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

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 \u3c 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

Exosome signalling in the kidney

Urine contains exosomes originating from the circulation and all cells lining the urinary tract. Exosomes are a route of inter-cellular communication along the nephron potentially able to transfer of protein and/or RNA. It is not known whether this is a regulated process analogous to other cell-to-cell signalling systems. The aims of this study were to develop nanoparticle tracking analysis (NTA) as a technique to quantify exosomes in urine. Secondly, the hormonal regulation of exosome uptake in vitro and in vivo was investigated. Thirdly, exosome excretion in a central diabetes insipidus (DI) patient and a patient group after radiocontrast exposure was measured to investigate exosome excretion along the kidney in injury. Using the fluorescent capabilities of NTA, urinary exosomes were quantified in urine samples. NTA was able to detect changes in aquaporin 2 levels in vitro and in vivo. Storage conditions for human urinary exosomes were also optimised using NTA. A kidney cortical collecting duct cell line (CCDs) was used to model regulation of exosome uptake in vitro. CCDs were stimulated with desmopressin, a vasopressin analogue, and uptake of fluorescently-loaded or microRNA-loaded exosomes was measured. Desmopressin stimulated exosome uptake into collecting duct cells via V2 receptor stimulation. Intra-cellular uptake of exosomes was confirmed by microRNA specific mRNA down-regulation. Mechanistically, exosome uptake in response to desmopressin required cyclic AMP production, was mediated by clathrin-dependent endocytosis and was selective for exosomes from kidney tubular cells. In mice, fluorescently-loaded exosomes were systemically injected before and after administration of the V2 antagonist, tolvaptan, and urinary exosome excretion was measured. Basally, 2.5% of injected exosomes were recovered in urine; tolvaptan treatment resulted in a 5-fold increase. By combining antibodies to nephron segment-specific proteins with NTA we measured human urinary exosome excretion in central diabetes insipidus (DI) and after radiocontrast exposure (n=37). In DI, desmopressin reduced the excretion of exosomes derived from upstream glomerular and proximal tubule cells. In patients exposed to radiocontrast, urinary exosomes from the glomerulus were positively correlated with the tubular injury markers KIM- 1 and NGAL. These findings therefore show that tubular exosome uptake is a specific, hormonally regulated process that is reduced with injury. Physiologically, exosomes are a mechanism of inter-cellular communication; therapeutically, exosomes represent a novel vehicle by which RNA therapy could be targeted for the treatment of kidney disease

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

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

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 MyoDC, Myf-5C, and Pax7C MSC populations were quantified on immunofluorescence-stained cryosections. Relative collagen protein expression was quantified by fluorescent Western Blotting. In both 25 and 43-daysold 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 MyoDC, Myf-5C:MyoDC MSC were larger and relative collagen protein expression was greater in WB-affected vs. unaffected broilers (P _ 0.05). Pax7C MSC relative to total cells was also increased as WB severity increased in 43-days-old broilers (P _ 0.05). Densities of Myf-5C (P = 0.092), MyoDC (P = 0.030), Myf5C:MyoDC (P = 0.046), and Myf-5C:MyoDC:Pax7C (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 WBaffected 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

Combining Maternal and Post-Hatch Dietary 25-Hydroxycholecalciferol Supplementation on Broiler Chicken Growth Performance and Carcass Characteristics

Dietary inclusion of the vitamin D3 (D3) metabolite, 25-hydroxycholecalciferol (25OHD3), was demonstrated to improve broiler growth performance and breast meat yield. To assess the effect of combined maternal (MDIET) and post-hatch (PDIET) dietary 25OHD3 inclusion on broiler growth performance and carcass characteristics, a randomized complete block design experiment with a 2 × 2 factorial treatment structure was conducted. From 25 to 38 weeks of age, broiler breeder hens were provided with 1 of 2 MDIET formulated to contain: 5000 IU D3 (MCTL), or 2240 IU of D3 + 2760 IU of 25OHD3 per kg of feed (M25OHD3). Their chick offspring (n = 448; 224 per MDIET) hatched from eggs collected from 37 to 38 weeks of age were reared in 16 replicate pens with 7 birds per pen and fed 1 of 2 PDIET in 3 phases up to day 40 formulated to contain: 5000 IU of D3 per kg of feed (PCTL), or 2240 IU of D3 + 2760 IU of 25OHD3 per kg of feed (P25OHD3). No additive or synergistic effects of combining 25OHD3 inclusion in MDIET and PDIET were observed. Broilers from 25OHD3-fed hens (M25OHD3) were heavier on day 40 than those from hens fed only D3 (MCTL; 2.911 vs. 2.834 kg; p = 0.040). Tender weight (123 vs. 117 g) and yield (5.63 vs. 5.44%) were greater in the M25OHD3 broilers than the MCTL broilers (p = 0.006). Broilers fed 25OHD3 (P25OHD3) tended to have heavier breasts (637 vs. 615 g; p = 0.050), bone-in wings (215 vs. 210 g; p = 0.070), and boneless thighs (279 vs. 270 g; p = 0.078) compared with those fed only D3 (PCTL). Neither MDIET nor PDIET altered the severity of Wooden Breast and White Striping (p ≥ 0.106). Overall, including 25OHD3 in either the maternal or broiler diet increased broiler meat yield

Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production ▿ †

Many bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production