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.

Internal organ and skeletal muscle development in commercial broilers with woody breast myopathy.

Increasing growth rate, body weight, and breast muscle yield have been linked to broiler muscle problems such as woody breast (WB). The aim of this study was to investigate the internal organ and skeletal muscle development of broilers with WB myopathy under dietary and Eimeria challenge treatments. A 3 diet (control, antibiotic, or probiotic) × 2 challenge (control or Eimeria) × 2 sex factorial arrangement of treatments was used in a randomized complete block design. Ross × Ross 708 chicks were randomly assigned to 96 floor pens with 12 treatment combinations (8 replicates per treatment). Internal organs were sampled on d 13 and 41. Skeletal muscles were sampled on d 41. Internal organ and skeletal muscle weights were analyzed using a 3-way analysis of variance (ANOVA). Relationships between WB and internal organ and skeletal muscle weights were analyzed using one-way ANOVA as all treatments were pooled together and regrouped according to WB scores. On d 41, absolute and relative heart weights were greater in males when they were averaged over diet and challenge treatments (P < 0.001 and P = 0.026, respectively). The birds with WB score 3 had greater absolute heart (P = 0.0002) and spleen weights (P = 0.016), but there was no difference in relative spleen weight (P > 0.05). When averaged over diet and challenge treatments, males have greater absolute duodenum, jejunum, and ileum weights (for all P < 0.0001). Compared with birds with normal breasts, the birds with WB scores 1, 2, and 3 had a greater live weight (for all P < 0.0001) and absolute and relative breast weights (for all P < 0.0001). The birds with WB score 1, 2, and 3 had greater (P < 0.0001) absolute but lower (P < 0.0001) relative drumstick, thigh, and wing weights. Results indicated that broilers with WB had lower relative proventriculus and gizzard weights and greater relative breast meat weight with lower relative drumstick, thigh, and wing muscle weights.

Effects of dietary bacitracin or Bacillus subtilis on the woody breast myopathy-associated gut microbiome of Eimeria spp. challenged and unchallenged broilers.

Study suggested that dysbiosis of the gut microbiota may affect the etiology of woody breast (WB). In the current study, the cecal microbiota and WB in chickens fed three different diets were investigated. A total of 504 male chicks were used in a randomized complete block design with a 3 (Diet) × 2 (Challenge) factorial arrangement of treatments with 6 replicates per treatment, 6 treatments per block, and 14 birds per treatment. The experimental diets were a control diet (corn-soybean meal basal diet), an antibiotic diet (basal diet + 6.075 mg bacitracin/kg feed), and a probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). On d 14, birds that were assigned to the challenge treatment received a 20 × live cocci vaccine. On d 41, breast muscle hardness in live birds was palpated and grouped into normal (NB) and WB phenotypes. Cecal contents were collected and their bacterial compositions were analyzed and compared. The genomic DNA of the cecal contents was extracted and the V3 and V4 regions of 16S rRNA gene were amplified and sequenced via an Illumina MiSeq platform. There were no differences (P > 0.05) in Shannon and Chao 1 indexes between the challenges, diets, and phenotypes (NB vs. WB). However, there was a difference (P = 0.001) in the beta diversity of the samples between the challenged and nonchallenged groups. Relative bacterial abundance differed (false discovery rate, FDR < 0.05) between the challenge treatments, but there were no significant differences (FDR > 0.05) among the three diets or two phenotypes. Predicted energy metabolism, nucleotide metabolism, and amino acid and coenzyme biosynthesis activities only differed (q-value < 0.05) between challenged and nonchallenged groups. The cocci challenge altered the gut microbial composition on Butyricicoccus pullicaecorum, Sporobacter termitidis, and Subdoligranulum variabile, but the dietary antibiotic and probiotic treatments did not impact gut microbial composition. No strong association was found between WB myopathy and gut microbial composition in this study.

Evaluation of Euthanasia Methods on Behavioral and Physiological Responses of Newly Hatched Male Layer Chicks.

Newly hatched male layer chicks are considered as "by-products" in the egg industry and must be humanely euthanized at the hatchery. Instantaneous mechanical destruction (maceration) is the predominant euthanasia method applied in poultry hatcheries and is approved by the American Veterinary Medical Association (AVMA). However, maceration is not perceived by the public to be a humane means of euthanasia. The effects of alternative euthanasia methods, including carbon dioxide (CO2) or nitrogen (N2) inhalation, and a commercial negative pressure stunning system on behavioral and physiological responses of day-of-hatch male layer chicks, were evaluated in a field trial. Chick behaviors, including ataxia, loss of posture, convulsions, cessation of vocalization, and cessation of movement, were monitored. Serum hormones were assessed at the end of each of the alternative euthanasia treatments, including a control group allowed to breathe normal atmospheric air. The N2 method induced unconsciousness and death later than the CO2 and negative pressure methods, and increased serum corticosterone concentrations of neonatal chicks. Carbon dioxide inhalation increased serotonin concentrations as compared to controls, as well as the N2 and the negative pressure methods. The behavioral and physical responses observed in this study suggest that both CO2 inhalation and negative pressure stunning can be employed to humanely euthanize neonatal male layer chicks.

Effects of broiler genetic strain and dietary amino acid reduction on meat yield and quality (part II).

Genetic selection and advances in nutrition have improved broiler growth performance. However, meat quality issues have gained preference over increased growth rate. These meat quality issues may be reduced by lowering dietary amino acid (AA) content. In the present study, 5 common commercial broiler strains were fed either a control or an AA-reduced diet. The control diet was formulated to contain the highest digestible AA (lysine, total sulfur AA, and threonine) levels recommended for the 5 strains. The AA-reduced diet was formulated to contain 20% lower levels of these 3 digestible AA than in the control diet. This resulted in a 5 (strains) × 2 (AA levels) factorial arrangement. A total of 1,280 straight run broilers were randomly allocated to 8 replicate blocks. The AA reduction decreased absolute breast weights of 3 strains on day 42 and 2 strains on day 56, and decreased absolute weights of tender, wing, drumstick, and thigh on both day 42 and 56 for all 5 strains. However, the absolute fat pad weight and relative fat pad and thigh weights to BW were increased in the AA reduction treatments on both day 42 and 56. The AA reduction contributed to the lower breast meat pH on both day 42 and 56, which may have been directly related to decreased severe woody breast myopathy (WBM) incidence on day 42 and moderate WBM incidence on day 56. The severity of WBM was positively related to breast weight in all 10 treatments on both day 42 and 56, with the exception of birds in strain 3 on day 56 that were fed the AA-reduced diet. At the same time, AA reduction was more cost-effective when WBM incidence was considered in a theoretical model. In conclusion, WBM severity was associated with higher breast weight in birds of most strains fed either a control or AA-reduced diet. Dietary AA reduction decreased processing yields but decreased WBM incidence, which may be more economical.

Effects of broiler genetic strain and dietary amino acid reduction on (part I) growth performance and internal organ development.

Genetic selection in broilers has resulted in improved growth performance, meat yield, and feed conversion efficiency. However, consumers have become increasingly concerned about modern broiler welfare that is related to their rapid growth rate, which may be alleviated by nutrient dilution. This study was conducted to investigate the effects of dietary amino acid (AA) reduction on the growth performance and internal organ development of different genetic strains of broilers. A randomized completed block design with a factorial arrangement of 10 treatments (5 strains × 2 AA levels) was used. The 5 different strains of broilers were fed either a control diet, with digestible AA (lysine, total sulfur AA, and threonine) at the highest recommended levels for the 5 strains, or an AA-reduced diet, with the digestible AA being 20% lower than the control diet. Feed conversion ratio was increased by AA reduction in all 5 strains during day 0-14, 14-28, and 28-41 but was not affected from day 41-55. Body weight and feed intake responses to AA reduction varied in the different strains and ages of birds. Liver weight relative to BW on day 40, and weights of the duodenum and jejunum relative to BW on day 60 were increased by decreasing the dietary AA concentration. These results indicate that the birds had adjusted their organ growth and metabolism in response to increases in digestion, absorption, and utilization efficiency to accommodate a decrease in dietary AA content. Surprisingly, the cost of feed required to produce the same BW was decreased in 4 of 5 strains on both day 41 and 55, which was largely because of the lower price of the diets containing reduced AA levels and the later compensatory growth experienced by the birds fed AA-reduced diets. In the future, when dietary AA levels need to be adjusted to control growth rate and improve welfare status, the genetic strain, age of the birds, and targeted goals need to be taken into consideration.

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.

Evaluation of Alternative Euthanasia Methods of Neonatal Chickens.

Hatched male layer chicks are currently euthanized by maceration in the United States. Public concerns on the use of maceration have led to the search for alternative methods. We hypothesized that gas inhalation and low atmospheric pressure stunning (LAPS) are viable and humane alternatives to instantaneous mechanical destruction. The objective of this study was to evaluate the physiological and behavioral responses of recently hatched male layer chicks when subjected to carbon dioxide, nitrogen inhalation, or LAPS. The study consisted of seven treatments: breathing air (NEG), 25% carbon dioxide (CO2), 50% CO2, 75% CO2, 90% CO2, 100% nitrogen (N2), or LAPS. Ten day-of-hatch, male layer chicks were randomly assigned to each treatment, and each treatment was replicated on ten different days. A custom-made vacuum system was used to reduce air pressure inside the chamber from 100.12 kPa to 15.3 kPa for the LAPS treatment. Serum corticosterone and serotonin levels were measured using commercially available competitive enzyme linked immunosorbent assay (ELISA). Latencies to loss of posture and motionlessness were determined from video recordings. The 25% and 50% CO2 treatments were discontinued after the first replication, as the majority of the chicks recovered. The chicks in the negative (NEG) group had significantly higher levels of corticosterone than the other four euthanasia treatments. On the other hand, the serotonin levels of chicks in the NEG group was significantly lower when compared to the other four euthanasia treatments. The latencies to loss of posture and motionlessness of chicks exposed to 75% and 90% CO2 were significantly shorter than those in the LAPS and N2 inhalation treatments. These data suggest that the stress responses of chicks to the CO2, N2, and LAPS treatments do not differ among each other. However, the CO2 inhalation method was faster in inducing loss of posture and motionlessness in chicks than the LAPS and N2 inhalation treatments.

Effects of different vaccine combinations against Mycoplasma gallisepticum on blood characteristics in commercial layer chickens.

Mycoplasma gallisepticum (MG) is a major and economically significant pathogen of avian species. When administered before lay, F-strain MG (FMG) can reduce egg production during lay, but the ts-11 strain of MG (ts11MG) does not exert this effect. Two trials were conducted to determine the effects of pre-lay vaccinations of ts11MG, MG-Bacterin (MGBac), or their combination, in conjunction with an FMG challenge overlay after peak production on the blood characteristics of commercial layers. In each trial, 160 mycoplasma-free Hy-Line W-36 layers were housed in negative-pressure biological isolation units (4 units per treatment, 10 birds per unit) from 9 through 52 wk of age (woa). The following vaccination treatments were administered at 10 woa: 1) Control (no vaccinations); 2) MGBac; 3) ts11MG; and 4) ts11MG and MGBac combination (ts11MG+MGBac). At 45 woa, half of the birds were challenged with a laboratory stock of high-passage FMG. Parameters measured in both trials were whole-blood hematocrit and serum concentrations of cholesterol (SCHOL), triglycerides, calcium, and total protein (STP). An age×treatment interaction (P=0.04) was observed for STP between 23 and 43 woa. The STP concentration in the ts11MG and ts11MG+MGBac groups was higher at 33 woa, but was lower at 43 woa, in comparison to the Control group. Also, at 38 woa, the STP of the ts11MG+MGBac group was higher than that of the MGBac group. Although use of the ts11MG vaccine alone or in combination with MGBac may influence circulating STP concentrations when administered before lay, it remains effective in protecting layers against the adverse effect of a post-peak challenge of FMG on egg production, as was observed in a previous companion study.