Insect Peptide CopA3 Mitigates the Effects of Heat Stress on Porcine Muscle Satellite Cells.

Heat stress inhibits cell proliferation as well as animal production. Here, we aimed to demonstrate that 9-mer disulfide dimer peptide (CopA3) supplementation stabilizes porcine muscle satellite cell (PMSC) proliferation and heat shock protein (HSP) expression at different temperatures. Therefore, we investigated the beneficial effects of CopA3 on PMSCs at three different temperatures (37, 39, and 41 °C). Based on temperature and CopA3 treatment, PMSCs were divided into six different groups including treatment and control groups for each temperature. Cell viability was highest with 10 µg/mL CopA3 and decreased as the concentration increased in a dose-dependent manner. CopA3 significantly increased the cell viability at all temperatures at 24 and 48 h. It significantly decreased apoptosis compared to that in the untreated groups. In addition, it decreased the apoptosis-related protein, Bcl-2-associated X (BAX), expression at 41 °C. Notably, temperature and CopA3 had no effects on the apoptosis-related protein, caspase 3. Expression levels of HSP40, HSP70, and HSP90 were significantly upregulated, whereas those of HSP47 and HSP60 were not affected by temperature changes. Except HSP90, CopA3 did not cause temperature-dependent changes in protein expression. Therefore, CopA3 promotes cell proliferation, inhibits apoptosis, and maintains stable HSP expression, thereby enhancing the heat-stress-tolerance capacity of PMSCs.

Comparative Analysis of Porcine Adipose- and Wharton's Jelly-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration, cell therapy, and cultured meat research owing to their ability to differentiate into various lineages including adipocytes, chondrocytes, and osteocytes. As MSCs display different characteristics depending on the tissue of origin, the appropriate cells need to be selected according to the purpose of the research. However, little is known of the unique properties of MSCs in pigs. In this study, we compared two types of porcine mesenchymal stem cells (MSCs) isolated from the dorsal subcutaneous adipose tissue (adipose-derived stem cells (ADSCs)) and Wharton's jelly of the umbilical cord (Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs)) of 1-day-old piglets. The ADSCs displayed a higher proliferation rate and more efficient differentiation potential into adipogenic and chondrogenic lineages than that of WJ-MSCs; conversely, WJ-MSCs showed superior differentiation capacity towards osteogenic lineages. In early passages, ADSCs displayed higher proliferation rates and mitochondrial energy metabolism (measured based on the oxygen consumption rate) compared with that of WJ-MSCs, although these distinctions diminished in late passages. This study broadens our understanding of porcine MSCs and provides insights into their potential applications in animal clinics and cultured meat science.

Effects of heat stress exposure on porcine muscle satellite cells.

Heat stress (HS) affects cell culture as well as animal production. Although there have been many reports on the disparate effects of heat stress, its effects on mammalian muscle stem cells are still unclear. In this study, we isolated porcine muscle satellite cells (PMSCs) from the femurs of 1-day-old piglets, and cultured them under three temperature conditions: 37 °C, 39 °C, and 41 °C. Exposure to HS not only decreased the viability and proliferation rates of PMSCs, but also regulated the cell cycle and induced apoptosis. High-temperature culture conditions decreased both protein and gene expression of Pax7, a proliferation and maintenance marker of muscle satellite cells, whereas it increased both protein and gene expression of MyoG, a differentiation marker, and promoted myotube formation in the early stage of differentiation induction. In addition, the protein and gene expression of several heat shock proteins (HSPs) in PMSCs increased due to heat treatment. In conclusion, HS induced the cell cycle arrest of PMSCs, thereby reducing the proliferation rate. In addition, high-temperature culture conditions promoted the formation of myotubes at the early stage of differentiation of PMSCs without additives.

COPA3 peptide supplementation alleviates the heat stress of chicken fibroblasts.

Heat stress inhibits cellular proliferation and differentiation through the production of reactive oxygen species. Under stress conditions, antioxidant drugs promote stable cellular function by reducing the stress level. We sought to demonstrate 9-mer disulfide dimer peptide (COPA3) supplementation stabilizes fibroblast proliferation and differentiation even under heat stress conditions. In our study, fibroblasts were assigned to two different groups based on the temperature, like 38°C group presented as Control - and 43°C group presented as Heat Stress-. Each group was subdivided into two groups depending upon COPA3 treatment, like 38°C + COPA3 group symbolized Control+ and the 43°C + COPA3 group symbolized as Heat Stress+. Heat stress was observed to decrease the fibroblast viability and function and resulted in alterations in the fibroblast shape and cytoskeleton structure. In contrast, COPA3 stabilized the fibroblast viability, shape, and function. Moreover, heat stress and COPA3 were found to have opposite actions with respect to energy production, which facilitates the stabilization of cellular functions by increasing the heat tolerance capacity. The gene expression levels of antioxidant and heat shock proteins were higher after heat stress. Additionally, heat stress promotes the mitogen-activated protein kinase/ extracellular signal-regulated kinase-nuclear factor erythroid 2-related factor 2 (MAPK/ERK-Nrf2). COPA3 maintained the MAPK/ERK-Nrf2 gene expressions that promote stable fibroblast proliferation, and differentiation as well as suppress apoptosis. These findings suggest that COPA3 supplementation increases the heat tolerance capacity, viability, and functional activity of fibroblasts.

Inhibition of GSK3ß Promotes Proliferation and Suppresses Apoptosis of Porcine Muscle Satellite Cells.

As the global population increases, interest in cultured meat (a new research field) is gradually increasing. The main raw material for the production of cultured meat is muscle stem cells called satellite cells isolated from livestock. However, how to mass proliferate and maintain satellite cells in vitro without genetic manipulation remains unclear. In the present study, we isolated and purified porcine muscle satellite cells (PMSCs) from the femur of a 1-day-old piglet and cultured PMSCs by treating them with an inhibitor (XAV939, Tankyrase (TNKS) inhibitor) or an activator (CHIR99021, glycogen synthase kinase 3 beta (GSK3ß) inhibitor) of Wnt signaling. The CHIR group treated with 3 µM CHIR99021 showed a significantly increased proliferation rate of PMSCs compared to the SC group (control), whereas the XAV group treated with 1 µM XAV939 showed a significantly decreased proliferation rate of PMSCs. CHIR99021 also inhibited the differentiation of PMSCs by reducing the expression of MyoD while maintaining the expression of Pax7 and suppressed apoptosis by regulating the expression of apoptosis-related proteins and genes. RNA sequencing was performed to obtain gene expression profiles following inhibition or activation of the Wnt signaling pathway and various signaling mechanisms related to the maintenance of satellite cells were identified. Our results suggest that inhibition of GSK3ß could dramatically improve the maintenance and mass proliferation ability of PMSCs in vitro by regulating the expression of myogenic markers and the cell cycle.

Cortisol differentially affects the viability and myogenesis of mono- and co-cultured porcine gluteal muscles satellite cells and fibroblasts.

Cortisol is a ubiquitously expressed stress hormone. In this study, we investigated the effects of exogenous cortisol on porcine gluteal muscles primary cultured satellite cells and fibroblasts. Satellite cells and fibroblasts were mono-or co-cultured, and cells in each type of culture were categorized into the control and cortisol-treated (treatment) groups. We selected 28 µmol mL-1 cortisol for treatment based on their efficacy. Cortisol treatment reduced viability of monocultured satellite cells and fibroblasts. In both monocultured and co-cultured cells, the nucleus in the treatment group was damaged than that control group. Moreover, the total cell cycle duration was shorter in the treatment group than the control group. PAX-7 expression was upregulated in the control group of co-cultured satellite cells and fibroblasts than those remaining groups. Moreover, MyoD expression was downregulated in the cortisol treated group of both mono-and co-cultured satellite cells compared with that in the control group. In co-cultured fibroblasts, MyoD and MyoG expression was upregulated than those remaining groups. The Cyto-C expression was upregulated in the treatment group compared to the control mono-and co-cultured both cells. These results suggest that the selected experimental dose of cortisol reduced cell viability and myogenesis-related gene expression in the monoculture compared to that in the co-culture of satellite cells and fibroblasts.

Altered relationship between gluconeogenesis and immunity in broilers exposed to heat stress for different durations.

This study determined the relationship between inflammation and gluconeogenesis level in broilers in different durations of heat stress. A total of 240 Ross 308 broilers were offered control and heat stress temperature from 21 to 35 d post-hatch, each experimental group had 8 replications, and each replication obtained 15 broilers. The temperature in the control (Ctrl) group and heat stress group were maintained at 24 ± 1°C and 34 ± 1°C, respectively throughout the experimental period. Based on the duration of heat stress, the heat stress group was divided into 2 subgroups, like, 7-d heat stress (28-day-old broiler) designated ST group and 14-d heat stress (35-day-old broiler) designated the LT group. The ad libitum commercial feed and fresh water were provided to all experimental broilers during the experiment. The growth performance of experimental broilers was calculated at 35 d. However, the liver and blood samples were collected from the Ctrl group in 21 d, as well as these samples were collected from the heat stress ST and LT groups in 28-d and 35-d, respectively. Obvious gene expression of immunity, gluconeogenesis, glycogenolysis, and glycogenesis, as well as glucose-6-phosphate dehydrogenase and adenosine triphosphate was determined in the liver sample. The blood glucose concentration and histopathology of the liver was also examined in the different grouped broilers. Body weight, weight gain, and feed intake significantly decreased in the 35-d heat stress group than the Ctrl group. However, the feed conversion ratio increased at the 35-d heat stress group than the Ctrl group. The amount of glucose-6-phosphate dehydrogenase was significantly higher in ST and LT groups than Ctrl, whereas the blood glucose level was downregulated in the LT group. The amount of adenosine triphosphate was significantly decreased in the LT group than the Ctrl and ST groups. Heat stress acts as an impediment to the general relation between gluconeogenesis and immunity, as well as changes cellular structure. This experiment contributed to the establishment of a relationship between gluconeogenesis and immunity, which affects the growth performance of broilers during heat stress.

Growth factors improve the proliferation of Jeju black pig muscle cells by regulating myogenic differentiation 1 and growth-related genes.

OBJECTIVE: The growth rate of pigs is related to differentiation and proliferation of muscle cells, which are regulated by growth factors and expression of growth-related genes. Thus, the objective of this study was to establish optimal culture conditions for Jeju black pig (JBP) muscle cells and determine the relationship of various factors involved in muscle growth with the proliferation of JBP muscle cells. METHODS: Muscles were taken from the femur skeletal muscle of JBP embryos. After isolation of the muscle cells, cells were cultured in a 6-well plate under four different culture conditions to optimize culture conditions for JBP muscle cells. To analyze proliferation rate of JBP muscle cells, these muscle cells were seeded into 6-well plates at a density of 1.5×105 cells per well and cultured for 3 days. Western blot and quantitative real-time polymerase chain reaction were applied to verify the myogenic differentiation 1 (MyoD) expression and growth-related gene expression in JBP muscle cells, respectively. RESULTS: We established a muscle cell line from JBP embryos and optimized its culture conditions. These muscle cells were positive for MyoD, but not for paired box 7. The proliferation rate of these muscle cells was significantly higher in a culture medium containing bFGF and epidermal growth factor + basic fibroblast growth factor (EGF+bFGF) than that without a growth factor or containing EGF alone. Treatment with EGF and bFGF significantly induced the expression of MyoD protein, an important transcription factor in muscle cells. Moreover, we checked the changes of expression of growth-related genes in JBP muscle cells by presence or absence of growth factors. Expression level of collagen type XXI alpha 1 gene was changed only when EGF and bFGF were added together to culture media for JBP muscle cells. CONCLUSION: Concurrent use of EGF and bFGF increased the expression of MyoD protein, thus regulating the proliferation of JBP muscle cells and the expression of growth-related genes.

Modulatory effect of heat stress on viability of primary cultured chicken satellite cells and expression of heat shock proteins ex vivo.

Satellite cells promote muscle repairing and muscle growth. Thereby the intention of the present study was to investigate the beneficial effects of heat stress at different time intervals on chicken satellite cells' viability. Satellite cells were isolated from 1-day-old chicks and treated at two different temperatures (37 °C and 41 °C) for various time periods (6 h, 12 h, 24 h, 48 h, and 72 h). Both temperatures significantly increased cell viability after 24 h and 48 h. After 12 h, cell viability was significantly increased at 41 °C compared to 37 °C. However, more apoptotic cells were observed at end of the experiment of 41 °C compared to 37 °C. In addition, more live cells were found at early of experimental period at 41 °C than 37 °C. Additionally, protein and mRNA expression of HSP70, HP60 and HSP47 were significantly upregulated throughout the experimental period at temperature of 41 °C compared to those at 37 °C. These results indicate that cell viability and expression of heat stress related proteins/genes are induced by high temperature of 41 °C via heat stress pathway whereas activation of heat stress related proteins/genes are lower at 37 °C. Thus, 41 °C can trigger satellite cells' viability essential for better cell survival than 37 °C at early incubation time.

Chronic heat stress regulates the relation between heat shock protein and immunity in broiler small intestine.

Chronic heat stress is considered to decrease the immune functions which makes negative effect on broiler growth performance. Here, we investigated the relationship between chronic heat stress, growth performance, and immunity in the small intestine of broilers. The study included two groups (control and heat stressed group) with eight replications per group. Ten broilers of 20-day aged were allocated in each replication. On day 35, the treatment group was subdivided into two groups based on their body weights (heavy and low body weight). Although, there was only the control and treatment group on day 28. The growth performance decreased and expression of heat shock protein 70 (HSP70), HSP60, and HSP47 increased on days 28 and 35 in the chronic heat stress group as compared with those in the control group. The expression levels of HSPs were significantly higher in the low body weight group than in the control group. The genes HSP70 and HSP60 were significantly associated with pro- and anti-inflammatory cytokines in the small intestine of the broilers of the treatment group. Thus, HSP70 and HSP60 activated the adaptive immunity in the small intestines of the broilers from the treatment group to allow adaptation to chronic heat stress environment.

Direct exposure to mild heat stress stimulates cell viability and heat shock protein expression in primary cultured broiler fibroblasts.

Fibroblasts produce collagen which is mainly essential for repairing tissue damage and maintaining the structural integrity of tissues. However, studies have given scientific evidence about harmful effect of thermal manipulation in fibroblast. Therefore, the aim of this study was to determine the mild heat stress temperature which increased broiler fibroblast viability. The experiment was divided into two groups (37 °C and 41 °C), and each group was divided into five subgroups based on different incubation times (6 h, 12 h, 24 h, 48 h, and 72 h) with three replications. In experimental group (41 °C), fibroblast viability increased significantly in 12 h but decreased in 72 h compared with control (37 °C). At 41 °C, live cell increased significantly in 24 h and then declined in 48 h as well as 72 h than control. Moreover, the S phase lengthened in shorter incubation time of experimental group compared with control. Protein and mRNA (HSP70, HSP60, and HSP47) expressions were significantly higher at 41 °C compared with 37 °C, but at the end of the experiment, HSP expression level was higher in both groups. Finally, this study recommended 41 °C as a mild heat stress temperature for increasing broiler fibroblast viability.

Acute Heat Stress Induces the Differential Expression of Heat Shock Proteins in Different Sections of the Small Intestine of Chickens Based on Exposure Duration.

In this study, we examined the protein and gene expression of heat shock proteins (HSPs) in different sections of the small intestine of chickens. In total, 300 one-day-old Ross 308 broiler chicks were randomly allocated to the control and treatment groups. The treatment group was divided into four subgroups, according to the duration of acute heat exposure (3, 6, 12, and 24 h). The influence of heat stress on the protein and gene expression of HSP70, HSP60, and HSP47 in different sections of the small intestine of chickens was determined. The protein expression of HSP70 and HSP60 was significantly higher at 6 h in the duodenum and jejunum and 12 h in the ileum. The HSP47 protein expression was significantly higher at 3 h in the duodenum and ileum and at 6 h in the jejunum. The gene expression levels of HSP70, HSP60, and HSP47 were significantly higher at the 3 h treatment group than the control group in the duodenum, jejunum, and ileum. The glutamate pyruvate transaminase and glutamate oxaloacetate transaminase levels were significantly higher at 12 and 24 h in the serum of the blood. Acute heat stress affected the expression of intestinal proteins and genes in chickens, until the induction of heat tolerance.

Effects of In Ovo Supplementation with Nanonutrition (L-Arginine Conjugated with Ag NPs) on Muscle Growth, Immune Response and Heat Shock Proteins at Different Chicken Embryonic Development Stages.

The aim of the study was to analyze the in ovo injection of inorganic and organic synthesized silver nanoparticles (Ag NPs) using Brassica oleracea L. var. capitate F. rubra (BOL) conjugation with L-Arginine (L-Arg) on the immune, muscle growth, survivability and hatchability of broiler chickens. The conjugation of L-Arg (100 µg) with 1000 µg of Ag NPs synthesized by (BOL)-extract and L-Arg (100 µg) conjugated with 100 µg of Ag NPs inorganic synthesized were injected into fertile eggs at 8 d, 14 d and 18 d of incubation. Survival and hatching rate were significantly improved in the dose of L-Arg (100 µg) with 1000 µg (BOL-Ag NPs) and L-Arg (100 µg) with 100 µg (C-Ag NPs) on 14 d injection whereas it was decreased on 8 d or 18 d injection. Moreover, the protein expression of muscle development markers such as myogenin and myoD were significantly uprelated in 14 d of incubation whereas the heat shock proteins (HSPs), such as HSP-60 and HSP-70, were significantly upregulated in 18 d incubation. In addition, the liver function marker of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were significantly decreased and the immunoglobulin (IgM) levels were increased in a 14 d incubation period in serum at the same concentration.

Effects of white, yellow, and blue colored LEDs on milk production, milk composition, and physiological responses in dairy cattle.

Light emitting diode (LED) is more energy efficient than incandescent or fluorescent light. This study was to evaluate effects of different colored LEDs on milk production, milk composition, and physiology of Holstein cow. According to milk production and parity, cows (n = 186) were allotted to four treatments: control (natural daylight), white, yellow, and blue LED groups. Of these, 40 cows that had passed 57 day-in-milk were used. Yellow and blue LED groups demonstrated greater rates of decline in milk production than control and white LED groups. At the finish point, milk fat, protein, and lactose contents were the lowest in the blue LED group, whereas milk-urea-nitrogen levels were the highest in the yellow and blue LED groups. Extended exposure to blue LED light lowered antioxidant enzyme activity and insulin-like growth factor-1 levels. Prolactin concentrations were higher in the white and blue LED groups than in the control. Cortisol level was the highest in the blue LED group among the groups. Nonesterified fatty acid levels in the yellow and blue LED groups decreased to the greatest extent compared to the start point. These results suggest that blue LED light can decrease milk production and generate more stress than white and yellow LED lights.

Heat Treatment at an Early Age Has Effects on the Resistance to Chronic Heat Stress on Broilers.

This study was conducted to investigate the effects of early heat conditioning on growth performance, liver-specific enzymes (GOT and GPT), neuro-hormones (dopamine and serotonin), stress hormones (corticosterone), and the expression of HSPs (heat shock proteins), HSFs (heat shock factors), and pro-inflammatory cytokines under chronic high temperature. Broilers were raised with commercial feed and supplied with water ad libitum under conventional temperature. We separated the broilers into three groups: the control without any heat exposure (C), chronic heat-stressed group (CH), and early and chronic heat-stressed group (HH). At 5 days of age, the HH group was exposed to high temperatures (40 °C for 24 h), while the remaining groups were raised at a standard temperature. Between days 6 and 20, all three groups were kept under optimal temperature. From 21 to 35 days, the two heat-stressed groups (CH and HH) were exposed to 35 °C. Groups exposed to high temperature (CH and HH) showed significantly lower body weight and feed intake compared to the control. GOT and GPT were lower expressed in the CH and HH groups than the control group. In addition, the protein expressions of HSPs were down-regulated by chronic heat stress (CH and HH groups). The gene expressions of HSP60 and HSF3 were significantly down-regulated in the CH and HH groups, while HSP70 and HSP27 genes were up-regulated only in the HH group compared with the control group. The expression of pro-inflammatory cytokine genes was significantly up-regulated in the HH group compared with the control and CH groups. Thus, exposure of early Heat stress (HS) to broilers may affect the inflammatory response; however, early heat exposure did not have a positive effect on chronic HS of liver enzymes and heat shock protein expression.