Hypoxia promotes proliferation and inhibits myogenesis in broiler satellite cells.

Breast muscle myopathies in broilers compromise meat quality and continue to plague the poultry industry. Broiler breast muscle myopathies are characterized by impaired satellite cell (SC)-mediated repair, and localized tissue hypoxia and dysregulation of oxygen homeostasis have been implicated as contributing factors. The present study was designed to test the hypothesis that hypoxia disrupts the ability of SC to differentiate and form myotubes, both of which are key components of myofiber repair, and to determine the extent to which effects are reversed by restoration of oxygen tension. Primary SC were isolated from pectoralis major of young (5 d) Cobb 700 chicks and maintained in growth conditions or induced to differentiate under normoxic (20% O2) or hypoxic (1% O2) conditions for up to 48 h. Hypoxia enhanced SC proliferation while inhibiting myogenic potential, with decreased fusion index and suppressed myotube formation. Reoxygenation after hypoxia partially reversed effects on both proliferation and myogenesis. Western blotting showed that hypoxia diminished myogenin expression, activated AMPK, upregulated proliferation markers, and increased molecular signaling of cellular stress. Hypoxia also promoted accumulation of lipid droplets in myotubes. Targeted RNAseq identified numerous differentially expressed genes across differentiation under hypoxia, including several genes that have been associated with myopathies in vivo. Altogether, these data demonstrate localized hypoxia may influence SC behavior in ways that disrupt muscle repair and promote the formation of myopathies in broilers.

Technical note: an optimized method to isolate, purify, and differentiate satellite cells from broiler chicks.

Development and maintenance of healthy muscle fibers rely on the myogenic potential of satellite cells (SC), muscle stem cells that proliferate and differentiate to form myotubes. Satellite cells are indispensable for post-hatch muscle growth as well as muscle repair and regeneration when myofibers are damaged. Pectoralis major of young broiler chicks (5-d olds) is a readily available source of SC, which can be used in vitro to elucidate cellular and molecular mechanisms responsible for muscle growth and regeneration in broilers. Here, we optimized a method for efficient isolation, purification, and differentiation of SC, from young broiler chicks. This procedure includes a simple method that allows SC to be purified from other muscle cell types that can impede the fidelity of follow-on experiments, particularly highly sensitive measures such as RNAseq. The methods for culturing and differentiating SC into multinucleated myotubes were also optimized by testing serum types, concentrations, and the effects of chicken embryo extract. Using the isolation procedure, a highly pure SC population (94.6 ± 2.11% Pax7+) with high viability and yield was obtained, and their capacity to differentiate into myotubes was confirmed. Enrichment for SC and myogenic capacity were maintained through multiple passages and after cryopreservation. Analysis of gene expression over the first 48 h of differentiation confirmed that SC exhibited the expected molecular signature of myogenesis. Taken together, this method simplifies the ability to isolate and maintain a relatively pure population of SC with strong myogenic potential from young broiler chicks, and should support downstream applications for assessing the impact of nutrients, metabolites, and other physiological cues on muscle growth and development in broilers.

The purpose of this study is to optimize the isolation and culture method for muscle stem cells, called satellite cells, in the breast muscle of meat-type chicken (Broiler). Satellite cells play pivotal roles in muscle development, growth, and muscle regeneration. One challenge of primary cell isolation is efficient and effective purification from a mixture of cell types, while maintaining viability and achieving high yield. Here, we developed a protocol enabling high yield and purity of primary satellite cells isolation and optimized culture conditions. We obtained about 95% purified satellite cells with high viability, and this method is simple and cost-effective compared to alternative cell sorting techniques that require cell-labeling steps and expensive equipment. Thus, this optimized satellite cell isolation and culture may be valuable for in vitro studies of cellular and molecular mechanisms responsible for muscle diseases (e.g., white striping and wooden breast) and the nutritional regulation of muscle development in broiler.

Isolation of Preadipocytes from Broiler Chick Embryos.

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

Identification of candidate proteins regulated by long-term caloric restriction and feed efficiency in longissimus dorsi muscle in Korean native steer.

We aimed to investigate candidate proteins related to long-term caloric restriction and feed efficiency in bovine longissimus dorsi muscle (LM). A total of 31 Korean native steers were randomly distributed to ad libitum (n = 16) or caloric restriction group (n = 15) to conduct two feeding trials for 13 mon. In the first trial (10-18 mon of age), steers were fed with 100% ad libitum (NEg = 0.63 Mcal/kg) or caloric restriction (80% of the previous day's feed intake of ad libitum group). In the second trial (18-23 mon of age), the energy value of 100% ad libitum diet was 1.13 Mcal/kg NEg and those in caloric restriction group diet was 0.72 Mcal/kg NEg. At the endpoint of this experiment, in each group, 6 animals were selected with high (n = 3) or low feed efficiency (n = 3) to collect muscle tissue samples (6 animals/group). From muscle tissues of 23 mo of age, we excavated 9 and 12 differentially expressed (two-fold or more) proteins in a nutritional group and feed efficiency group using two-dimensional electrophoresis, respectively. Of these proteins, heat shock protein beta-6 was up-regulated in both the caloric restriction and the low feed efficiency group. In bovine embryonic fibroblasts, the mRNA expression of heat shock protein beta-6 increased after adipogenic differentiation, however, decreased after myogenic differentiation. Our data provide that heat shock protein beta-6 may be an adipogenic protein involved in the mechanism of caloric restriction and feed efficiency in the LM of the steer.

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice.

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

Administration of encapsulated L-tryptophan improves duodenal starch digestion and increases gastrointestinal hormones secretions in beef cattle.

OBJECTIVE: This study investigated the effects of oral administration of rumen-protected L-tryptophan (RPL-T) on duodenal starch digestion and gastrointestinal hormones (GIH) secretions using Hanwoo beef steers as the animal models. METHODS: Four steers (423±24 kg) fitted with ruminal and duodenal cannulas were employed in a crossover design replicated twice. Treatments were control (basal diet) and RPL-T (basal diet+191.1 mg/kg body weight [BW]) group. Blood and duodenal samples were collected to measure serum GIH levels and pancreatic α-amylase activity at day 0, 1, 3, and 5 (-30, 30, 90, 150, and 210 min) of the study. Samples from each segment of the gastrointestinal tract were collected via ruminal and duodenal cannulas and were used to determine soluble protein and the starch digestion rate at days 6 (-30, 180, 360, and 540 min) and 8 (-30, 90, 270, and 450 min) of the experiment. RESULTS: No significant difference in ruminal pH, NH3-N, and total volatile fatty acid including the levels of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and the acetate-to-propionate ratio was observed between groups (p>0.05). Crude protein uptake was higher and feces starch content was lower in RPL-T group than the control group (p<0.05). The D-glucose contents of feces in RPL-T group decreased at day 5 compared to those in the control group (p<0.05), however, no change was found at day 0, 1, or 3 compared to the control group (p>0.05). Serum cholecystokinin (CCK), melatonin, duodenal pancreatic α-amylase activity, and starch digestion were significantly higher in RPL-T group than the control group (p<0.05). CONCLUSION: Taken together, oral administration of RPL-T at the rate of 191.1 mg/kg BW consistently increased CCK concentration, pancreatic α-amylase activity in duodenal fluids, and starch digestion rate in the small intestine and thus found to be beneficial.

Radical Response: Effects of Heat Stress-Induced Oxidative Stress on Lipid Metabolism in the Avian Liver.

Lipid metabolism in avian species places unique demands on the liver in comparison to most mammals. The avian liver synthesizes the vast majority of fatty acids that provide energy and support cell membrane synthesis throughout the bird. Egg production intensifies demands to the liver as hepatic lipids are needed to create the yolk. The enzymatic reactions that underlie de novo lipogenesis are energetically demanding and require a precise balance of vitamins and cofactors to proceed efficiently. External stressors such as overnutrition or nutrient deficiency can disrupt this balance and compromise the liver's ability to support metabolic needs. Heat stress is an increasingly prevalent environmental factor that impairs lipid metabolism in the avian liver. The effects of heat stress-induced oxidative stress on hepatic lipid metabolism are of particular concern in modern commercial chickens due to the threat to global poultry production. Chickens are highly vulnerable to heat stress because of their limited capacity to dissipate heat, high metabolic activity, high internal body temperature, and narrow zone of thermal tolerance. Modern lines of both broiler (meat-type) and layer (egg-type) chickens are especially sensitive to heat stress because of the high rates of mitochondrial metabolism. While this oxidative metabolism supports growth and egg production, it also yields oxidative stress that can damage mitochondria, cellular membranes and proteins, making the birds more vulnerable to other stressors in the environment. Studies to date indicate that oxidative and heat stress interact to disrupt hepatic lipid metabolism and compromise performance and well-being in both broilers and layers. The purpose of this review is to summarize the impact of heat stress-induced oxidative stress on lipid metabolism in the avian liver. Recent advances that shed light on molecular mechanisms and potential nutritional/managerial strategies to counteract the negative effects of heat stress-induced oxidative stress to the avian liver are also integrated.

Intravenous administration of L-tryptophan stimulates gastrointestinal hormones and melatonin secretions: study on beef cattle.

This study aimed to determine the effective dose of intravenous administration of L-tryptophan (L-T) on gastrointestinal hormones (GIH) secretions and melatonin using Hanwoo cattle. Three steers (362 ± 23 kg) fitted with indwelling jugular vein catheters were assigned in a 3 × 3 Latin square design. Treatments were intravenous administration of saline (control), 28.9 mg L-T/kg body weight (BW; low) and 57.8 mg L-T/kg BW (high) L-T for 1 day with 7 days of adaptation. Samples were collected after adaptation period at -60, 0, 30, 60, 90, 120, 150, 180, 240, and 300 min of sampling day. The levels of serum cholecystokinin (CCK) and secretin were higher (p < 0.05) in the high L-T group than those in the other groups. Serum Melatonin (MEL) levels were increased upon L-T administration (p < 0.05) in the high L-T group. Taken together, the effective dose of L-T administration was defined at 57.8 mg L-T/kg BW in order to stimulate increase of GIH and MEL.

Effects of dietary phytoncides extracted from Korean pine (Pinus koraiensis) cone on performance, egg quality, gut microflora, and immune response in laying hens.

This study was conducted to evaluate the effects of dietary phytoncides extracted from discarded Korean pine cones (Pinus koraiensis) on the performance, egg quality, immune response and gut microflora in laying hens. A total of 400 Hy-Line brown laying hens (50-week old) were allotted into four dietary treatments including a control diet or a diet supplemented with phytoncides at 0.002%, 0.004% and 0.008%. During the 6 weeks of experimental feeding, 0.008% of dietary phytoncides improved egg production, feed conversion ratio (p < 0.05), but not feed intake, egg weight or feed efficiency. Although dietary phytoncides had no effect on egg quality, decreases in Haugh units depending on storage periods were improved by 0.008% of dietary phytoncides (p < 0.05). To investigate the roles of dietary phytoncides on the alteration of the immune response during inflammation, lipopolysaccharide (LPS) or saline was intraperitoneally injected into 10 hens per diet group on the end date of the experimental feeding period. Serum immunoglobulins and splenic cytokine expression at mRNA levels were then measured at 4 hr postinjection. Although the levels of IgA were decreased by LPS injection in all dietary groups, dietary phytoncides at 0.008% showed a higher level of IgA by LPS (p < 0.05). Interestingly, although LPS injection resulted in an enhanced expression of proinflammatory cytokines such as IL-1ß and IL-6, dietary phytoncides at 0.008% showed less increased levels of them (p < 0.05). Gut microflora was examined from 10 hens per diet group at the end of the experimental period. While the number of Lactobacillus spp. was increased (p < 0.05), Escherichia coli counts in the cecal contents were decreased by 0.008% of dietary phytoncides. Taken together, these results demonstrate that dietary supplementation of 0.008% phytoncides improved the egg production, immune responses during inflammation and gut microflora in laying hens.

Physiological concentrations of trans-11 18:1 vaccenic acid suppress pro-inflammatory markers under acute inflammation in isolated ICR mice splenocytes.

Direct influences of dietary trans-11 18:1 vaccenic acid (TVA) at physiological concentrations of 50-200 µM were evaluated for cell growth, cytotoxic activity, and cytokine production in leukocytes isolated from the mouse spleen. TVA supplementation for 24 h induced growth of splenocytes at concentrations of 50-200 µM, except for 100 µM. The cytokines TNFα, IFNγ, and IL-10 of splenocytes were stimulated by 100 µM TVA. Induced production of TNFα in splenocytes challenged with lipopolisaccharides was suppressed by 100 µM TVA. Physiological levels of TVA had direct effects on growth and cytokine production in splenocytes. Further in vivo studies are needed to improve understanding of the precise influence of trans fatty acids on production of pro-inflammatory markers under acute inflammation conditions.

Chemerin is a novel regulator of lactogenesis in bovine mammary epithelial cells.

Chemerin is a chemoattractant cytokine (chemokine) produced by adipocytes and hepatocytes; it regulates insulin sensitivity and adipocyte differentiation. The objective of this study was to investigate the effect of chemerin on the expression of genes related to lactogenesis and the regulators of chemerin signaling in a bovine mammary epithelial cell line (MAC-T). Two types of chemerin receptors, chemokine like-receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2), were detected in cultured MAC-T cells, whereas chemerin was not detected. G protein-coupled receptor 1 (GPR1), another receptor of chemerin, was undetectable in MAC-T cells. Chemerin upregulated transcript expression of CMKLR1, CCRL2, and genes associated with fatty acid synthesis, glucose uptake, insulin signaling, and casein synthesis in MAC-T cells. Lactogenic hormones (insulin, growth hormone, and prolactin) downregulated the expression of CMKLR1 in MAC-T cells. Adiponectin suppressed CMKLR1 expression. TNF-α suppressed CMKLR1, but induced CCRL2 expression. These data suggest chemerin is a novel regulator of lactogenesis via its own receptor in bovine mammary epithelial cells.

Effects of dietary trans-9 octadecenoic acid, trans-11 vaccenic acid and cis-9, trans-11 conjugated linoleic acid in mice.

The aim of the present study was to investigate the effects of dietary trans fatty acids in mice. Following the administration of a 0.5/100 g diet of trans-9 octadecenoic acid (EA), trans-11 vaccenic acid (TVA) or cis-9, trans-11 conjugated linoleic acid (CLA) for 4 weeks, the body weights and the weights of the liver, testis and mediastinal adipose tissue (MAT) of the animals gradually decreased (P<0.05). The EA group exhibited the lowest levels of magnesium and triglycerides (P<0.05). CLA increased villus length (P<0.05), while EA and TVA decreased villus length (P<0.05). The TVA group exhibited the lowest levels of low-density lipoprotein and tumor necrosis factor-α (P<0.05). Taken together, EA, TVA and CLA affected the physiological conditions of mice differently. The potential effects of three well-known fatty acids, including trans-9 octadecenoic acid (EA), trans-11 vaccenic acid (TVA) and cis-9, trans-11 conjugated linoleic acid (CLA), in animals or humans remain to be elucidated. Therefore, in the present study, 32 animals were randomly divided into four groups and administered a 0.5/100 g diet of EA, TVA or CLA for 4 weeks. The results demonstrated that the body weights and the weights of the liver, testis and mediastinal adipose tissue (MAT) of the animals gradually decreased (P<0.05). Blood was collected individually via the external jugular veins and the EA group exhibited the lowest levels of magnesium and triglycerides (P<0.05). CLA increased villus length (P<0.05), while EA and TVA decreased villus length (P<0.05). The TVA group exhibited the lowest levels of low-density lipoprotein and tumor necrosis factor-α (P<0.05). Taken together, EA, TVA and CLA affected the physiological conditions of mice differently and these may further our understanding of the various effects of these fatty acids on animals and humans.