) in SPF ECEs. A control group inoculated in ovo with APV alone was also established. Each treatment consisted of three replicates. Parameters including embryo survival, CAM lesions, and average number of pock lesions were determined.Results:In ovo inoculation of MR and RG ethanolic extracts was not harmful to the ECEs and did not induce CAM lesions. The average number of pock lesions in the control group (C1, C2, C3, and P) was 35, 14, 10, and 17, respectively, whereas in all treatment groups, the number was 0, except in the 5% RG group of C1, which had a value of 10.Conclusion:In ovo inoculation of 1.5% and 3% MR, 5% and 10% RG, and the combination of 1.5% MR plus 5% RG ethanolic extracts at 0.1 mL/egg inhibit APV by reducing the number of pock lesions on the CAM of the ECE.

In ovo inoculation of probiotics for broiler chickens: Systematic review and meta-analysis

The objective of this study was to evaluate the effect of probiotic inoculation in fertilized eggs on hatching and posthatching characteristics of broilers. The search for scientific articles was conducted in December 2020 in eight databases (Embase, Google Scholar, SciELO, Science Direct, Scopus, Periodicos Capes, PubMed, and Web of Science) using the following combination of keywords: ((probiotic OR synbiotic) AND "in ovo"). Only articles that evaluated the effect of in ovo inoculation of probiotics, combined or not with other compounds, on hatching parameters (number of hatched eggs and hatching weight) and/or posthatching parameters (performance and carcass yield) in broilers were included. The meta-analysis was performed using the random-effects model to calculate the confidence interval (CI) of the differences between the group inoculated with probiotics and the control (vehicle inoculation). The degree of heterogeneity was assessed by meta-regression, and publication bias was assessed by funnel plots. Wide heterogeneity was observed (P < 0.01) in all analyzes. The overall analysis showed that probiotic inoculation did not influence (P > 0.05) number of hatched eggs, hatching weight, or feed intake after hatching but increased (P < 0.01) weight gain and reduced (P < 0.05) feed conversion ratio. Subgroup analysis showed that, for performance, the Bifidobacterium as probiotic brought the best results, while the yolk was best inoculation site. We conclude that probiotics can improve posthatching performance without influencing hatching parameters, depending on the type of microorganism, inoculation site, and broiler strain used.

Effects of in ovo injection of Manihot esculenta extract on hatchability and post-hatch performance of Sasso broiler chickens

Abstract Manihot esculenta leaf meal in feed at post-hatch improves growth and egg production. One may question the efficacy of Manihot esculenta leaf extract for in ovo feeding. This study was conducted to determine the effects of in ovo injection of Manihot esculenta extract on hatchability, growth performance and biochemical parameters of Sasso broiler chickens. The hatching eggs from 38-week-old broiler breeders were set and at d 18 of incubation, 600 eggs with evidence of living embryos were assigned to 4 treatment groups of 150 eggs each. These groups were: (1) eggs without in ovo injection (negative control); (2) eggs perforated but no injection of any additive (positive control); (3) eggs perforated and injected with 0.1Â ml of saline solution and (4) eggs perforated and injected with 0.1Â ml of Manihot esculenta extract. Data were collected on hatching performance. During the post hatch growth phase, performance traits such as feed intake, weight gain, and feed conversion ratio were collected and at 84 days of age, blood samples were taken for determination of serum total proteins, triglycerides, aspartate aminotransferase (ASAT) and alanine amino transaminase (ALAT). The results showed that hatchability was similar in the negative control and Manihot esculenta extract groups but significantly higher (PÂ <Â 0.05) than those of positive control and saline solution treatment groups. Chick quality was better in the groups with Manihot esculenta extract and saline solution application. Post-hatch, the body weight gain of birds from the Manihot esculenta extract group was higher than those in the groups with saline solution application and in the negative control and positive control groups. Manihot esculenta leaf extract increased significantly the triglyceride rate and reduced ALAT and ASAT concentration at hatch. It can be concluded that in ovo inoculation of 0.5Â μg/ml Manihot esculenta leaf extract on d 18 of incubation into the eggs air chamber improved hatchability, chick quality and post-hatch growth of broilers.

Calcium Carbonate Nanoparticles-Toxicity and Effect of In Ovo Inoculation on Chicken Embryo Development, Broiler Performance and Bone Status.

Simple SummaryIntensive selection in broiler chicken flocks has led do several leg disorders. The injection of nanoparticles, with high specificity to the bone, into the egg is a potential method to improve bone quality. The objective of our study was to evaluate the potential effect of calcium carbonate nanoparticles injected to the egg on chicken embryo development and bone quality of broiler chickens after 42 day of life. The calcium carbonate nanoparticles were not toxic to embryo and even improved the bone quality of embryos and later broilers without negative impact on production results. Thus, the application of calcium carbonate nanoparticles to the egg may be the potential solution for improving the bone mineralization of broiler chickens.The use of intensive selection procedure in modern broiler chicken lines has led to the development of several skeletal disorders in broiler chickens. Therefore, current research is focused on methods to improve the bone quality in birds. In ovo technology, using nanoparticles with a high specificity to bones, is a potential approach. The present study aimed to evaluate the effect of in ovo inoculation (IOI) of calcium carbonate nanoparticles (CCN) on chicken embryo development, health status, bone characteristics, and on broiler production results and bone quality. After assessing in vitro cell viability, the IOI procedure was performed with an injection of 500 μg/mL CCN. The control group was not inoculated with CCN. Hatchability, weight, and selected bone and serum parameters were measured in embryos. Part of hatchlings were reared under standard conditions until 42 days, and production results, meat quality, and bone quality of broilers were determined. CCN did not show cytotoxicity to cells and chicken embryo and positively influenced bone parameters of the embryos and of broilers later (calcification) without negatively affecting the production results. Thus, the IOI of CCN could modify the molecular responses at the stage of embryogenesis, resulting in better mineralization, and could provide a sustained effect, thereby improving bone quality in adult birds.

In ovo administration of Bacillus subtilis serotypes effect hatchability, 21-day performance, and intestinal microflora

Recent research has tried to maximize broiler chick health and performance by utilizing commercial in-feed probiotics to inoculate fertile hatching eggs, and thus expose birds earlier to beneficial bacteria. However, the in ovo inoculation of a specific serotype of Bacillus subtilis was detrimental for broiler hatchability. Therefore, the objective of this study was to determine if other B. subtilis serotypes negatively affect hatchability or if it is associated with a specific serotype. It was also of interest to determine if the B. subtilis serotype influence chick performance and intestinal microflora. On d18 of incubation, 1886 fertile broiler eggs were in ovo inoculated with the following treatments (T): T1 = Marek's vaccine (MV), T2 = MV + B. subtilis (ATCC 6051), T3 = MV + B. subtilis (ATCC 8473), and T4 = MV + B. subtilis (ATCC 9466). It should be noted that in a previous study, T2 was detrimental to hatchability. Inoculated eggs were transferred to 3 hatchers/T. At hatch, chicks were weighed, feather sexed, and hatch residue analysis was conducted. Male chicks were randomly assigned to 40 raised wire cage so that there were 10 birds/cage. On d 0, 7, 14, and 21 of the grow-out, chicks and feed were weighed to calculate performance data. On these days, the ileum and ceca were aseptically collected to enumerate total aerobes and coliforms. No differences were observed for percentage of mid dead embryos, cracked eggs, and cull chicks (P > 0.05). However, hatch of transfer was significantly reduced by T2 compared to T1, T3, and T4 (P < 0.001). T2 had significantly higher percentages of late dead embryos and pips when compared to the other treatments (P = 0.002 and P < 0.001, respectively). Chicks hatched from T2 were not vigorous and, thus, not used for the grow-out trial. No differences were observed for growth performance characteristics for any of the treatments (P > 0.05). For bacterial enumeration, the ileum had equal or fewer bacterial counts for T3 and T4 when compared to T1 on most sampling days, except on d21 where T4 had higher aerobic and coliform counts (P ≤ 0.0001). For the ceca, T3 and T4 had equal or fewer bacterial counts than T1 on every sampling day (P ≤ 0.0001). These data demonstrate that not all B. subtilis evaluated are detrimental to hatchability, but rather, serotype dependent. In addition, different B. subtilis serotypes can modify the intestinal microflora with potential to reduce pathogenic bacteria present in young broiler, without impacting overall performance.

Effects of Bacillus Subtilis Supplementation in Ovo or Water on Hatchability and Growth Performances of Broilers

The aim of the present study was to determine the best method as well as appropriate concentration of Bacillus subtilis (B.subtilis)that can be applied to maximize the hatching and growth performances of broiler chickens. A total of 480 Cobb500 broilers fertile hatching eggs were assigned to five experimental groups (T1, T2, T3, T4andT5) as follows: T1assigned to three subgroups; One: it Penetrated and sealed without injection (control negative); Two: Eggs injected with 0.1 ml of physiological saline 0.9 NaCl (control positive) and the third: contains eggs that didn’t receive any pre-hatch treatment.T2 and T3 are the groups injected by B.subtilis while T4 and T5 are the eggs that didn’t receive any pre-hatch treatment. On the 18thday of incubation, 100 μL of 107 and 1010 CFU of B. subtilis/egg or saline was injected into the air cell of T2, T3 and the control positive eggs; respectively. Hatched chicks were re- assigned according to way and concentration of B. subtilis application toT1,T2,T3,T4 and T5groups; control group with no probiotic treatment ,inovo inoculated by 107 CFU/egg, 1010 CFU/egg, water supplementation by107 CFU/ml and 107 CFU/ml then managed for 35 days. Results revealed that, neither in ovo inoculation nor watery administration of B.subtilis of different concentrations had significant effect on hatchability parameters of incubated broiler eggs except increment of sticky embryo percentages in all treated groups. Broilers that supplemented in ovo via 107B.subtilis/egg had better weekly body weight, highest RGR% and the lowest FCR through the experimental period. Irrespective of the method of B. subtilis supplementation to broiler,107concentration of B.subtilis showed the highest significant marketing weight (p

In Ovo Inoculation of Vitamin A Modulates Chicken Embryo Immune Functions.

Vitamin A mediates many important biological functions in humans and animals. Presence of vitamin A receptors on immune system cells emphasizes their role in immune functions. To assess the effects of in ovo inoculation of vitamin A on the immune system of chicken embryos, 18 days old embryonated eggs were inoculated with 3 different concentrations of retinoic acid (the active form of vitamin A) at 30, 90, and 270 μmol/egg via the amniotic sac. After 6, 18, and 24 h, the spleen and bursa of the embryos were collected for RNA extraction and real-time polymerase chain reaction. The results were dose dependant. After 24 h, inoculation with 270 μmol/egg downregulated relative expression of interferon IFN-α, IFN-β, IFN-γ, interleukin (IL)-1β, IL-2, CXCLi2, IL-12, and IL-13 compared to control in the spleen, indicating an anti-inflammatory effect at this concentration. In comparison, 90 μmol/egg induced greater expressions of the above genes at the same timepoint compared to the 270 μmol. The results of this study indicate that in ovo inoculation of vitamin A can modulate immune functions of the chicken embryo, which might be beneficial for induction of immune responses by in ovo vaccines.

Effects of in ovo probiotic administration on the incidence of avian pathogenic Escherichia coli in broilers and an evaluation on its virulence and antimicrobial resistance properties

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, which has been traditionally controlled by the prophylactic in-feed supplementation of antibiotics. However, antibiotics are being removed from poultry diets owing to the emergence of multidrug-resistant (MDR) bacteria. Therefore, alternatives to control APEC are required. This study aimed to evaluate the effects of in ovo inoculation of probiotics on the incidence of APEC in broilers and evaluate the virulence and antimicrobial resistance properties of the APEC isolates. On embryonic day 18, 4 in ovo treatments (T) were applied: T1 (Marek's vaccine [MV]), T2 (MV and Lactobacillus animalis), T3 (MV and Lactobacillus reuteri), and T4 (MV and Lactobacillus rhamnosus). A total of 180 male broilers per treatment were randomly placed in 10 pens. The heart, liver, spleen, and yolk sac were collected on day 0, 14, 28, and 42. Presumptive E. coli isolates were confirmed by real-time PCR. The positive isolates were screened for the APEC-related genes (iroN, ompT, hlyF, iss, and iutA), and E. coli isolates containing one or more of these genes were identified as APEC-like strains. A total of 144 APEC-like isolates were isolated from 548 organ samples. No differences (P > 0.05) among treatments were observed for the incidence of APEC-like strains in all organs when averaged over sampling days. However, when averaged over treatments, the incidence in the heart, liver, and yolk sac was different among sampling days; a significant increase was observed in these organs on day 14 compared with day 0. Twenty-five antimicrobial resistance genes were evaluated for all APEC-like isolates, and 92.4% of the isolates carried at least one antimicrobial resistance gene. Thirty-seven isolates were then selected for antimicrobial susceptibility testing; MDR strains accounted for 37.8% of the isolates. In conclusion, the in ovo inoculation of a single probiotic strain did not confer protection against APEC strains in broilers. The high prevalence of MDR isolates indicates that further research on antibiotic alternatives is required to prevent APEC infections in broilers.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

In ovo inoculation of an Enterococcus faecium–based product to enhance broiler hatchability, live performance, and intestinal morphology

Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium–based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 μL of Marek's vaccine (MV), 2) MV and 1.4 × 105 cfu GH/50 μL, 3) MV and 1.4 × 106 cfu GH/50 μL, 4) MV and 1.4 × 107 cfu GH/50 μL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 105 and 107 GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 107 GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 107 GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 105 and 107 GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study.

In ovo supplementation of chitooligosaccharide and chlorella polysaccharide affects cecal microbial community, metabolic pathways, and fermentation metabolites in broiler chickens

The chitooligosaccharide (COS) and chlorella polysaccharide (CPS) have been used as feed supplements in the poultry industry for improving growth performance and immunity. However, the benefits of these prebiotics on the gut health of chickens when used in early nutrition are unknown. This study evaluated the effects of in ovo feeding of COS and CPS on the cecal microbiome, metabolic pathways, and fermentation metabolites of chickens. A total of 240 fertile eggs were divided into 6 groups (n = 4; 10 eggs/replicate): 1) no-injection control, 2) normal saline control, 3) COS 5 mg, 4) COS 20 mg, 5) CPS 5 mg, and 6) CPS 20 mg injection. On day 12.5 of egg incubation, test substrate was injected into the amniotic sac of eggs in respective treatments. The hatched chicks were raised for 21 D under standard husbandry practices. On day 3 and 21, cecal digesta were collected to determine microbiota by shotgun metagenomic sequencing and short-chain fatty acids by gas chromatography. The cecal microbial composition was not different (P > 0.05) among the treatment groups on day 3 but was different (P < 0.05) on day 21. At the species level, the polysaccharide-utilizing bacteria including Lactobacillus johnsonii, Bacteroides coprocola, and Bacteroides salanitronis were higher in the COS group, whereas the relative abundance of some opportunistic pathogenic bacteria were lower than those in the CPS and control groups. At the functional level, the pathways of gluconeogenesis, L-isoleucine degradation, L-histidine biosynthesis, and fatty acid biosynthesis were enriched in the COS group. In addition, propionic acid content was higher (P < 0.05) in the COS group. A network based on the correlation between the COS and other factors was constructed to illuminate the potential action mechanism of the COS in chicken early nutrition. In conclusion, in ovo inoculation of COS 5 mg showed positive effects on the cecal microbiota, metabolic pathways, and propionic acid, thus can be used as in ovo feeding to modulate the gut health of chickens.

In ovo inoculation of duck embryos with different strains of Bacillus cereus to analyse their synergistic post-hatch anti-allergic potentialities.

Background Bacillus cereus is a Gram‐positive, facultative anaerobic bacteria with few strains reported to be used as probiotics for animals and birds in recent times if the doses are formulated properly.ObjectivesTo analyse the synergistic anti‐allergic potentiality of different Bacillus cereus strains on experimental in ovo and in vitro duck model, as probiotic immune stimulant.Materials and methodsDifferent strains of Bacillus cereus from 29 isolates were identified through 16S rRNA gene sequencing from the milk samples of buffalo breeds of South Asia. The probiotic properties were tested in aspects of gram staining, catalase test, coagulase, test, bile salt tolerance, pH tolerance and phenol tolerance test. MIC50 and MIC90 levels were profiled using nine different antibiotics, and antimicrobial activity against eight different enteric pathogens was assessed. Finally, the test strains of Bacillus cereus (Colony Forming Unit [CFU] 30X1011) were combined‐infused at different concentrations in embryonated duck eggs to assess the post‐hatch anti‐allergic effects against histamine‐induced allergic reaction and their immunoglobulin E (IgE) level was tested.ResultsMolecular identification confirmed the test strains as B. cereus HKS 1–1, B. cereus LOCK 1,002 and B. cereus BF2, which were all motile, spore‐forming, catalase‐positive and rod‐shaped. All were 0.3% bile salt, 0.4% phenol and pH tolerant. Two‐way ANOVA test P values revealed that B. cereus BF2 was statistically significant (p < .0014) in bile salt tolerance test. B. cereus HKS 1–1 was significant in phenol and pH tolerance at p < .0002 and p < .0489, respectively. Besides, the test strains showed antibiotic sensitivity and antimicrobial activity to different enteric pathogens. In vivo model referred the test strains as effective in partial allergy reduction at same CFU but at different concentrations with p < .0001 among the groups.ConclusionThe isolated and characterized strains of B. cereus showed partial immune‐stimulating potentiality against experimentally induced allergic reaction.

Effects of in ovo Inoculation of Multi-Strain Lactobacilli on Cytokine Gene Expression and Antibody-Mediated Immune Responses in Chickens.

This study was conducted to investigate the effects of various doses of a multi-strain lactobacilli mixture (Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus, and Lactobacillus johnsonii) on the innate and adaptive immune responses in broiler chickens. At embryonic day eighteen, 200 eggs were injected with PBS, or three different doses of a multi-strain lactobacilli mixture (1 × 105, 1 × 106, and 1 × 107 CFU/egg, P1, P2, and P3 respectively) along with a group of negative control. On days 5 and 10 post-hatch, cecal tonsil, bursa of fabricius, and spleen were collected for gene expression and cellular analysis. On days 14 and 21 post-hatch, birds were immunized intramuscularly with both sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH). Serum samples were collected on days 0, 7, 14, and 21 after primary immunization. The results demonstrated that lactobacilli inoculation increased the splenic expression of cytokines, including interferon (IFN)-α, IFN-β, IFN-γ, interleukin (IL)-8, and IL-12 on day 5 post-hatch compared to the control group (PBS). However, in cecal tonsils, lactobacilli treatment downregulated the expression of IL-6 on day 5 post-hatch and IL-2 and IL-8 on day 10 post-hatch. No significant differences were observed in the expression of cytokine genes in the bursa except for IL-13 which was upregulated in lactobacilli-treated groups P2 and P3 on days 5 and 10 post-hatch. Flow cytometry analysis showed that the percentage of KUL01, CD4+ and CD8+ splenocytes was not affected by treatments. In addition, no significant differences were observed for antibody titers against SRBC. However, lactobacilli treatment (P1, P2, and P3) was found to increase IgM titers on day 21 post-primary immunization compared to controls. Furthermore, in ovo injection of the highest dose of probiotics (1 × 107, P3) increased serum IgG titers against KLH on day 7 post-primary immunization. In conclusion, this study demonstrated that that in ovo administration of lactobacilli can improve antibody-mediated immune responses and differentially modulate cytokine expression in mucosal and systemic lymphoid tissues of chickens.

Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch

Pioneer colonization of the gastrointestinal tract (GIT) by bacteria is thought to have major influence on neonatal tissue development. Previous studies have shown in ovo inoculation of embryos with saline (S), species of Citrobacter (C, C2), or lactic acid bacteria (L) resulted in an altered microbiome on day of the hatch (DOH). The present study investigated GIT proteomic changes at DOH in relation to different inoculations. Embryos were inoculated in ovo with S or ∼102 cfu of C, C2, or L at 18 embryonic days. On DOH, the GIT was collected, and tissue proteins were extracted for analysis via tandem mass spectrometry. A total of 493 proteins were identified for differential comparison with S at P ≤ 0.10. Different levels were noted in 107, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine canonical pathways and biological functions related to these changes. Three members of the cytokine family (interleukin [IL]-1β, IL6, and Oncostatin M) were predicted to be activated in C2, indicated with Z-score ≥ 1.50, which suggested an overall proinflammatory GIT condition. This was consistent with the activation of the acute-phase response signaling pathway seen exclusively in C2 (Z-score = 2.00, P < 0.01). However, activation (Z-score = 2.00) of IL-13, upregulation of peroxiredoxin-1 and superoxide dismutase 1, in addition to activation of nitric oxide signaling in the cardiovascular system of the L treatment may predict a state of increased antioxidant capacity and decreased inflammatory status. The nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response (Z-score = 2.00, P < 0.01) was predicted to be upregulated in C which suggested that chicks were in an inflammatory state and associated oxidative stress, but the impact of these pathways differed from that of C2. These changes in the proteome suggest that pioneer colonizing microbiota may have a strong impact on pathways associated with GIT immune and cellular development.

Effect of in Ovo Inoculation of Bifidobacterium spp. on Growth Performance, Thyroid Activity, Ileum Histomorphometry, and Microbial Enumeration of Broilers.

Early colonization by beneficial bacteria stimulates the function and development of the digestive tract, on which the performance and vitality of broilers rely. This experiment evaluated the effects of in ovo injection of bifidobacteria on the performance, thyroid activity, bacterial enumeration, and ileal histomorphometry of broiler chickens. A total of 360 fertile eggs were inoculated into the yolk sac, on day 17 of embryogenesis, and allocated to six experimental groups: negative control (not injected, G1), positive control (injected with sterilized distilled water, G2), and groups inoculated with 2 × 108 CFU of Bifidobacterium bifidum, G3; B. animalis, G4; B. longum, G5; or B. infantis, G6. The results revealed an increase (P < 0.01) in body weight and weight gain in all treated groups increases of at least 5.38 and 8.27%, respectively, compared with the control. Feed consumption was not affected during all experimental periods, while the feed conversion ratio (FCR) was enhanced (P < 0.01) only for the overall experimental period (1-28 days of age). The G3 birds recorded the lowest FCR (1.38), while the highest was observed in G1 birds (1.57). Serum concentrations of thyroxin and triiodothyronine were elevated (P < 0.05) with probiotic inoculation. The antioxidant status and immune response of bifidobacteria injected birds were improved; the serum contents of superoxide dismutase and immunoglobulins Y, M, and A were increased (P < 0.05 and P < 0.01), while the malondialdehyde content was decreased (P < 0.01). Ileal architecture was improved in the bifidobacteria treated groups; the highest values of villus height and the villus height/crypt depth ratio were recorded in G3 (936.6 and 11.80) compared with those of G1 (537.1 and 6.93). Moreover, ileal lactic acid bacteria and Bifidobacterium spp. counts increased by at least 10.64 and 51.75%, while total coliform and bacterial counts reduced by at least 15.46 and 15.18%, respectively, compared with those of the control. In conclusion, all tested strains of bifidobacteria enhanced broiler growth performance, ileal function, and thyroid hormone metabolism without obvious differences among them.

Assessment of In Ovo Administration of Bifidobacterium bifidum and Bifidobacterium longum on Performance, Ileal Histomorphometry, Blood Hematological, and Biochemical Parameters of Broilers.

Bifidobacterium is one of the most promising probiotics which was recently used as an alternative growth promoter in poultry. This trial was considered to evaluate the potential effects of in ovo administration of Bifidobacterium bifidum and Bifidobacterium longum on growth performance and biochemical parameters of broilers. Three hundred broiler breeder eggs were incubated and candled individually at day 17 of embryogenesis; then, eggs that had live embryos were randomly divided into six groups. The first and second groups were of intact (without injection) and vehicle-injected (sterile distilled water) control groups, respectively. Other groups were in ovo injected with 1 × 109 and 1 × 107CFU B. bifidum, and 1 × 109 and 1 × 107CFU B. longum, respectively. Hatchability was significantly (P < 0.001) affected by in ovo inoculation of bifidobacteria strains. Body weight gain and feed conversion ratio were significantly improved (P = 0.014 and 0.019, respectively) in all treatment groups. Protein fractions, aspartate aminotransferase, and alanine aminotransferase activities were not significantly altered by bifidobacteria strains in ovo injection. Regarding ileal histomorphometry parameters, a significant elevation in villus height and villus height/crypt depth ratio values were recorded while crypt depth was insignificantly affected by in ovo treatments. It could be concluded that the tested manual method of in-yolk sac injection at day 17 of incubation is more favorable in easier delivering of bifidobacteria strains which improve broiler growth performance and ileal development without any undesirable effects on blood indices and liver and renal functions.