Effects of light intensity and reduction of starter diet digestible lysine and metabolizable energy on broiler chicken growth performance, breast meat yield, and meat quality defects.

The etiology of Wooden Breast (WB) is unknown; therefore, it is difficult to produce broiler flocks with similar proportions of WB-affected and unaffected birds. Because WB has been detected as early as 15 d posthatch, the objective of this randomized complete block experiment with a 2 × 2 factorial treatment arrangement was to determine whether combining the effects of light intensity (LI) and early nutrient reduction strategies could reliably produce WB-affected and normal broilers to further investigate the physiological mechanisms underlying WB. On day of hatch, male, Ross 708 × Yield Plus broilers (n = 384; 16 birds per pen; 3 replicate blocks) were randomly allotted to floor pens in the same facility and exposed to either 2 (LOWLI) or 30 (HIGHLI) lux of light from d 0 to 35. Birds were fed either a commercial starter diet (CON) or the CON diet with a 10% reduction in both ME and digestible lysine (dLys; RED) from d 0 to 14 and then a common grower diet from d 15 to 35. Broiler growth performance, breast yield, and incidence and severity of WB and White Striping (WS) were assessed. Data were analyzed as a 2-way ANOVA with SAS PROC GLIMMIX and means separated at P < 0.05 with PDIFF. No interaction among LI and diet was observed (P > 0.05). Broilers reared with HIGHLI were heavier on d 35 and consumed more feed in all phases compared with broilers reared under LOWLI (P ≤ 0.0096). Broilers reared under LOWLI gained less BW from d 15 to 35 and d 0 to 35 compared with broilers reared under HIGHLI (P = 0.0073). Broilers fed the RED starter diet consumed more feed and had higher FCR from d 0 to 14 compared with broilers fed the CON diet (P ≤ 0.0012). In conclusion, combining reductions in LI and starter diet ME and dLys did not produce the hypothesized reductions in breast yield and incidence and severity of WB or WS.

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells.

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96 h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168 h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96 h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

Evaluation of Increasing Concentrations of Supplemental Choline Chloride on Modern Broiler Chicken Growth Performance and Carcass Characteristics.

Choline has been demonstrated to partially substitute methionine in broiler chicken diets due to their interconnected biosynthesis pathways. Yet, research on the impacts of dietary choline supplementation on modern strains of high-yielding broilers is limited. The objective was to evaluate the effect of increasing additions of choline chloride on the performance and carcass characteristics of broilers fed reduced methionine diets and reared under summer environmental conditions. Ross 708 x Yield Plus male broilers were reared for 41 days on used litter in floor pens (n = 2232; 31 birds per pen). Birds were fed one of six corn and soybean meal-based, reduced methionine diets containing 0, 400, 800, 1200, 1600, or 2000 mg of added choline chloride per kg of feed. Diets were provided in three phases. On day 43, 10 birds per pen were processed. Increasing dietary choline resulted in similar body weight gain, reduced feed intake, and improved feed efficiency. Choline chloride supplementation linearly increased both breast and carcass yields while concomitantly increasing the incidence and severity of wooden-breast-affected fillets. These results indicate that supplementing reduced-methionine broiler diets with choline chloride during high environmental temperatures may improve feed efficiency and increase carcass and breast yields but may also increase wooden breast.

Effect of combined maternal and post-hatch dietary 25-hydroxycholecalciferol supplementation on broiler chicken Pectoralis major muscle growth characteristics and satellite cell mitotic activity.

Skeletal muscle growth is largely dependent on the proliferation and differentiation of muscle-specific stem cells known as satellite cells (SC). Previous work has shown that dietary inclusion of the vitamin D3 metabolite, 25-hydroxycholecalciferol (25OHD3), also called calcidiol, can promote skeletal muscle growth in post-hatch broiler chickens. Improving vitamin D status of broiler breeder hens by feeding 25OHD3 in addition to vitamin D3 has also been shown to positively impact progeny. Yet, whether combined pre- and post-hatch supplementation with 25OHD3 produces an additive or synergistic SC-mediated, skeletal muscle growth response remains unanswered. To evaluate the effect of combined maternal and post-hatch dietary 25OHD3 supplementation on the growth and SC mitotic activity of the Pectoralis major (PM) muscles in broiler chickens, a randomized complete block design experiment with the main effects of maternal diet (MDIET) and post-hatch diet (PDIET) arranged in a 2 × 2 factorial treatment structure was conducted. From 25 to 36 wk of age, broiler breeder hens were fed 1 of 2 MDIET formulated to provide 5,000 IU D3 (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3). Their male broiler chick offspring (n = 400) hatched from eggs collected from 35 to 36 wk of age were reared in raised floor pens. Broilers were fed 1 of 2 PDIET formulated to provide 5,000 IU of D3 per kg of feed (PCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (P25OHD3). Muscle was collected at days 4, 8, 15, 22, and 29 and stored until immunofluorescence analysis. Data were analyzed as a 2-way ANOVA with SAS GLIMMIX. Dietary 25OHD3 was effectively transferred from hen plasma to egg yolks (P = 0.002) and to broiler progeny plasma (days 4 to 22; P ≤ 0.044). Including 25OHD3 in either MDIET or PDIET altered PM hypertrophic growth prior to day 29 (P ≥ 0.001) and tended to reduce Wooden Breast severity (P ≤ 0.089). Mitotic SC populations were increased in PM of MCTL:P25OHD3 and M25OHD:PCTL-fed broilers at d 4 (P = 0.037). At d 8, the PM mitotic SC populations were increased 33% by P25OHD3 (P = 0.054). The results of this study reveal that combined maternal and post-hatch 25OHD3 supplementation does not produce additive or synergistic effects on SC-mediated broiler muscle growth. However, vitamin D status improvement through dietary 25OHD3 inclusion in either the maternal or post-hatch diet stimulated broiler breast muscle growth by increasing proliferating SC populations.

Skeletal muscle growth is largely dependent on the proliferation and differentiation of muscle-specific stem cells known as satellite cells (SC). Previous work has shown that dietary inclusion of the vitamin D3 metabolite, 25-hydroxycholecalciferol (25OHD3), also called calcidiol, can promote skeletal muscle growth in post-hatch broiler chickens. Improving vitamin D status of broiler breeder hens by feeding 25OHD3 in addition to vitamin D3 has also been shown to positively impact progeny. Yet, whether combined pre- and post-hatch supplementation with 25OHD3 produces an additive or synergistic SC-mediated, skeletal muscle growth response remains unanswered. The results of this study reveal that combined maternal and post-hatch 25OHD3 supplementation does not produce additive or synergistic effects on SC-mediated broiler muscle growth. However, vitamin D status improvement through dietary 25OHD3 inclusion in either the maternal or post-hatch diet stimulated broiler breast muscle growth by increasing proliferating SC populations. Overall, this work answers not only practical questions for the broiler industry regarding the possible benefits of combining maternal and post-hatch dietary 25OHD3 supplementation but also improves our understanding of vitamin D's role in pre- and post-hatch broiler skeletal muscle growth.

Dietary 25-Hydroxyvitamin D3 Supplementation Modulates Intestinal Cytokines in Young Broiler Chickens.

Vitamin D signaling is important for intestinal homeostasis. An increase in vitamin D receptors in immune cells can modulate cell phenotype and cytokine secretion. Cytokines regulate both pro- (interleukin 17; IL-17) and anti-inflammatory (IL-10) responses triggered by external stimuli. Inflammation in intestinal tissues can disrupt the structure and the remodeling of epithelial tight junction complexes, thus, compromising the protective barrier. The objective of the study was to determine the impact of dietary supplementation with 25-hydroxycholecalciferol (25OHD3), a hydroxylated metabolite of vitamin D, on intestinal cytokine abundance and epithelial barrier integrity over time in broilers. A randomized complete block design experiment was conducted to evaluate the effect of dietary 25OHD3 inclusion on relative protein expression of the cytokines, IL-17 and IL-10, and tight junction proteins, Zona Occludens 1 (ZO-1), and Claudin-1 (CLD-1), in broiler chicken duodenum and ileum from 3 to 21 days post-hatch. On day 0, male chicks (n = 168) were randomly assigned to raised floor pens. Experimental corn-soybean meal-based treatments were as follows: (1) a common starter diet containing 5,000 IU of D3 per kg of feed (VITD3) and (2) a common starter diet containing 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (25OHD3) fed from days 0 to 21. On days 3, 6, 9, 12, 15, 18, and 21, 12 birds per treatment were euthanized to collect tissue samples for quantitative, multiplex, and fluorescent Western blot analysis. Target proteins were quantified using Image Quant TL 8.1 and expressed relative to total protein. Feeding 25OHD3 post-hatch decreased ileal IL-10 (anti-inflammatory) protein expression in 21-day-old broilers compared with VITD3 only (P = 0.0190). Broilers fed only VITD3 post-hatch had greater IL-17 (pro-inflammatory) protein expression in the ileum at 18 and 21 days-of-age (P = 0.0412) than those that fed 25OHD3. Dietary inclusion of 25OHD3 lowered the abundance of key inflammatory cytokines in the ileum of young broilers.

Effect of Different Basal Culture Media and Sera Type Combinations on Primary Broiler Chicken Muscle Satellite Cell Heterogeneity during Proliferation and Differentiation.

The objective of this experiment was to access primary satellite cell (SC) proliferation and differentiation when cultured in different combinations of basal media and sera due to little consistency being published on the optimal culture media for primary broiler chicken satellite cells. Cells were cultured in one of three different basal media: McCoy's 5A, high glucose Dulbecco's Modified Eagle's medium (DMEM), and low glucose DMEM. Media were supplemented with 15% chicken serum (CS) or a combination of 5% horse serum (HS) + 10% CS during proliferation while 3% HS or 3% CS were added to the media during differentiation. Cultures were immunofluorescence stained for myogenic regulatory factors (MRF) at 48, 72, and 96 h post-plating for proliferation (Pax7, MyoD, and Myf-5) and 96 h post-proliferation during differentiation (Pax7 and MyoD), including MF20 to assess fusion. Cells cultured in Dulbecco's Modified Eagle's medium tended to have higher proportions of myogenic cells expressing MRF during proliferation and promoted fusion into myotubes compared with McCoy's 5A during differentiation. Culturing primary SC in low glucose media, glucose concentrations similar to circulating glucose concentrations in broilers, HSCS during proliferation and CS during differentiation, appears to be optimal for promoting broiler chicken satellite cell proliferation and differentiation.

Combined Maternal and Post-Hatch Dietary Supplementation of 25-Hydroxycholecalciferol Alters Early Post-Hatch Broiler Chicken Duodenal Macrophage and Crypt Cell Populations and Their Mitotic Activity.

The previous work has demonstrated that maternal supplementation of the circulating metabolite of vitamin D3 (D3), 25-hydroxycholecalciferol (25OHD3), enhances the immunocompetence of broiler chick offspring. In post-hatch broiler diets, 25OHD3 has been shown to affect intestinal morphology and improve the immune status of broilers. An experiment with a 2 × 2 factorial treatment arrangement was conducted to assess the effects of combining maternal (MDIET) and post-hatch (PDIET) dietary 25OHD3 inclusion on duodenal crypt and macrophage cell populations and mitotic activity in young broiler chickens. All diets were formulated to provide 5,000 IU of vitamin D. Broiler breeder hens were offered 1 of 2 MDIET: 5,000 IU D3 per kg of feed (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3) from week 25 to 41. Male broiler offspring (n = 480) hatched from eggs collected during week 41 of breeding age were allotted in raised floor pens (4 birds per pen from day 0 to 7 and individually allotted from day 8 to 21). Chicks were fed 1 of 2 PDIET (starter day 0 to 21): 5,000 IU D3 per kg of feed (PCTL) or 2,240 IU D3 + 2,760 IU 25OHD3 (P25OHD3). DUO samples (n = 12 birds per treatment per day) were collected on days 3, 6, 9, 12, 15, 18, and 21 for cryohistological and immunofluorescence analysis to facilitate the enumeration of the total macrophages, CD80+ macrophages (pro-inflammatory macrophages), and mitotically active cells (BrdU+) to calculate the proportion of proliferating cells (PPC) per duodenal crypt. Bird age impacted crypt PPC with the greatest PPC per duodenal crypt observed on days 3 and 9, and the lowest PPC per crypt was observed on day 21 (P < 0.0001). Broilers from the M25OHD3:PCTL treatment had a greater PPC (P =.002) than birds from the MCTL:PCTL treatment at day 3. An interaction among MDIET and PDIET was observed for proliferating macrophages at day 21 (P = 0.029) where M25OHD3:P25OHD3 birds had more proliferating macrophages than M25OHD3:PCTL-fed birds. These results indicate that combined MDIET and PDIET 25OHD3 supplementation may alter early post-hatch duodenal development and innate immunity.

Phytogenic Water Additives Improve Broiler Growth Performance via Modulation of Intermediary Metabolism-Related Signaling Pathways.

A ban on the use of antibiotic growth promoters (AGPs) has fueled and promoted scientific research towards the identification of reliable and effective alternatives. The supplementation of phytogenics AV/SSL12 (AVSSL) and Superliv Gold (SG) in water has been shown to improve broiler feed efficiency (FE) via modulation of hypothalamic neuropeptides. However, their effects on peripheral metabolic pathways are still unknown. The present study was undertaken to determine the effects of AVSSL and SG on lipid and protein metabolism-associated pathways in various tissues. Day-old male Cobb 500 chicks (n = 288) were randomly assigned to 3 treatment groups, with 8 replicates of 12 birds each. The treatment groups were fed a basal diet and supplemented with AVSSL or SG in the drinking water at a rate of 2, 4, and 7 mL/100 birds/d during the starter, grower, and finisher phases, respectively. The control group were fed a basal diet with no additive supplementation. On d 35, liver, adipose, and muscle tissue were collected from one bird per pen (8 birds/group). Data were analyzed using Student's T-test to compare one treatment group to the control using Graph Pad Prism version 6.0 for Windows. In the liver, the levels of phosphorylated acetyl-CoA carboxylase alpha (ACCα) were significantly increased in both the AVSSL and SG groups compared to the control. The hepatic expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP) was significantly downregulated in both treated groups compared to the control. AVSSL supplementation downregulated the hepatic expression of SREBP-2 and adiponectin (AdipoQ), while SG administration upregulated hepatic AdipoR1/R2 mRNA abundances compared to the untreated group. Both AVSSL and SG treatments upregulated hepatic stearoyl-CoA desaturase-1 (SCD-1) gene expression compared to their untreated counterparts. In the adipose tissue, the levels of phosphorylated hormone-sensitive lipase (HSL) at Ser855/554 site were increased in both the AVSSL and SG groups compared to the control. However, ATGL protein expression was decreased in SG compared to the untreated group. In the muscle, the levels of phosphorylated mechanistic target of rapamycin (mTOR) were increased in the AVSSL, but decreased in the SG group compared to the control. Collectively, these data indicate that supplementation of the phytogenics AVSSL and SG in water reduced hepatic lipogenesis-related proteins and increased adipose tissue lipolysis- and muscle protein synthesis-associated targets, which might explain, at least partially, the improvement in FE observed in previous research.

Phytogenic feed additives improve broiler feed efficiency via modulation of intermediary lipid and protein metabolism-related signaling pathways.

Diets enriched with phytogenic feed additives (PFA) such as AV/HGP/16 premix (AVHGP), Superliv concentrate premix (SCP), and bacteriostatic herbal growth promotor (BHGP) with essential oils have been shown to improve feed efficiency (FE) in broilers. This FE improvement was achieved via modulation of hypothalamic neuropeptides, which results despite feed intake reduction, in increased breast yield without changes in body weight compared to the control group. To gain further insights into the mode of action of these PFA, the present study aimed to determine the potential involvement of signaling pathways associated with lipid and protein metabolism. One day-old male Cobb 500 chicks were randomly assigned into 1 of 4 treatments, comprising 8 replicates per treatment in a completely randomized design. The dietary treatments included a basal diet (control) or 0.55 g/kg diet of AVHGP, SCP, or BHGP. The birds had ad libitum access to water and feed. On day 35, after blood sampling, the liver, abdominal adipose tissue (AT), and breast muscle samples were collected. The levels of phosphorylated mechanistic target of rapamycin (mTOR)Ser2481 as well as its levels of mRNA and those of its downstream mediator RPS6B1 were significantly upregulated in the muscle of the PFA-fed groups compared with the control group. In the liver, the phosphorylated levels of acetyl-CoA carboxylase alpha at Ser79, the rate-limiting enzyme in fat synthesis, was significantly induced in the PFA-fed groups compared with the control group, indicating a lower hepatic lipogenesis. The hepatic expression of hepatic triglyceride lipase (LIPC) and adipose triglyceride lipase (ATGL) was significantly upregulated in the AVHGP-fed group compared with the control group. These hepatic changes were accompanied by a significant downregulation of hepatic sterol regulatory element-binding protein cleavage-activating protein in all the PFA groups and an upregulation of peroxisome proliferator-activated receptor alpha and gamma in the SCP-fed compared with the control group. In the AT, the mRNA abundances of ATGL and LIPC were significantly increased in both SCP- and BHGP-fed birds compared with the control group. Together these data indicate that PFA improve FE via modulation of muscle mTOR pathway and hepatic lipolytic/lipogenic programs, thus, favoring muscle protein synthesis and lowering hepatic lipogenesis.

Characterization of Pectoralis Major Muscle Satellite Cell Population Heterogeneity, Macrophage Density, and Collagen Infiltration in Broiler Chickens Affected by Wooden Breast.

Muscle satellite cells (MSCs) are myogenic stem cells that play a critical role in post-hatch skeletal muscle growth and regeneration. Activation of regeneration pathways to repair muscle fiber damage requires both the proliferation and differentiation of different MSC populations as well as the function of resident phagocytic cells such as anti-inflammatory and pro-inflammatory macrophages. The Wooden Breast (WB) phenotype in broiler chickens is characterized by myofiber degeneration and extensive fibrosis. Previous work indicates that the resident MSC populations expressing the myogenic regulatory factors, Myf-5 and Pax7 are larger and more proliferative in broilers severely affected with WB vs. unaffected broilers. To further characterize the cellular and molecular changes occurring in WB-affected muscles, samples from pectoralis major (PM) muscles with varying severity of WB (WB score 0 = normal; 1 = mildly affected; 2 = severely affected) were collected at 25 and 43 days post-hatch (n = 8 per score per age) and processed for cryohistological and protein expression analyses. Collagen per field and densities of macrophages and MyoD+, Myf-5+, and Pax7+ MSC populations were quantified on immunofluorescence-stained cryosections. Relative collagen protein expression was quantified by fluorescent Western Blotting. In both 25 and 43-days-old broilers, the proportion of collagen per field (P ≤ 0.021) and macrophage density (P ≤ 0.074) were greater in PM exhibiting severe WB compared with normal. At day 43, populations of MyoD+, Myf-5+:MyoD+ MSC were larger and relative collagen protein expression was greater in WB-affected vs. unaffected broilers (P ≤ 0.05). Pax7+ MSC relative to total cells was also increased as WB severity increased in 43-days-old broilers (P ≤ 0.05). Densities of Myf-5+ (P = 0.092), MyoD+ (P = 0.030), Myf5+:MyoD+ (P = 0.046), and Myf-5+:MyoD+:Pax7+ (P = 0.048) MSC were greater in WB score 1 birds compared with WB score 0 and 2 birds. Overall, alterations in the resident MSC and macrophage populations and collagen protein content were observed in WB-affected muscle. Further investigation will be required to determine how these changes in cell population kinetics and local autocrine and paracrine signaling are involved in the apparent dysregulation of muscle maintenance in WB-affected broilers.