Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

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.

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.

The potential for inoculating Lactobacillus animalis and Enterococcus faecium alone or in combination using commercial in ovo technology without negatively impacting hatch and post-hatch performance.

The poultry industry has recently undergone transitions into antibiotic free production, and viable antibiotic alternatives, such as probiotics, are necessary. Through in ovo probiotic inoculation, beneficial microflora development in the gastrointestinal tract may occur prior to hatch without negatively impacting chick performance. Therefore, the objective of the present study was to observe the impacts of the injection of probiotic bacteria individually or combined into fertile broiler hatching eggs on hatch and live performance characteristics. A total of 2,080 fertile broiler hatching eggs were obtained from a commercial source. On day 18 of incubation, 4 in ovo injected treatments were applied: 1.) Marek's Disease (HVT) vaccination, 2.) L. animalis (∼106 cfu/50µl), 3.) E. faecium (∼106 cfu/50µl), and 4.) L. animalis + E. faecium (∼106 cfu & ∼106 cfu/50µl each). On day of hatch, hatchability and hatch residue data were recorded. A portion of male chicks from each treatment were placed in a grow-out facility for a 21 d grow-out (18 chicks/pen × 10 pens/treatment = 720 male chicks) with a corn and soy bean meal-based diet without antibiotics or antibiotic alternatives. Performance data and gastrointestinal samples were collected on days 0, 7, 14, and 21. Results indicated no differences in all hatch parameters between treatments (P > 0.05) except for % pipped, where the L. animalis treatment had lower % pipped eggs compared to the HVT control and E. faecium treatments (P = 0.04). No differences were observed in body weight gain or mortality (P > 0.05). Probiotic treatments altered gastrointestinal tissue length, weight, and pH. This resulted in all in ovo injected probiotic treatments increasing feed conversion ratio (FCR) from days 7 to 14 as compared to the control (P = 0.01). Differences in FCR were not observed in any other week of data collection (days 0 to 7, 14 to 21, or 0 to 21; P > 0.05). Although probiotics altered live performance from days 7 to 14, these data suggest that in ovo inoculations of L. animalis and E. faecium in combination are viable probiotic administration practices that potentially improve hatch characteristics and gastrointestinal tract development.

Effects of Bacillus subtilis and coccidial vaccination on cecal microbial diversity and composition of Eimeria-challenged male broilers.

In a companion study, the effects of dietary antibiotic alternative and coccidial vaccination on the growth performance of male broilers have been reported. In this paper, the effects of dietary probiotics and coccidial vaccination on diversity and composition of cecal microbiota were investigated using a 3 (diets) × 2 (vaccinated or non-vaccinated) factorial setting of treatments. Three diets, including a corn and soybean-meal control diet, an antibiotic diet (a control diet supplemented with bacitracin and salinomycin), and a probiotic diet (a control diet supplemented with Bacillus subtilis) were provided to broiler chicken from day 0 to 42. To simulate an Eimeria challenge in the field, all chicks were gavaged with a 20× dose of commercial coccidial vaccine containing live Eimeria oocysts on day 14. Cecal contents were collected on day 42. High-throughput sequencing of the 16S rRNA gene was used to determine microbial diversity and composition. Coccidial vaccination to broilers reduced bacterial diversity (Shannon index) of the cecal microbiota. There was a significant interaction between the dietary additive and coccidial vaccination on the observed bacterial species number. Diets supplemented with B. subtilis increased bacterial species of non-vaccinated broilers but decreased bacterial species of vaccinated broilers. In contrast, diets supplemented with antibiotics reduced bacterial species of broilers from both groups. Interactions between dietary additive and coccidial vaccination were also observed on microbial composition. Vaccinated broilers fed the B. subtilis diet exhibited the lowest Firmicutes percentage and highest Bacteroidetes percentage within the microbial community. In addition, vaccinated broilers fed the B. subtilis diet exhibited the highest Rikenella microfusus percentage. From this study, the coccidial vaccination on the day of hatch reduced the microbial diversity of broilers at a later age. The inclusion of B. subtilis-probiotics in the feed of vaccinated broilers may reduce microbial diversity in cecal content by increasing the proportion of a predominant bacterial species, R. microfusus, in the microbial community.

Effects of coccidial vaccination and dietary antimicrobial alternatives on the growth performance, internal organ development, and intestinal morphology of Eimeria-challenged male broilers.

Effects of the coccidial vaccination and dietary antimicrobial alternatives on growth performance, internal organ development, and intestinal morphology of male broilers subjected to an Eimeria challenge were determined. A total of 1,120 one d-old Ross × Ross 708 male broilers were randomly distributed to 80 floor pens (10 treatments, 8 replication pens/treatment, and 14 chicks/pen). A 2 × 5 factorial arrangement of treatments was used to determine the main and interaction effects of the coccidial vaccination (vaccinated or non-vaccinated) and the dietary additive [1) corn and soybean-meal basal diet, 2) basal diet + antimicrobials (bacitracin and salinomycin), 3) basal diet + probiotics (3 Bacillus subtilis strains), 4) basal diet + prebiotics (mannan-oligosaccharides and ß-glucans), and 5) basal diet + probiotics + prebiotics]. To mimic the Eimeria challenge, all chicks were gavaged with a 20x dose of a different coccidial vaccine (live Eimeria oocysts) at Day 14. The coccidial vaccination decreased Day 0-14 and 29-42 BW gain (BWG) and subsequently decreased Day 0-56 BWG. Broilers fed diets with antimicrobials exhibited the lowest feed conversion ratio (FCR) during the periods of Day 0-14 and 15-28, the shallowest ileal crypt depth on Day 28, and the lowest relative duodenum weight on Day 28 and 42. The Pre+Pro diets helped the broilers to reach a lower overall FCR than did the Pro alone diets and helped the broilers reach a FCR similar to that of the Anti diets. However, broilers fed diets supplemented with prebiotics and probiotics exhibited the deepest intestinal crypt depth on Day 28. There was no interaction between coccidial vaccination and dietary additive on growth performance or any carcass yield. In conclusion, antimicrobial additives might reduce the intestinal size of broilers; whereas prebiotic and B. subtilis-based probiotic additives might promote the growth of several digestive organs. Prebiotics can be safely used with B. subtilis-probiotics in broiler feed without compromising feed conversion ability.

Effects of Bacillus subtilis and zinc on the growth performance, internal organ development, and intestinal morphology of male broilers with or without subclinical coccidia challenge.

Effects of antibiotic (bacitracin), anticoccidial (narasin), and alternative (Bacillus subtilis and zinc) feed additives on growth performance, internal organ development, and intestinal morphology of commercial broilers with or without subclinical coccidia challenge were determined. A total of 1,344 1-day-old male Ross × Ross 708 broilers were randomly distributed into 12 treatments (6 diets × 2 challenge treatments, 8 replication pens/treatment) in 96 floor pens. The 6 dietary treatments were as follows: a control diet (corn and soybean-meal basal diet), a probiotic diet (basal diet + Bacillus subtilis), a zinc diet (basal diet + 100 ppm zinc), a probiotic and zinc combined diet, an anticoccidial diet (basal diet + narasin), and a practical diet (basal diet + narasin + bacitracin). On day 21, each chick in the challenge treatment was gavaged with a 10× dose of a commercial vaccine containing live Eimeria oocytes, whereas each chick in the non-challenge treatment was gavaged with equivalent distilled water. The subclinical coccidia challenge increased the relative weights of pancreas and decreased the ileal crypt depth of broilers at 26 d of age, increased feed conversion ratios from day 15 to 28 and 29 to 40, and increased the relative weights of duodenum and bursa on day 54. As compared to other diets, anticoccidial and practical diets increased BW gain and decreased feed conversion ratio from day 15 to 28, and increased the day 40 carcass weights. As compared to control diets, probiotic diets decreased BW gain and increased the mortality from day 15 to 28; however, probiotic diets did not affect the overall growth performance from day 0 to 54 or carcass yield on day 54. Growth measurements during periods of day 29 to 40 and day 41 to 54 were not affected by any feed additive. From this study, a subclinical coccidia challenge enlarged specific internal organs and compromised the feed conversion ability of broilers. Dietary Bacillus subtilis did not affect overall growth rate or carcass yield of broilers under subclinical coccidia challenge.