Jersey steer ruminal papillae histology and nutrigenomics with diet changes.

The transition from a high forage to a high concentrate diet is an important milestone for beef cattle moving from a stocker system to the feedlot. However, little is known about how this transition affects the rumen epithelial gene expression. This study assessed the effects of the transition from a high forage to a high concentrate diet as well as the transition from a high concentrate to a high forage diet on a variety of genes as well as ruminal papillae morphology in rumen fistulated Jersey steers. Jersey steers (n = 5) were fed either a high forage diet (80% forage and 20% grain) and transitioned to a high concentrate diet (20% forage and 80% grain) or a high concentrate diet (40% forage and 60% grain) and transitioned to a high forage diet (100% forage). Papillae from the rumen were collected for histology and RT-qPCR analysis. Body weight had a tendency for significant difference (p = .08). Histological analysis did not show changes in papillae length or width in steers transitioning from a high forage to a high concentrate diet or vice versa (p > .05). Genes related to cell membrane structure (CLDN1, CLDN4, DSG1), fatty acid metabolism (CPT1A, ACADSB), glycolysis (PFKL), ketogenesis (HMGCL, HMGCS2, ACAT1), lactate/pyruvate (LDHA), oxidative stress (NQO1), tissue growth (AKT3, EGFR, EREG, IGFBP5, IRS1) and the urea cycle (SLC14A1) were considered in this study. Overall, genes related to fatty acid metabolism (ACADSB) and growth and development (AKT3 and IGFBP5) had a tendency for a treatment × day on trial interaction effect. These profiles may be indicators of rumen epithelial adaptations in response to changes in diet. In conclusion, these results indicate that changes in the composition of the diet can alter the expression of genes with specific functions in rumen epithelial metabolism.

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 arginine, glycine + serine concentrations, and guanidinoacetic acid supplementation in vegetable-based diets for chickens.

The study investigated guanidinoacetic acid (GAA) supplementation with varying dietary digestible arginine (Arg) and glycine+serine (Gly+Ser) concentrations in the starter phase, exploring respective carry-over effects on growth performance, blood chemistry, incidence of pectoral myopathies and proximate composition in broilers. A total of 2,800 one-day-old male broiler chicks were distributed in a central composite design with 2 factors and double experimental mesh, represented by supplementation or omission of 0.6 g per kg of GAA, with a central point represented by 107% of Arg and 147% of Gly+Ser, 4 factorial points (combinations of Arg/Gly+Ser concentrations: 96.4/132.5%; 117.6/132.5%; 96.4/161.5%, and 117.6/132.5%), and 4 axial points (combinations of axial points estimated for Arg and Gly+Ser, with the central points of 92/147%; 122/147%; 107/126.5, and 107/167.5%), totaling 18 treatments, 4 repetitions to factorial and axial points, 24 replicates to the central point, and 25 birds per pen. Feed conversion ratio (FCR) from d 1 to 10 had a linear response (P = 0.009) for the decreasing Arg content and a quadratic response (P = 0.047) for Gly+Ser concentrations. Broilers supplemented GAA had lower FCR compared with nonsupplemented groups from d 1 to 10 (P = 0.048) and d 1 to 42 (P = 0.026). Aspartate aminotransferase (AST) exhibited increasing and decreasing linear effects as a function of Arg (P = 0.008) and Gly+Ser (P = 0.020) concentrations, respectively. Guanidinoacetic acid decreased serum AST (P = 0.028). Guanidinoacetic acid reduced moderate + severe (P = 0.039) and mild (P = 0.015) Wooden Breast scores. The occurrence of normal White Striping increased (P = 0.002), while severe score was reduced (P = 0.029) with GAA supplementation. In conclusion, increased digestible Arg:Lys and 14% and 6% above the recommendations (107% and 147%), respectively, provided improved FCR during the starter phase. Dietary GAA supplementation (0.6 g per kg) improved FCR, reduced severity of breast myopathies and appears to have reduced muscle damage in broilers fed plant-based diets.

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.

Feeding Ractopamine Improves the Growth Performance and Carcass Characteristics of the Lard-Type Mangalica Pig.

Mangalica pigs are gaining popularity within the U.S. as a niche breed, given their reputation for superior-quality pork. However, slow growth rates, a poor lean yield, and excessive adiposity limit the widespread adoption of Mangalica. To determine if feeding the metabolic modifier, ractopamine hydrochloride (RAC), would improve growth performance without impairing pork quality in the Mangalica, pigs were fed either 0 or 20 mg per kg RAC for 21 days. At 24 h postharvest, pork quality and carcass composition measurements were recorded; then, primal cuts were fabricated and assessed. RAC increased ADG (p < 0.04) and gain efficiency (p < 0.03) by 24% and 21%, respectively. RAC increased Loin Eye Area (p < 0.0001) by 21% but did not impact the 10th rib fat depth (p > 0.90) or marbling score (p > 0.77). RAC failed to alter any primal cut weights. Feeding RAC lowered b* values (p < 0.04) and tended to lower L* values (p < 0.08) while not affecting a* values (p > 0.30), suggesting RAC darkened loin color. Finally, RAC decreased cook yield percentage (p < 0.02) by 11% without impacting Warner-Bratzler Shear Force (p > 0.31). These data support the hypothesis that feeding RAC to Mangalica improves growth performance without impairing pork quality in this breed.

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.

Dietary Protein Source and Litter Condition Alter Broiler Chicken Intestinal Macrophage and Mitotically Active Cell Populations.

As antibiotic-free (ABF) broiler production continues to increase, understanding the development and local immune response in the intestines of ABF broilers is essential. Mitotically active cells, the majority of which will become enterocytes, help maintain the intestinal epithelial barrier. Macrophages prevent pathogen invasion by their phagocytic activity, functioning as immune response amplifying cells to aid in the recruitment of additional immune cells, and stimulating cytokine production in other adjacent cells. The objective of this experiment was to evaluate commonly used practical production practices on intestinal cell mitotic activity and local intestinal immunological responses. A randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. The 3 dietary protein sources were: soybean meal (SBM), a mix of 50% poultry by-product meal and 50% feather meal (PFM), and porcine meat and bone meal (MBM) and broilers were reared on either new litter (NL) or used litter (UL). On d 3, 8, 11, 15, and 21, 6 birds per treatment from 6 blocks (total n = 36 per d) were randomly selected for sampling. Broilers were injected intraperitoneally with 5'-bromo-2'-deoxyuridine (BrdU) 1 h prior to sampling to label mitotically active cells. Samples were analyzed using cryohistology and immunofluorescence to determine the density of mitotically active cells and macrophages. Mitotically active cell and macrophage densities changed in both the duodenum and ileum over time. Neither dietary protein source nor litter condition affected mitotically active cell or macrophage densities in the duodenum on d 11 and 21 or in the ileum on d 3, 8, 11, and 15. However, on d 3 and 15 in the duodenum (P ≤ 0.0126) and d 21 in the ileum (P ≤ 0.0009), broilers reared on UL had greater mitotically active cell densities than those reared on NL. On d 8 in the duodenum, broilers fed MBM had increased macrophage density compared with those fed PFM and SBM (P ≤ 0.0401). These results indicate dietary protein source and litter condition may impact the physiology of the broiler small intestine, though additional work with this model is necessary to understand the underlying mechanisms.

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.

Efficacy and safety of a novel source of dietary 25-hydroxycholecalciferol in growing pigs.

A randomized complete block design experiment was conducted to determine the safety and efficacy of supplementation of increasing concentrations of a novel, bacterial fermentation-derived vitamin D source on growth performance and tissue deposition of 25-hydroxycholecalciferol (25OHD3) in growing swine. Dietary treatments were as follows: commercial control with vitamin D3 (CON) at NRC recommended concentrations and three diets composed of CON + increasing inclusions (25, 50, and 250 µg/kg equivalent) of 25OHD3 from a novel source (CON + 25; CON + 50; and CON + 250, respectively). Pigs (n = 144) were assigned to 24 pens which were allotted to one of the four dietary treatments and fed for 42 d. Blood samples were collected for 25OHD3 concentration determination and individual body weights (BW) were measured on experimental day 0, 39, and 63. On day 42, tissues from 48 pigs (12 pigs per dietary treatment) were analyzed for 25OHD3 concentration. No differences were observed in growth performance. Day 39 serum 25OHD3 concentrations were greatest in CON + 250-fed pigs and linearly decreased as dietary 25OHD3 inclusion decreased (P < 0.0001). On day 42, tissue 25OHD3 concentrations increased linearly as 25OHD3 increased in the diet (P < 0.0001). On day 63, 21 d after dietary 25OHD3 withdrawal, serum 25OHD3 concentrations of all 25OHD3-fed pigs decreased to that of or within 2.76 ±â€…0.89 ng/mL of CON-fed pigs which demonstrates that feeding 250 µg/kg 25OHD3 is well tolerated by growing pigs and will clear the body within 21 d.

Pigs require several essential nutrients to meet their needs for maintenance, growth, reproduction, and other functions. It is important to provide these nutrients to the animals properly to assure their health and wellbeing as well as the profitability of production. Vitamin D is a nutrient that plays an important role in bone development and mineralization since it regulates calcium and phosphorus metabolism. Vitamin D has also been reported to aid in additional functions including immunity. Vitamin D can be synthetized in plants and is also produced in humans and animals when ultraviolet rays from sunlight strike the skin and lead to vitamin D synthesis. In pigs, vitamin D requirements can also be satisfied by dietary sources. The objective of this experiment was to determine the efficacy and safety of supplementation of a novel, bacterial fermentation-derived vitamin D source on growth and tissue accumulation in growing swine. Based on the results obtained, it can be concluded that concentrations of vitamin D in serum and tissue samples increased as dietary vitamin D supplementation increased, but did not alter growth performance, nor did there appear to be any safety issues with feeding up to 250 µg per kg feed of this vitamin D source to growing pigs.

Impacts of Increasing Additions of Choline Chloride on Growth Performance and Carcass Characteristics of Broiler Chickens Reared to 66 Days of Age.

The most recent research cited by the NRC Nutrition Requirements of Poultry to establish choline recommendations was published in 1987, so choline guidelines for modern broilers are outdated and may be insufficient to optimize growth. The objective was to determine the effect of additional dietary choline chloride supplementation on growth performance and carcass characteristics of modern broilers reared for 66 days. As-hatched Ross 708 × Yield Plus broiler chicks (n = 2160; 30 birds per pen) were randomly allotted to one of six experimental corn and soybean meal-based diets formulated to contain an additional 0, 400, 800, 1200, 1600, or 2000 mg of choline chloride above the choline content of the basal diet ingredients. Diets were fed in four phases, and birds were processed at day 66 of age. Growth performance and breast myopathy incidence was not impacted by added choline. While there were differences in breast, wing, thigh, and drum yields, the effects of added choline were not linear. Supplemental choline chloride was not beneficial for growth performance but did impact the carcass characteristics of modern, large frame broilers reared for 66 days.

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.

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.

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.

Effect of stocker management program on beef cattle skeletal muscle growth characteristics, satellite cell activity, and paracrine signaling impact on preadipocyte differentiation.

The objective of this study was to determine the effect of different stocker management programs on skeletal muscle development and growth characteristics, satellite cell (SC) activity in growing-finishing beef cattle as well as the effects of SC-conditioned media on preadipocyte gene expression and differentiation. Fall-weaned Angus steers (n = 76; 258 ± 28 kg) were randomly assigned to 1 of 4 stocker production systems: 1) grazing dormant native range (NR) supplemented with a 40% CP cottonseed meal-based supplement (1.02 kg · steer-1 · d-1) followed by long-season summer grazing (CON, 0.46 kg/d); 2) grazing dormant NR supplemented with a ground corn and soybean meal-based supplement fed at 1% of BW followed by short-season summer grazing (CORN, 0.61 kg/d); 3) grazing winter wheat pasture (WP) at high stocking density (3.21 steers/ha) to achieve a moderate rate of gain (LGWP, 0.83 kg/d); and 4) grazing winter WP at low stocking density (0.99 steers/ha) to achieve a high rate of gain (HGWP, 1.29 kg/d). At the end of the stocker (intermediate harvest, IH) and finishing (final harvest, FH) phases, 4 steers / treatment were harvested and longissimus muscles (LM) sampled for cryohistological immunofluorescence analysis and SC culture assays. At IH, WP steers had greater LM fiber cross-sectional area than NR steers; however, at FH, the opposite was observed (p < 0.0001). At IH, CORN steers had the lowest Myf-5+:Pax7+ SC density (p = 0.020), while LGWP steers had the most Pax7+ SC (p = 0.043). At FH, CON steers had the highest LM capillary density (p = 0.003) and their cultured SC differentiated more readily than all other treatments (p = 0.017). At FH, Pax7 mRNA was more abundant in 14 d-old SC cultures from HGWP cattle (p = 0.03). Preadipocytes exposed to culture media from proliferating SC cultures from WP cattle isolated at FH had more PPARγ (p = 0.037) and less FABP4 (p = 0.030) mRNA expression compared with NR cattle. These data suggest that different stocker management strategies can impact skeletal muscle growth, SC function, and potentially impact marbling development in growing-finishing beef cattle.

Skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt nonmyogenic fates.

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD(iCre/+);R26R(EYFP/+) muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.

Transforming growth factor beta1 is up-regulated by activated Raf in skeletal myoblasts but does not contribute to the differentiation-defective phenotype.

The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafER(DD)) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafER(DD) myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-beta(1) (TGF-beta(1)) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-beta(1) autocrine loop. However, extinguishing the biological activity of TGF-beta(1) does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis.