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In the commercial table egg industry, avian pathogenic E. coli (APEC) can lead to significant economic loss and bird mortality. The Poulvac E. coli vaccine (PECV) (Zoetis, Parsippany, NJ) may be administered to poultry post hatch via coarse spray or in drinking water to protect against APEC infections. The purpose of this experiment was to in ovo vaccinate commercial layers with various doses of the PECV to evaluate hatchability and post hatch development of the chicks through 21 d of age. Eggs were either non-injected or vaccinated at 18 d of incubation with a diluent-injected control or one of the PECV treatments which included a full dose (2.65 × 10 7 E. coli CFU/egg) or dilutions of the full dose to produce 2.65 × 10 5 , 2.65 × 10 3 , or 2.65 × 10 1 CFU/egg. Mean hatch of injected eggs was significantly (P < 0.0001) affected by treatment, with all the PECV treatments significantly decreasing hatch. Mean chick BW was determined on d of hatch and at 1, 2, and 3 wk post hatch, and mean chick length was determined at 3 wk post hatch. Chick BW was significantly (all P < 0.0001) different between treatments at 1, 2, and 3 wk of age, with the chicks that had received diluent alone having the highest BW, and the chicks that had received the full dose having the lowest BW. Chick length was significantly (P = 0.045) different between treatments, with the chicks in the full dose treatment having a shorter body length than the chicks in the 2 control groups. Cumulative mortality from 0 to 21 d of growout was 5.41% in the full dose treatment, whereas no mortalities were observed in the other treatment groups. While layer chicken embryos were able to survive to 3 wk post hatch after having received the PECV by in ovo injection, the full dose of the vaccine increased cumulative chick mortality and decreased chick BW through 3 wk post hatch., Competing Interests: Disclosures The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Katie Elliott reports equipment, drugs, or supplies was provided by Boehringer Ingelheim. Katie Elliott reports equipment, drugs, or supplies was provided by Zoetis Animal Health Co. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Investigations were performed to determine the systemic immune and small intestine (SI) morphological responses of Ross 708 broilers to the Marek's Disease vaccine (MDV) administered alone or in conjunction with the in ovo and dietary administration of calcifediol (25OHD 3 ). Live embryonated hatching eggs were assigned at random to 3 in ovo treatments at 18 d of incubation. Pre-specified in ovo treatments were: commercial MDV-alone-injected (50 µL) or commercial MDV containing 1.2 (MDV+25OHD 3 -1.2) or 2.4 (MDV+25OHD 3 -2.4) μg of 25OHD 3 . A noninjected control treatment was also included. For the growing phase, broilers received a commercial diet containing 250 IU of vitamin D 3 /kg (control) or a commercial diet supplemented with 2,760 IU of 25OHD 3 /kg (Hy-D diet). For determination of serum IgG, nitric oxide, and α-1-acid glycoprotein (AGP) at 14 and 40 d of age (doa), blood was collected from 1 bird per pen (48 total). In the duodenum, jejunum, and ileum of the same bird, villus length (VL), crypt depth (CD), VL to CD ratio (VCR), and villus surface area were also determined. There were no significant dietary x in ovo treatment interactions for any of the variables examined. However, birds fed Hy-D diets had lower serum AGP levels at 14 doa when compared to those fed un-supplemented commercial diets. Additionally, at 40 doa, birds in the MDV+25OHD 3 -1.2 and MDV+25OHD 3 -2.4 treatments experienced a decrease in serum AGP in comparison to those belonging to the noninjected and MDV-alone treatment groups. A higher jejunal VCR was observed at 14 and 40 doa in birds that belonged to the MDV+25OHD 3 -1.2 treatment when compared to those in the noninjected and MDV-alone treatment groups, and dietary Hy-D increased the VL of the duodenum and jejunum in birds at 14 and 40 doa when compared to those fed the commercial diet. In conclusion, both dietary or in ovo administration of 25OHD 3 lowered inflammatory reactions and improved the SI morphology of broilers that were in ovo-injected with the MDV., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Influences the Marek's disease vaccine (MDV) alone or combined with the in ovo and dietary administration of 25-hydroxycholecalciferol (25OHD 3 ) on the expression of genes associated with the breast muscle deposition, adaptive and innate immunity, and antioxidant and vitamin D activities of 14 d-old broilers were investigated. Four in ovo treatments were: noninjected; commercial MDV-alone-injected (50 μl); or 50 μl of MDV containing 1.2 (MDV+25OHD 3 -1.2) or 2.4 (MDV+25OHD 3 -2.4) μg of 25OHD 3 . Two dietary treatments were a commercial diet containing 25OHD 3 (250 IU)/kg of feed (control) or the same diet supplemented with additional 25OHD 3 ( 2,760 IU)/kg of feed (Hy-D diet). One bird per pen (48 total) was sampled at 14 d for determination of the expression of genes involved with the muscle deposition (MyoD1, MyoG, Pax3, and Mrf4), immunity (INF-γ, IL-10, IL-8, IL-1β, and TGF-β4), antioxidant capacity (SOD1, SOD2, GSH-P1, GSH-P7, and CAT), and vitamin D activity (VDR, 1α-hydroxylase, and 24-hydroxylase) in the spleen and pectoralis major (P.major) muscle. The treatment differences were considered significant at P ≤ 0.05. In the P. major, Mrf4 and MyoG were up-regulated in Hy-D-fed birds. Also, the in ovo and dietary 25OHD 3 sources individually increased SOD2 gene expression in the P. major. In the spleen, the expressions of IL-1β and IL-8 were down-regulated and IL-10 and TGF-β4 gene expressions were up-regulated in Hy-D-fed birds than those commercial-fed broiler. In ovo and dietary 25OHD 3 sources enhanced vitamin D gene (1α-hydroxylase and 24-hydroxylase) activities in the breast and spleen. In ovo x dietary treatment interactions were significant for the MyoD1, IL-8, Pax3, TGF-β4 genes of the P. major, in which the combined MDV with 1.2 μg of 25OHD 3 enhanced their expressions in birds fed the Hy-D diet. In conclusion, both 25OHD 3 sources promoted the expression of genes associated with immunity and P. major growth. It is recommended that both 25OHD 3 sources can be used to promote the gene expression of 14-day-old broilers in the spleen and breast muscle when MDV administered in ovo., Competing Interests: DISCLOSURES There is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The effects of the Marek's disease vaccine (MDV) on the live performance, breast meat yield, and incidence of woody breast myopathy (WBM) of Ross 708 broilers were investigated when administered alone or in conjunction with in ovo and dietary supplemental 25-hydroxycholecalciferol (25OHD 3 ). At 18 d of incubation (doi), four in ovo injection treatments were randomly assigned to live embryonated Ross 708 broiler hatching eggs: (1) non-injected; (2) commercial MDV alone; or MDV containing either (3) 1.2 or (4) 2.4 μg of 25OHD 3 . An Inovoject multi-egg injector was used to inject a 50 μL solution volume into each egg. The birds were provided a commercial diet that contained 250 IU of cholecalciferol/kg of feed (control) or a commercial diet that was supplemented with an additional 2760 IU of 25OHD 3 /kg of feed (HyD-diet). In the growout period, 14 male broilers were placed in each of 48 floor pens resulting 6 replicated pens per in ovo x dietary treatment combination. Live performance variable were measured at each dietary phases from 0 to 14, 15 to 28, and 29 to 40 d of age (doa). At 14 and 40 doa, pectoralis major (P. major) and pectoralis minor (P. minor) muscles were determined for one bird within each of the six replicate pens. At 41 doa, WBM incidence was determined. No significant main or interaction effects occurred for WBM among the dietary or in ovo injection treatments. However, in response to in ovo 25OHD 3 supplementation, BW and BWG in the 29 to 40 doa period and BWG and FCR in the 0 to 40 doa period improved. In addition, at 40 and 41 doa, breast meat yield increased in response to in ovo and dietary 25OHD 3 supplementation. Future research is needed to determine the possible reasons that may have been involved in the aforementioned improvements.

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-1β, 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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The combined effects of the in ovo injection of commercial Marek's disease vaccine (MDV) and various levels of 25-hydroxyvitamin D 3 (25OHD 3 ) on the hatch variables, immunological measurements, and gene expression of Ross 708 hatchling broilers were investigated. A total of 5 in ovo injection treatments that were applied at 18 d of incubation (doi) included: 1) noninjected (control); or a 50 μL solution volume of 2) MDV alone; or MDV combined with 3) 0.6 μg of 25OHD 3 ; 4) 1.2 μg of 25OHD 3 ; or 5) 2.4 μg of 25OHD 3 . At hatch, hatchability of set and live embryonated eggs, hatchling body weight, hatch residue analysis, serum IgY and alpha-1 acid glycoprotein (AGP) concentrations, and the expression of genes related to immunity (INFα, INFβ, INFγ, TLR-3, and TLR-21) and vitamin D 3 activity (1 α-hydroxylase, 24 hydroxylase, and vitamin D receptor) were determined. No significant treatment differences were observed for hatchability of set and live embryonated eggs, or for serum IgY and AGP concentrations. However, hatchling body weight was higher when MDV was combined with either 1.2 or 2.4 μg of 25OHD 3 than when MDV was provided alone or in combination with 0.6 μg of 25OHD 3 . Also, in comparison to the noninjected treatment group, the expression of the genes for 1 α-hydroxylase and 24 hydroxylase was improved when MDV was combined with either 1.2 or 2.4 μg of 25OHD 3 . Lastly, expression of the genes linked to viral detection (TLR-3) and antibody production (INF-β) was increased in those treatments that contained any level of 25OHD 3 . These results indicate that in comparison to controls, the effects of MDV were observed to be greater on hatchling BW and splenic gene expression when it was administered in combination with the 1.2 or 2.4 μg doses of 25OHD 3 . Further research is needed to determine the posthatch effects of the administration of various levels of 25OHD 3 in combination with MDV., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In the commercial egg industry, avian pathogenic Escherichia coli (APEC) can lead to significant economic loss. The Poulvac E. coli vaccine (PECV) is a commercially available attenuated live vaccine commonly applied via spray or drinking water to protect against losses associated with colibacillosis. The PECV has not been tested in layer hatching eggs using in ovo injection. Therefore, the purpose of this experiment was to determine the effects of injecting 50 μL of different doses of the PECV into Hy-Line W-36-layer hatching eggs on the hatchability and quality characteristics of hatchlings. At 18 d of incubation (DOI), treatments included 1 noninjected and 1 diluent-injected control. Furthermore, PECV treatments included a full dose (4.4 × 10 8 E. coli CFU) or serial dilutions of the full dose to produce 4.4 × 10 6 , 4.4 × 10 4 , or 4.4 × 10 2 CFU doses of E. coli. In ovo injections targeted the amnion. Percent hatchability of live embryonated eggs (HI), percent residue eggs, hatchling mortality, and female chick whole and yolk-free BW, relative yolk sac weight, and body length were among the variables examined. Treatment significantly (P < 0.0001) affected HI, with HI being highest in the control groups (97.3% in the noninjected and 94.2% in the diluent-injected), and with HI values being 89.0, 88.9, 84.4, and 71.2% in the 4.4 × 10 2 , 4.4 × 10 4 , 4.4 × 10 6 , and 4.4 × 10 8 CFU E. coli dose treatments, respectively. The percentage of live embryos that did not complete hatch but that pipped internally (P = 0.024) or externally (P < 0.0001) were significantly affected by treatment, with percentages being highest in the 4.4 × 10 8 CFU treatment. Female chick body length was significantly (P < 0.0001) affected by treatment and was longer in both control groups and in the 1 × 10 2 CFU E. coli treatment in comparison to all other treatments. Yolk-free female chick BW was significantly (P = 0.034) affected by treatment and was lower in the 4.4 × 10 6 CFU and 4.4 × 10 8 CFU treatments when compared to the diluent-injected control group. An increase in the E. coli concentration administered in the amnion of embryonated layer hatching eggs at 18 DOI decreased hatch success and female chick yolk-free BW and body length., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The current commercial broiler embryo experiences a rapid metabolism and growth rate and may produce more heat than those of the past. Consequently, it may be beneficial to lower standard incubation temperature for commercial broiler hatching eggs. The purpose of this experiment was to determine if lowering incubation temperature at 12 d of incubation (DOI) would affect embryo temperature (ET) in embryonated Ross 708 broiler hatching eggs. From 0 to 12 DOI, eggs were incubated under standard conditions (37.50°C dry bulb, 29.76°C wet bulb). At 12 DOI, temperature transponders were aseptically placed on the inner air cell membrane to measure air cell temperature (ACT) as an estimate of ET in 120 eggs. The eggs were then randomly assigned to 4 separate still-air incubators, each containing 30 eggs. Two replicate incubators were maintained at a standard (STRT; 37.5°C) or low (LTRT; 35.6°C) temperature treatment between 12 and 21 DOI. A significant positive correlation existed between incubator air temperature (IAT) and ACT across temperature treatment and in the STRT. However, IAT was not significantly correlated with ACT in the LTRT. A significantly higher ACT was observed in the STRT than in the LTRT for 88% of the readings taken between 12 and 21 DOI. Percent egg weight loss (PEWL) between 13 and 17 DOI did not significantly differ between temperature treatments. From 13 to 17 DOI, there was a significant positive correlation between PEWL and ACT in the STRT, however, no significant correlation occurred between PEWL and ACT in the LTRT. Percent hatch of fertile eggs containing live embryos at 12 DOI was 93.3% in the STRT and 100% in the LTRT. However, time of hatch occurred 14 to 19 h later and hatchling BW was lower in the LTRT than in the STRT. Although lowering IAT at 12 DOI may improve hatchability, an associated delay in hatch and a decrease in hatchling BW may not be commercially acceptable., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In ovo administration as a possible alternative method of 6/85 MG vaccination was assessed. After 18 days of incubation (doi), the eggs were administered a particular dosage of a live attenuated 6/85 MG vaccine in either the air cell (AC) or amnion (AM). The treatments included non-injected eggs and eggs injected into the AC or AM with diluent alone as controls. Treatments also included eggs injected with diluent, which contained 1.73 × 10 2 , or 1.73 × 10 4 CFU of 6/85 MG. Hatchability of viable injected eggs (HI) and residual embryonic mortality were determined at 22 doi. At hatch and at three weeks posthatch, one hatched chick per treatment replicate was bled and swabbed for the detection of 6/85 MG in the choanal cleft using PCR, serum plate agglutination (SPA), and ELISA methods. The results show that AC in ovo injection of 6/85 MG had no negative impacts on HI or on the live performance of pullets, but that it failed to provide adequate protection ( p ≤ 0.0001) in hatchlings or three-week-old pullets. The 1.73 × 10 4 6/85 MG CFU dosage injected into the AM decreased the hatchability of injected eggs containing viable embryos (HI; p = 0.009) and was associated with a significant increase in late dead mortality ( p = 0.001). Hatchling and three-week-old chick mortalities ( p = 0.008) were significantly greater in the 1.73 × 10 4 CFU-AM treatment group in comparison with the other treatment groups. In addition, the 1.73 and 1.73 × 10 2 6/85 MG-AM treatments had no negative effects on the hatching process or on posthatch growth, and the 1.73 × 10 2 6/85 MG-AM treatment was more effective in the protection of pullets against MG ( p ≤ 0.0001) as compared with the low dosage and non-injected treatment groups. Further research is needed to examine the influence of the 6/85 MG in ovo vaccine on layer immune competence.

Effects of the in ovo injection of various levels of L-ascorbic acid (L-AA) on the performance and corneal erosion incidence in Ross 708 broilers exposed to 50 parts per million (ppm) of atmospheric ammonia (NH 3 ) after hatch were determined. A total of 1440 Ross 708 broiler embryos were randomly assigned to 4 treatments: non-injected (control), 0.85% sterile saline-injected (control), or saline containing 12 or 25 mg of L-AA. At hatch, 12 male chicks were randomly assigned to each of 48 battery cages with 12 replicate cages randomly assigned to each treatment group. All birds were exposed to 50 ppm of NH 3 for 35 d and the concentration of NH 3 in the battery cage house was recorded every 20 s. Mortality was determined daily, and mean body weight (BW), BW gain (BWG), average daily BW gain (ADG), and feed intake, as well as feed conversion ratio (FCR), were determined weekly. From 0 to 35 d of post-hatch age (doa), six birds from each cage were selected and sampled for eye erosion scoring. Incidences of corneal erosion were significantly higher at 21 and 28 doa in comparison to those at 14 and 35 doa, and at 21 doa, birds in the saline-injected group exhibited a higher incidence of corneal erosion compared to all other treatment groups. The in ovo injection of 12 mg of L-AA increased BWG ( p = 0.043) and ADG ( p = 0.041), and decreased FCR ( p = 0.043) from 0 to 28 doa in comparison to saline-injected controls. In conclusion the in ovo administration of 12 mg of L-AA may have the potential to improve the live performance of broilers chronically exposed to high aerial NH 3 concentrations, but further study is needed to determine the physiological and immunological factors that may contribute to this improvement.

Effects of the dietary and in ovo administration of L-ascorbic acid (L-AA) on the performance, plasma nitric oxide, and eye L-AA concentrations of Ross 708 broilers were investigated. At 17 days of incubation, live embryonated hatching eggs were randomly assigned to a non-injected or sham-injected (100 μL of saline) control group, or a group injected with either 12 or 25 mg of L-AA suspended in 100 μL of saline. Chicks received a commercial diet with or without 200 mg/kg of supplemental L-AA and were randomly assigned to each of 6 replicate floor pens in each in ovo injection-dietary treatment combination. Weekly live performance variables through 14 days of post hatch age (doa) and the eye weights in both sexes at 0, 7, and 14 doa were determined. At 0 and 14 doa, plasma nitric oxide levels and eye L-AA concentrations of one bird of each sex in each pen were determined. Dietary supplemental L-AA decreased feed intake and growth between 0 and 7 doa, but from 8 to 14 doa; all birds fed supplemental L-AA had a lower feed conversion ratio. At 14 doa, male chicks had higher eye L-AA concentrations and lower plasma nitric oxide levels when treated in ovo with 12 mg of L-AA. In conclusion, dietary L-AA may be used to improve feed conversion in the second week of broiler post hatch growth. However, the in ovo administration of 12 mg of L-AA can increase male eye L-AA concentrations and is effective in reducing their general inflammatory response.

Effects of the in ovo administration of two vitamin D 3 sources (vitamin D 3 (D 3 ) and 25-hydroxyvitamin D 3 (25OHD 3 )) on the expression of D 3 activity- and immunity-related genes in broilers subjected to a coccidiosis infection were investigated. At 18 d of incubation (doi), five in ovo injection treatments were administrated to live embryonated Ross 708 broiler hatching eggs: non-injected (1) and diluent-injected (2) controls, or diluent injection containing 2.4 μg of D 3 (3) or 2.4 μg of 25OHD 3 (4), or their combination (5). Birds in the in ovo-injected treatments were challenged at 14 d of age (doa) with a 20× dosage of a live coccidial vaccine. At 14 and 28 doa, the expression of eight immunity-related genes (IL-2, IL-6, IL-10, TLR-4, TLR-15, MyD88, TGF-β4, and IFN-γ) and four D 3 activity-related genes (1α-hydroxylase, 25-hydroxylase, 24-hydroxylase, and VDR ) in the jejunum of one bird in each treatment-replicate group were evaluated. No significant treatment effects were observed for any of the genes before challenge. However, at 2 weeks post-challenge, the expression of 1α-hydroxylase, TGF-β4, and IL-10 increased in birds that received 25OHD 3 alone in comparison to all the other in ovo-injected treatment groups. Additionally, the expression of 24-hydroxylase and IL-6 decreased in birds that received 25OHD 3 in comparison to those injected with diluent or D 3 alone. It was concluded that the in ovo injection of 2.4 μg of 25OHD 3 may improve the intestinal immunity as well as the activity of D 3 in Ross 708 broilers subjected to a coccidiosis challenge.

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 × 10 8 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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The transmission of the ts-11 strain of Mycoplasma gallisepticum (MG) vaccine (ts-11MGV) between incubated eggs and between hatchlings that was administrated via in ovo injection, and its subsequent effects on their posthatch performance were evaluated. Marek's disease diluent alone (sham-injected) or containing either 3.63 × 10 1 , 10 2 , 10 3 , or 10 4 cfu of ts-11MGV was manually in ovo-injected into the amnion on 18 days of incubation. Egg residue analysis, percentage incubational egg weight loss, hatchability of viable injected eggs, and hatchling body weight (BW) were assessed. Selected hatchlings from each treatment replicate group were swabbed in the choanal cleft for MG DNA detection. Female chick live performance was also assessed through 21 days of posthatch age. Unexposed control sentinel chicks were allocated to each treatment replicate group to assess horizontal transmission. Birds were later swabbed and bled respectively, for detection of MG DNA and IgM production at 21 days posthatch. In all birds, no MG DNA was detected and SPA tests for IgM were negative. Among all variables, only 0 to 21 day BW gain was significantly affected by treatment and was lower in the 3.63 × 10 4 ts-11 MGV treatment in comparison to all the other treatments. Because ts-11MGV does not exhibit vertical or horizontal transmission capabilities under commercial conditions, it may not be a good candidate for in ovo injection.

In broilers challenged with coccidiosis, effects of in ovo vitamin D 3 (D 3 ) and 25-hydroxyvitamin D 3 (25OHD 3 ) administration on their inflammatory response and small intestine morphology were evaluated. At 18 d of incubation (doi), a 50 μL volume of the following 5 in ovo injection treatments was administrated: non-injected (1) and diluent injected (2) controls, or diluent injection containing 2.4 μg D 3 (3) or 2.4 μg 25OHD 3 (4), or their combination (5). Four male broilers were randomly allocated to each of eight isolated replicate wire-floored battery cages at hatch, and birds were challenged at 14 d of age (doa) with a 20x live coccidial vaccine dosage. One bird from each treatment-replicate (40 birds in each of 8 replicates per treatment) was bled at 14 and 28 doa in order to collect blood for the determination of plasma IL-1β and nitric oxide (NO) concentrations. The duodenum, jejunum, and ilium from those same birds were excised for measurement of villus length, crypt depth, villus length to crypt depth ratio (VCR), and villus surface area. In ovo injection of 2.4 μg of 25OHD 3 resulted in a reduction in plasma NO levels as compared to all other treatments at 28 doa. Additionally, duodenal VCR increased in response to the in ovo injection of 25OHD 3 when compared to the diluent, D 3 alone, and the D 3 + 25OHD 3 combination treatments at two weeks post-challenge (28 doa). Therefore, it can be concluded that 2.4 μg of 25OHD 3 , when administrated in ovo at 18 doi, may be used to decrease the inflammatory reaction as well as to enhance the small intestine morphology of broilers during a coccidiosis challenge.

Effects of the in ovo injection of various concentrations of L-ascorbic acid (L-AA) on the hatchability and retention levels of L-AA in the serum of broiler embryos were investigated. A total of 960 Ross 708 broilers hatching eggs were randomly divided into four treatment groups: non-injected control, saline-injected control, and saline containing either 12 or 25 mg of L-AA. At 18 days of incubation (doi), injected eggs received a 100 μL volume of sterile saline (0.85%) alone or containing one of the two L-AA levels. Percentage egg weight loss was also determined from 0 to 12 and 12 to 18 doi. Hatch residue analysis was conducted after candling to determine the staging of embryo mortality. At approximately 21 doi, hatchability of live embryonated eggs (HI) and hatchling body weight (BW) were determined. Blood samples were taken at 6 and 24 h after L-AA in ovo injection to determine serum L-AA concentrations. Serum L-AA concentrations, HI, and hatchling BW did not differ among all treatment groups. However, chicks in the non-injected group had a higher ( p = 0.05) embryonic mortality at hatch in comparison to those in the 12 mg of L-AA in saline and saline alone treatment groups. These results suggest that the in ovo injection of high levels of L-AA (12 and 25 mg) does not negatively affect HI or serum concentrations of L-AA but has the potential to promote embryonic livability. Further research is needed to determine the retention time of L-AA in the other tissues of broilers, including the cornea of the eye, in response to different levels of supplemental L-AA.

Previous trials in which layers were in ovo-vaccinated against strain F Mycoplasma gallisepticum (FMG) showed that nearly 50% of the birds produced IgM antibody against FMG at 6 wk of age (WOA). Standard FMG vaccination application at 9 or 10 woa, result in this percentage at approximately 15 woa. This study investigated when FMG in ovo-vaccinated birds initiate a humoral immune response prior to 6 wk, and if sex influences this response. Hy-Line W-36 embryonated eggs were either not vaccinated (controls) or in-ovo vaccinated with a 50 µL volume of a 10 -6 dilution of Poulvac MycoF vaccine (Zoetis). For each treatment group, 384 straight-run chicks were reared. At hatch and at 2, 3, 5, 7, 14, 21, and 28 d post-hatch, 54 birds per treatment were individually weighed and a blood sample was collected for Mycoplasma gallisepticum (MG) IgM antibody detection. ELISA was run on blood samples at 14, 21, and 28 d to distinguish IgG antibody production. At each age, BW was not different between vaccinated and control chicks (all P > 0.19). Males, however, outweighed females starting at d 5 (P = 0.02). Mortality was 1.0% for the control birds and 12.2% for the FMG birds during the first 2 wk. The majority (72.3%) of the mortalities in the FMG group were male. The percentage of control and FMG in ovo-vaccinated birds with IgM antibody production was 0% and 1.9% on d 7, 0% and 31.5% on d 14, 1.9% and 55.9% on d 21, and 0% and 60.6% on d 28, respectively. IgG antibody production in the FMG in ovo-vaccinated birds was 0.0% at 14 d, 2.9% at 21 d, and 21.2% at 28 d of age. All control birds tested negative for FMG-IgG production. In conclusion, the earliest detection of MG antibodies after in ovo vaccination with live FMG occurred at 7 d. Male layer chickens were more susceptible to the effects of an in ovo FMG vaccine than females., (Published by Elsevier Inc.)

Effects of the in ovo administration of vitamin D 3 (D 3 ) and 25-hydroxyvitamin D 3 (25OHD 3 ) on broiler intestinal lesion incidence, performance and breast meat yield after a coccidiosis challenge were investigated. On each of 10 incubator tray levels, 10 Ross 708 broiler hatching eggs were randomly assigned to each of the following 5 in ovo injection treatments administrated at 18 d of incubation (doi): 1) noninjected; 2) diluent; diluent containing either 3) 2.4 μg D 3 (D 3 ), 4) 2.4 μg 25OHD 3 (25OHD 3 ), or 5) 2.4 μg D 3  + 2.4 μg 25OHD 3 (D 3 +25OHD 3 ). A 50 μL solution volume was injected into each egg using an Inovoject multi-egg injector. Four male chicks were randomly assigned to each of 80 battery cages in each of 2 rooms. Half of the treatment-replicate cages (8) in each room were challenged with a 20× live coccidial vaccine at 14 d of age (doa). One randomly selected bird from each of 4 treatment-replicate cages was scored for coccidiosis lesions before and 2 wk after challenge. Mean BW, BW gain (BWG), feed intake, and feed conversion ratio were determined for all birds from 0 to 14, 15 to 28, and 29 to 41 doa. Carcass weight, and the absolute and relative (% of carcass weight) weights of carcass parts were determined in 3 birds per treatment-replicate cage at 42 doa. Hatchability of live embryonated injected eggs and hatch residue were not affected by treatment. Across challenge treatment, birds in the 25OHD 3 treatment group experienced an increase in BWG between 29 and 41 doa when compared to the D 3 or diluent-injected birds. Furthermore, pectoralis major muscle percentage tended (P = 0.059) to increase in birds belonging to the 25OHD 3 treatment in comparison to birds in the D 3 or diluent-injected treatments. These results indicate that regardless of challenge treatment, 2.4 μg of 25OHD 3 may increase the BWG and breast meat yield of birds relative to those that only received an injection of commercial diluent., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Effects of the in ovo-injection of vitamin D 3 (D 3 ) and 25-hydroxyvitamin D 3 (25OHD 3 ) on broiler performance, carcass characteristics, and woody breast myopathy (WBM) incidence were investigated. Live embryonated Ross 708 broiler hatching eggs (2,880) were randomly assigned to one of the following in ovo injection treatments: (1) diluent (50 μL); diluent (50 μL) containing either (2) 2.4 μg D 3 ; (3) 2.4 μg 25OHD 3 ; or (4) 2.4 μg D 3  + 2.4 μg 25OHD 3 . Eggs were injected at 18 d of incubation (doi) using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly placed in each of 6 replicate pens belonging to each in ovo injection and, dietary treatment combination. Birds were fed either a commercial diet or a diet restricted in calcium and phosphorous (ReCaP) content by 20% for the starter, grower and finisher dietary phases. Broiler performance was determined in each dietary phase and breast muscle yield was also determined at 14 and 40 d of age (doa). At 41 and 46 doa, birds were processed for determination of WBM, carcass weight, and the absolute and relative (% of carcass weight) weights of various carcass parts. Compared to birds fed the commercial diet, birds fed ReCaP diets experienced a reduction in performance from 14 to 40 doa, in breast meat yield at 41 and 46 doa, and in WBM at 41 and 46 doa. At 14 and 40 doa, breast meat yield in birds that received an in ovo injection of 25OHD 3 alone was higher compared to birds that received diluent alone or a combination of D 3 and 25OHD 3 . Lower WBM incidence in ReCaP-fed birds was associated with a lower breast weight. An increase in breast meat yield in response to 25OHD 3 alone may be due to improved immunity and small intestine morphology. However, further study is needed to determine the aforementioned effects., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Effects of the in ovo injection of vitamin D 3 (D 3 ) and 25-hydroxyvitamin D 3 (25OHD 3 ) on the immunity and small intestine morphology of broilers fed calcium and phosphorus-restricted diets were investigated. At 18 d of incubation (doi), live embryonated Ross 708 broiler hatching eggs were in ovo-injected with a 50 μL solution of one of the following treatments using an Inovoject multiegg injector: 1) diluent (control); diluent containing either 2) 2.4 μg D 3 ; 3) 2.4 μg 25OHD 3 ; or 4) 2.4 μg D 3  + 2.4 μg 25OHD 3 . At hatch, 18 randomly selected male broilers belonging to one of the 4 in ovo injection treatments were placed in each of 12 floor pens and were fed either a commercial diet or a diet restricted by 20% in calcium and available phosphorus (ReCaP) content for the starter, grower and finisher dietary phases. Concentrations of plasma IgG and IgM at 14 d of age (doa) and α-1-acid glycoprotein at 40 doa were determined. Bursa, liver, spleen, duodenum, jejunum, and ileum weights were recorded at 7, 14, and 40 doa and small intestine histology was evaluated at 14 and 40 doa. Blood and organ samples were randomly collected from 1 bird in each of the 6 replicate pens within each of the 8 (4 in ovo x 2 dietary) treatment groups. Plasma IgG levels were higher in 25OHD 3 than in diluent or D 3 in ovo-injected birds. At 14 doa, a higher jejunal villus length (VL) to crypt depth (CD) ratio (RVC) was observed in birds that were in ovo-injected with 25OHD 3 alone as compared to all other in ovo injection treatments. At 40 doa, ileal VL increased and jejunal CD decreased in commercial diet-fed birds compared to ReCaP diet-fed birds. In conclusion, the in ovo injection of 25OHD 3 alone increased the immune response and improved the small intestine morphology and subsequent nutrient uptake of Ross 708 broilers. However, a ReCaP diet was observed to be detrimental to their small intestine morphology., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (CO 2 ) or nitrogen (N 2 ) 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 N 2 method induced unconsciousness and death later than the CO 2 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 N 2 and the negative pressure methods. The behavioral and physical responses observed in this study suggest that both CO 2 inhalation and negative pressure stunning can be employed to humanely euthanize neonatal male layer chicks.

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Use of a live coccidiosis vaccine has become an increasingly common method to control coccidiosis, especially in antibiotic-free broiler production. The Inovocox EM1 vaccine (EM1) is recommended for the vaccination of embryonated broiler hatching eggs between 18.0 and 19.0 d of incubation (doi). This allows for earlier acquisition of immunity to wild-type coccidia. However, it is unclear whether the difference in embryo age at the time of in ovo injection can influence the effect of the vaccine during grow-out as well as if the growth performance of broiler chickens is affected. Therefore, the objective of the study was to evaluate the effects of 2 injection ages (18.5 and 19.0 doi) and 3 injection types (noninjected, diluent, and vaccine) in a 3 × 2 factorial design, consisting of 10 replicates per treatment (60 treatment-replicate groups). There was a significant effect of injection age on BW at 0, 14, and 35 d after hatch, with a difference in the BW of birds belonging to the 18.5 and 19.0 doi groups up to day 35 after hatch. There was a significant effect of injection type on BW gain, feed intake, and FCR between 0 and 28 d after hatch. Between 0 and 35 d, FCR was lower in the vaccine-injected group in comparison with the noninjected and diluent control groups. Furthermore, total intestine coccidia and lesion indices were higher in the vaccine-18.5 treatment group in comparison with the diluent-18.5 treatment group at 28 d. In conclusion, hatchling weight was affected by injection age, and this subsequently affected growth performance. Furthermore, intestinal coccidia cycling peaked at 28 d, resulting in a reduction in growth performance through 28 d and subsequent compensatory growth by 35 d. There was no significant difference in coccidiosis cycling between the vaccine-18.5 and vaccine-19.0 doi treatment combination groups., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Effects of the in ovo injection of organic microminerals (OM) (zinc, manganese, and copper) and posthatch holding time (HT) on the daily body temperature (bt) of broilers during grow out were determined. The hatching eggs from a Ross 708 breeder flock at 32 wk of age were incubated under standard commercial conditions. At 17 d of incubation, eggs were randomly allocated to 3 in ovo OM injection treatment (TRT) groups, and at 21 d of incubation, male hatchlings were randomly allocated to 2 posthatch HT treatment groups. Eggs were either not injected or were in ovo injected with diluent only or diluent containing the OM mixture. A 0-hour HT group had immediate access to water and feed, and a 24-hour HT (24HT) group contained birds that were kept in transport baskets in their pens without access to feed and water for 24 h before being released. Fifteen male birds were placed in each of 36 litter floor pens in a temperature-controlled facility. Approximately 2 birds in each of 6 replicate pens belonging to each TRT-HT combination had temperature transponders inserted subcutaneous in the mid-dorsal region of the neck. All birds were brooded under standard commercial conditions and had ad libitum access to feed and water after their respective HT. The bt of the same birds were determined daily at the same time each day beginning at hatch and ending on 39 d of posthatch age (AGE). There were no significant main or interactive effects involving TRT or HT for bt. However, there was a significant (P ≤ 0.0001) main effect because of AGE. A general increase in bt occurred during the 39 d grow out period. At hatch, bt was 40.54 ± 0.056°C and at AGE 39 was 41.46 ± 0.055°C. Under standard brooding conditions, a general increase in bt occurred in the Ross 708 broilers. However, these birds did not exhibit a significant bt response to TRT or a 24HT before placement., (Copyright © 2020. Published by Elsevier Inc.)

The effects of the in ovo administration of vitamin D 3 (D 3 ) and its metabolite, 25-hydroxyvitamin D 3 (25OHD 3 ) , on the performance, breast meat yield, and inflammatory responses of broilers fed commercial diets were investigated. Live embryonated Ross 708 broiler hatching eggs were randomly assigned to one of the following 5 in ovo injection treatments at 18 d of incubation: 1) noninjected; 2) diluent; diluent containing 3) 2.4-μg D 3 , 4) 2.4-μg 25OHD 3 , or 5) 2.4-μg D 3  + 2.4-μg 25OHD 3 . A 50-μL solution volume of each prespecified treatment was injected into each egg using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly assigned to each of 30 floor pens. The BW, BW gain, feed intake, and feed conversion ratio of the birds were determined in each dietary phase. At 14, 28, and 39 d of posthatch age (doa), plasma α-1-acid glycoprotein (AGP) levels in 1 bird in each of 6 replicate pens per treatment were determined at 14 and 39 doa. The pectoralis major and minor weights of those same birds were also determined. The remaining birds were processed at 43 doa, and the weights of their processing parts were determined. At 39 doa, the in ovo injection of 25OHD 3 alone decreased plasma AGP concentrations in comparison with the noninjected, diluent, and D 3 -alone treatment groups. In addition, birds that received 25OHD 3 alone had a greater BW at 42 doa than birds in the noninjected, diluent, and D 3 -alone treatment groups. At 39 and 43 doa, breast meat yield was increased in response to the in ovo injection of 25OHD 3 alone in comparison to all other treatments. These results indicate that the in ovo injection of 2.4 μg of 25OHD 3 resulted in an improvement in the performance and inflammatory responses of broilers. A reduction in the inflammatory response subsequent to the in ovo injection of 2.4 μg of 25OHD 3 may have led to an increase in broiler performance., (Copyright © 2020. Published by Elsevier Inc.)

Effects of dietary Original XPC (XPC) on 17 selected blood variables in commercial layer pullets challenged with the virulent, low-passage R strain of Mycoplasma gallisepticum (R low MG) were investigated. Hy-Line W-36 pullets sourced from M. gallisepticum-clean layer breeders were fed a basal diet with XPC (1.25 kg/metric ton) or without from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with R low MG. Blood samples were taken immediately before the initial R low MG challenge at 8 woa and again at 12 woa (4 wk after challenge). At 8 woa, blood pH was lower and glucose concentration was higher in the preassigned challenge treatment groups. At 12 woa, the concentration of oxygen dissolved in the blood was significantly lower in the R low MG-challenged group than the unchallenged group of birds regardless of dietary treatment. The R low MG challenge significantly increased blood carbon dioxide partial pressure, calcium, sodium, anion gap, osmolality, glucose, and corticosterone levels but significantly decreased blood oxygen partial pressure, oxyhemoglobin concentration, concentration of oxygen dissolved in the blood, chloride, and pH levels. Because blood pH and glucose concentration at 8 woa were examined before challenge, their baseline values were biased with respect to challenge treatment before treatment was applied. However, the lack of a significant main effect due to diet at 8 woa for blood pH and glucose concentration, along with the other 15 blood variables, indicate that the baseline data with respect to dietary treatment were unbiased, allowing for real dietary effects to be accurately assessed. In conclusion, layer pullets challenged with R low MG undergo a stress response associated with changes in various physiological blood variables, and a decrease in pH and increase in carbon dioxide partial pressure, in association with a lack of change in bicarbonate, indicates that the stress response caused by the R low MG challenge was associated with respiratory acidosis. Nevertheless, feeding XPC did not influence the effects of challenge treatment on these postchallenge physiological blood values., (Copyright © 2020. Published by Elsevier Inc.)

Effects of the in ovo injection of vitamin D 3 (D 3 ) and 25-hydroxycholecalciferol (25OHD 3 ) on broiler embryo serum 25OHD 3 concentrations, hatchability, and hatchling somatic characteristics were determined. Eggs from a 35-wk-old commercial Ross 708 broiler breeder flock were set in a single-stage incubator with 11 treatments represented on each of 8 incubator tray levels (blocks). Each treatment group within a flat on each tray level contained 30 eggs. Control treatments were noninjected and diluent injected. Vitamin treatments were commercial diluent containing 0.6 μg D 3 , 0.6 μg 25OHD 3 , 0.6 μg D 3 + 0.6 μg 25OHD 3 , 1.2 μg D 3 , 1.2 μg 25OHD 3 , 1.2 μg D 3 + 1.2 μg 25OHD 3 , 2.4 μg D 3 , 2.4 μg 25OHD 3 , or 2.4 μg D 3 + 2.4 μg 25OHD 3 . At 432 h of incubation (hoi), 50-μL solution volumes were injected. Blood samples were collected at 462 hoi for serum 25OHD 3 analysis, and hatchability of injected live embryonated eggs (HI) was determined at 492 and 516 hoi. At 516 hoi, hatchling yolk-free BW and weights of the liver and yolk sac were determined. Percentage of yolk moisture and dry mater was calculated. At 492 and 516 hoi, HI did not differ between treatments. Embryos that received 1.2 μg or more of either vitamin D 3 source alone or in combination had higher serum 25OHD 3 concentrations than those that were injected with diluent alone or diluent containing 0.6 μg of D 3 . Hatchlings that received 1.2 or 2.4 μg of 25OHD 3 had higher percentage of yolk dry matter or lower percentage of yolk moisture levels than noninjected controls and those that received D 3 alone at any level. These results indicate that the in ovo injection of either vitamin D 3 source at levels equal to or higher than 1.2 μg resulted in serum 25OHD 3 concentrations that were higher than that of noninjected controls. In addition, the in ovo injection of 1.2 μg or higher of either vitamin D 3 source did not negatively affect broiler HI or chick quality., (Copyright © 2020. Published by Elsevier Inc.)

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., (Copyright © 2020. Published by Elsevier Inc.)

Effects of dietary Original XPC (XPC) in commercial layer pullets challenged with the virulent, low passage R strain of Mycoplasma gallisepticum (R low MG) were investigated. Hy-Line W-36 pullets sourced from MG-clean breeders were fed a basal diet with or without (CON) XPC (1.25 kg/metric ton) from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with R low MG. Body weight was recorded at 3, 8, and 12 woa, and ovary, ceca, and bursa weights were recorded at 3 and 12 woa. Blood samples were taken immediately before the initial R low MG challenge at 8 woa and again at 12 woa to test for IgM and IgG antibody production against MG. All birds were evaluated for MG lesion scores at 12 woa. Regardless of challenge, inclusion of XPC in the diet did not significantly alter BW at 3 or 8 woa or relative organ weights at 3 or 12 woa. However, at 12 woa, BW of XPC-fed birds, regardless of challenge was significantly (P = 0.0038) heavier than CON by 25.7 g. All birds tested negative for MG antibodies before the 8 woa challenge. Respective percentage serum plate agglutination and ELISA positive birds at 12 woa were 0 and 0% (CON, nonchallenged), 1.4 and 0% (XPC, nonchallenged), 100 and 47.2% (CON, challenged), and 100 and 50.0% (XPC, challenged). Diet did not significantly affect ELISA titers, but they were significantly (P < 0.0001) increased due to challenge. Furthermore, lesion scores were significantly higher for R low MG-challenged birds (P = 0.0012), and dietary treatment with XPC in challenged birds numerically reduced MG lesion scores from 0.278 to 0.194. In conclusion, although dietary XPC did not significantly alter the humoral immune response, antibody titer levels, or severity of MG lesions in layer pullets that were or were not challenged with R low MG, it led to an increase in their rate of growth through 12 woa., (Copyright © 2020. Published by Elsevier Inc.)

The effects of in ovo injected vitamin D 3 source on eggshell temperature (ET) and performance of broilers through 14 D of age (doa) were investigated. Eggs from a 35-wk-old commercial Ross 708 broiler breeder flock were set in a single-stage incubator with 4 treatments representing each of 12 incubator tray levels (blocks). At 432 h of incubation (hoi), noninjected and diluent-injected (50 μL) groups were control treatment groups. Vitamin treatments in the commercial diluent were as follows: 2.4 μg of vitamin D 3 (D 3 ) or 25-hydroxylcholecalciferol (25OHD 3 ). After injection, ET readings were recorded (435, 441, 453, 459, and 465 hoi) by infrared thermometry. Hatchability, hatchling BW, and percentage of male and female hatchlings were determined at 502 hoi. Equal numbers of male and female chicks were placed in each pen and grown out for 14 doa. On a per-pen basis, BW was recorded after hatching at day 7 and 14 doa, and BW gain, average daily BW gain, feed intake (FI), and feed conversion ratio (FCR) were calculated between 0 to 14 doa. The ET of eggs significantly fluctuated during the postinjection time period; however, the type of vitamin D 3  source injected did not affect ET. Nevertheless, the injection of 25OHD 3 resulted in a lower late embryo mortality than the diluent and D 3 injection treatments. In addition, birds that received 25OHD 3 had a lower FI and FCR than birds in all other treatments. In conclusion, the in ovo injection of 25OHD 3 has the potential to improve early posthatch broiler performance without affecting ET., (Copyright © 2019. Published by Elsevier Inc.)

Control of coccidiosis in broiler chickens continues to pose challenges to commercial poultry producers, especially in an era of increased consumer demand for antibiotic-free broiler production. As a result, coccidiosis vaccines are now commonly used in rotation programs to achieve effective coccidiosis control. Inovocox EM1 vaccine (EM1) is a coccidiosis vaccine that allows for earlier immune acquisition through oocyst cycling, which reduces the effects of wild-type coccidia. The EM1 vaccine is administered to embryonated broiler hatching eggs between 18 and 19 D of incubation (doi). In the U.S., commercial broiler hatcheries vaccinate embryonated eggs at either 18.5 or 19 doi. However, it is unclear whether a difference in embryo age at the time of in ovo injection can impact the actual site of vaccine delivery. In addition, it is unclear where oocysts eventually become localized within the embryo following the in ovo injection of EM1. Therefore, the objective of this study was to determine the effects of stage of embryonic development on the actual deposition site of the EM1 vaccine oocysts when they are in ovo injected and to subsequently investigate the movement and eventual location of EM1 oocysts after in ovo injection. Because all eggs were injected at the same time, a 12-h difference in set time was a means to derive 18.5 and 19.0 incubation age of injection (IAN) treatments. The experimental design was a 3 injection treatment (noninjected, diluent-injected, and vaccine-injected) × 2 IAN factorial. There was a significant main effect of IAN on site of vaccine oocysts delivery, and subsequent hatching chick quality. Qualitative histological evaluation revealed the oral uptake of vaccine oocysts through the amnion, with their subsequent presence in the gizzard and intestinal lumen by 24 to 36 h postinjection. In conclusion, physiological development influenced the site of injection, and oocysts imbibed along with the amniotic fluid in late stage broiler embryos are subsequently transported to the gastrointestinal tract., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In ovo vaccination is currently being considered as a means of delivery for live Mycoplasma gallisepticum (MG) vaccines. This study was performed to determine the transmissibility of strain F MG (FMG) from in ovo-vaccinated chicks to non-vaccinated pen mates. Eggs from an MG clean flock were incubated together for 18 D, at which point all live embryonated eggs were either not injected or administered a dilution of an FMG vaccine at 106 CFU per dose, 1 × 104 CFU per dose, 1 × 102 CFU per dose, or 1 CFU per dose. Non-injected eggs were hatched in a separate incubator. Ten non-injected, sentinel birds, and 1 in ovo-vaccinated FMG chick were placed in each of 32 isolation units located in 2 replicate rooms (8 replicates per dose). At 6 wk of age, surviving birds that had been vaccinated in ovo were removed, swabbed for FMG detection by PCR, and bled for serum plate agglutination (SPA) and ELISA testing for the presence of antibodies against MG (1, 2, 6, and all 8 in ovo-vaccinated chicks in the 106, 104, 102, and 1 CFU dosages). At 12 wk of age, the remaining sentinel birds were likewise sampled. No sentinel birds died. The in ovo-vaccinated birds that survived to 6 wk were serologically positive except for 5 birds in the 1 CFU treatment. The percentages of MG-positive sentinel birds and sentinel birds with antibody production against MG at 12 wk from each unit were not different between all MG dosages (P = 0.48, PCR; P = 0.77, SPA; P = 0.85, ELISA). Body weights of the in ovo-vaccinated chicks at 6 wk of age (P = 0.43) and the sentinel birds at 12 wk of age (P = 0.95) were each not affected by FMG treatment. These findings indicate that layer chickens in ovo vaccinated with a live-attenuated FMG vaccine were capable of transmitting FMG to other chicks with which they were in direct contact., (© 2019 Poultry Science Association Inc.)

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., (© 2019 Poultry Science Association Inc.)

The current study was designed to investigate the effects of the in ovo injection of different dosages of L-ascorbic acid (AA) on posthatch growth performance, carcass characteristics, plasma antioxidant capacity, and meat quality in broiler chickens. A total of 2,220 Ross 708 broiler hatching eggs containing live embryos at 17 D of incubation were subjected to 1 of 6 treatments (trt): non-injected control, saline-injected control, or saline containing 3, 6, 12 or 36 mg of AA. An Inovoject m semi-automatic multi-egg injector was used to inject a 100 μL volume of sterile saline (0.85%) alone or containing the different AA dosage into each egg. After hatch, 14 male hatchlings from each trt group were randomly selected and placed in each of 10 replicate floor pens for growth performance evaluation through 45 D posthatch. Chicks from the 3 and 6 mg AA trt groups had a higher average daily BW gain and a higher feed intake compared to the saline-injected control group during the grower phase. Chicks in the 12 mg AA trt group exhibited a better feed efficiency in the late finisher phase and the entire growing phase than those in the saline-injected control group. Higher thigh and leg percentages were observed in chicks from the 3 and 6 mg AA trt groups than in the non-injected control group. Compared to the non-injected or saline-injected control groups, birds in the 12 mg AA trt group were observed to have a lower plasma malondialdehyde content during the grower and finisher phases, and breast muscle tenderness was elevated in chicks from the 36 mg AA trt group. Taken together, these results suggest that the in ovo injection of AA (3 to 12 mg per egg) have lasting positive effects on the posthatch growth, leg muscle development, and systemic antioxidant capacity of broilers. Higher injected dosages of AA (36 mg per egg) may also have the potential to improve broiler meat quality., (© 2019 Poultry Science Association Inc.)

This study was carried out to determine the effect of supplementing the semen extender with calcitriol on in vitro sperm characteristics in Chukar partridges. A total of 60 male Chukar partridges were habituated for semen collection by abdominal massage. Pooled ejaculates from several males were extended (1 to 5 v/v ratio) in the Sexton's diluent containing 0, 24, 48, 96, or 192 μg calcitriol/mL. These concentrations represented 0-, 2-, 4-, 8-, and 16-fold levels of the mean seminal calcitriol concentration, respectively. A total of 12 subsamples from each treatment group were kept at 4 to 5°C or 19 to 24°C for 4, 24, or 48 h. The percentages of motile sperm, live sperm, abnormal sperm, incidence of hypoosmotic swelling (HOS), and thiobarbituric acid reactive species (TBARS) concentrations were determined. The data were analyzed by the xtmixed procedure of STATA software. The percentages of motile sperm, live sperm, abnormal sperm, and seminal TBARS were affected by calcitriol (P < 0.05). There was no effect of treatments on HOS (P > 0.05). There was an interaction effect between calcitriol, storage time, and storage temperature on sperm motility, sperm viability, and seminal TBARS. Supplementation of the diluent with 96 μg calcitriol/mL resulted in the highest sperm motility at 4°C. Also, the same treatment group recorded the highest sperm viability and lowest seminal TBARS at 19 to 24°C. Supplementing the diluent with calcitriol had beneficial effects on spermatozoa; however, the fertility rate of spermatozoa extended in calcitriol-supplemented diluent needs to be determined before the procedure can be recommended for use in artificial insemination programs., (© 2019 Poultry Science Association Inc.)

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 β-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., (© 2018 Poultry Science Association Inc.)