Monitoring of Chicken RNA Integrity as a Function of Prolonged Postmortem Duration

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect A260/A280 and A260/A230 (p≥0.05), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples (p≥0.05), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing

Thermal impacts on transcriptome of Pectoralis major muscle collected from commercial broilers, Thai native chickens and its crossbreeds

Objective The main objective of this study was to define molecular mechanisms associated with thermal stress responses of chickens from commercial broilers (BR, Ross 308), Thai native chickens (NT) and crossbreeds between BR×NT (H75). Methods Twenty days before reaching specific market age, chickens from each breed were divided into control and thermal-stressed groups. The stressed groups were exposed to a cyclic thermal challenge (35°C±1°C for 6 h, followed by 26°C±1°C for 18 h) for 20 days. Control group was raised under a constant temperature of 26°C±1°C. Pectoralis major (n = 4) from each group was collected for transcriptome analysis using HiSeq Illumina and analysis of glycogen and lactate. Gene expression patterns between control and thermal-stressed groups were compared within the same breeds. Results Differentially expressed transcripts of 65, 59, and 246 transcripts for BR, NT, and H75, respectively, were revealed by RNA-Seq and recognized by Kyoto encyclopedia of genes and genomes database. Pathway analysis underlined altered glucose homeostasis and protein metabolisms in all breeds. The signals centered around phosphatidylinositol 3-kinase (PI3K)/Akt signaling, focal adhesion, and MAPK signaling in all breeds with slight differences in molecular signal transduction patterns among the breeds. An extensive apoptosis was underlined for BR. Roles of AMPK, MAPK signaling and regulation of actin cytoskeleton in adaptive response were suggested for H75 and NT chickens. Lower glycogen content was observed in the breast muscles of BR and NT (p<0.01) compared to their control counterparts. Only BR muscle exhibited increased lactate (p<0.01) upon exposure to the stress. Conclusion The results provided a better comprehension regarding the associated biological pathways in response to the cyclic thermal stress in each breed and in chickens with different growth rates

Insights Into Transcriptome Profiles Associated With Wooden Breast Myopathy in Broilers Slaughtered at the Age of 6 or 7 Weeks

open9siThis research was financially supported by Cluster and Program Management, National Science and Technology Development Agency (Thailand; project number P15-50668), and by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (Thailand; P20-50946 and P21-50165).Transcriptomes associated with wooden breast (WB) were characterized in broilers at two different market ages. Breasts (Pectoralis major) were collected, 20-min postmortem, from male Ross 308 broilers slaughtered at 6 and 7 weeks of age. The breasts were classified as “non-WB” or “WB” based on palpation hardness scoring (non-WB = no abnormal hardness, WB = consistently hardened). Total RNA was isolated from 16 samples (n = 3 for 6 week non-WB, n = 3 for 6 week WB; n = 5 for 7 week non-WB, n = 5 for 7 week WB). Transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique, and compared between non-WB and WB samples of the same age. Among 6 week broilers, 910 transcripts were differentially expressed (DE) (false discovery rate, FDR &lt; 0.05). Pathway analysis underlined metabolisms of glucose and lipids along with gap junctions, tight junction, and focal adhesion (FA) signaling as the top enriched pathways. For the 7 week broilers, 1,195 transcripts were identified (FDR &lt; 0.05) with regulation of actin cytoskeleton, mitogen-activated protein kinase (MAPK) signaling, protein processing in endoplasmic reticulum and FA signaling highlighted as the enriched affected pathways. Absolute transcript levels of eight genes (actinin-1 – ACTN1, integrin-linked kinase – ILK, integrin subunit alpha 8 – ITGA8, integrin subunit beta 5 – ITGB5, protein tyrosine kinase 2 – PTK2, paxillin – PXN, talin 1 – TLN1, and vinculin – VCL) of FA signaling pathway were further elucidated using a droplet digital polymerase chain reaction. The results indicated that, in 6 week broilers, ITGA8 abundance in WB was greater than that of non-WB samples (p &lt; 0.05). Concerning 7 week broilers, greater absolute levels of ACTN1, ILK, ITGA8, and TLN1, accompanied with a reduced ITGB5 were found in WB compared with non-WB (p &lt; 0.05). Transcriptional modification of FA signaling underlined the potential of disrupted cell-cell communication that may incite aberrant molecular events in association with development of WB myopathy.openMalila, Yuwares; Uengwetwanit, Tanaporn; Thanatsang, Krittaporn V.; Arayamethakorn, Sopacha; Srimarut, Yanee; Petracci, Massimiliano; Soglia, Francesca; Rungrassamee, Wanilada; Visessanguan, WonnopMalila, Yuwares; Uengwetwanit, Tanaporn; Thanatsang, Krittaporn V.; Arayamethakorn, Sopacha; Srimarut, Yanee; Petracci, Massimiliano; Soglia, Francesca; Rungrassamee, Wanilada; Visessanguan, Wonno

Transcriptional Profiles of Skeletal Muscle Associated With Increasing Severity of White Striping in Commercial Broilers

Development of the white striping (WS) abnormality adversely impacts overall quality of broiler breast meat. Its etiology remains unclear. This study aimed at exploring transcriptional profiles of broiler skeletal muscles exhibiting different WS severity to elucidate molecular mechanisms underlying the development and progression of WS. Total RNA was isolated from pectoralis major of male 7-week-old Ross 308 broilers. The samples were classified as mild (n = 6), moderate (n = 6), or severe (n = 4), based on number and thickness of the white striations on the meat surface. The transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique. Gene expression patterns of each WS severity level were compared against each other; hence, there were three comparisons: moderate vs. mild (C1), severe vs. moderate (C2), and severe vs. mild (C3). Differentially expressed genes (DEGs) were identified using the combined criteria of false discovery rate 64 0.05 and absolute fold change 651.2. Differential expression of 91, 136, and 294 transcripts were identified in C1, C2, and C3, respectively. There were no DEGs in common among the three comparisons. Based on pathway analysis, the enriched pathways of C1 were related with impaired homeostasis of macronutrients and small biochemical molecules with disrupted Ca2+-related pathways. Decreased abundance of the period circadian regulator suggested the shifted circadian phase when moderate WS developed. The enriched pathways uniquely obtained in C2 were RNA degradation, Ras signaling, cellular senescence, axon guidance, and salivary secretion. The DEGs identified in those pathways might play crucial roles in regulating cellular ion balances and cell-cycle arrest. In C3, the pathways responsible for phosphatidylinositol 3-kinase-Akt signaling, p53 activation, apoptosis, and hypoxia-induced processes were modified. Additionally, pathways associated with a variety of diseases with the DEGs involved in regulation of [Ca2+], collagen formation, microtubule-based motor, and immune response were identified. Eight pathways were common to all three comparisons (i.e., calcium signaling, Ras-associated protein 1 signaling, ubiquitin-mediated proteolysis, vascular smooth muscle contraction, oxytocin signaling, and pathway in cancer). The current findings support the role of intracellular ion imbalance, particularly Ca2+, oxidative stress, and impaired programmed cell death on WS progression

Monitoring of white striping and wooden breast cases and impacts on quality of breast meat collected from commercial broilers ()

Objective This study aimed at investigating white striping (WS) and wooden breast (WB) cases in breast meat collected from commercial broilers. Methods A total of 183 breast samples were collected from male Ross 308 broilers slaughtered at the age of 6 weeks (n = 100) and 7 weeks (n = 83). The breasts were subjected to meat defect inspection, meat quality determination and histology evaluation. Results Of 183, 4 breasts from 6-week-old broilers were classified as non-defective while the others exhibited the WS lesion. Among the 6-week-old birds, the defective samples from the medium size birds (carcass weight ≤2.5 kg) showed mild to moderate WS degree with no altered meat quality. Some of the breasts from the 6-week-old birds with carcass weight above 2.5 kg exhibited WB in accompanied with the WS condition. Besides of a reduction of protein content, increases in collagen matter and pH values in the defective samples (p<0.05), no other impaired quality indices were detected within this group. All 7-week-old broilers yielded carcasses weighing above 2.5 kg and showed abnormal characteristics with progressive severity. The breasts affected with severe WS and WB showed the greatest cook loss, hardness, springiness and chewiness (p<0.05). Development of WB induced significantly increased drip loss in the samples (p<0.05). Histology indicated necrotic events in the defective myofibers. Based on logistic regression, increasing percent breast weight by one unit enhanced the chance of WS and WB development with advanced severity by 50.9% and 61.0%, respectively. Delayed slaughter age from 6 to 7 weeks increased the likelihood of obtaining increased WS severity by 56.3%. Conclusion Cases of WS and WB defects in Southeast Asia have been revealed. Despite few cases of the severe WS and WB, such abnormal conditions significantly impaired technological properties and nutritional quality of broiler breasts

Development of chitin nanofiber coatings for prolonging shelf life and inhibiting bacterial growth on fresh cucumbers

Abstract The widespread usage of petroleum-based polymers as single-use packaging has had harmful effects on the environment. Herein, we developed sustainable chitin nanofiber (ChNF) coatings that prolong the shelf life of fresh cucumbers and delay the growth of pathogenic bacteria on their surfaces. ChNFs with varying degrees of acetylation were successfully prepared via deacetylation using NaOH with treatment times of 0–480 min and defibrillated using mechanical blending. With longer deacetylation reaction times, more acetamido groups (–NHCOCH3) in chitin molecules were converted to amino groups (–NH2), which imparted antibacterial properties to the ChNFs. The ChNF morphologies were affected by deacetylation reaction time. ChNFs deacetylated for 240 min had an average width of 9.0 nm and lengths of up to several μm, whereas rod-like structured ChNFs with a mean width of 7.3 nm and an average length of 222.3 nm were obtained with the reaction time of 480 min. Furthermore, we demonstrated a standalone ChNF coating to extend the shelf life of cucumbers. In comparison to the rod-like structured ChNFs, the 120 and 240-min deacetylated ChNFs exhibited a fibril-like structure, which considerably retarded the moisture loss of cucumbers and the growth rate of bacteria on their outer surfaces during storage. Cucumbers coated with these 120 and 240-min deacetylated ChNFs demonstrated a lower weight loss rate of ⁓ 3.9% day−1 compared to the uncoated cucumbers, which exhibited a weight loss rate of 4.6% day−1. This protective effect provided by these renewable ChNFs holds promising potential to reduce food waste and the use of petroleum-based packaging materials

Absolute expressions of hypoxia-inducible factor-1 alpha (HIF1A) transcript and the associated genes in chicken skeletal muscle with white striping and wooden breast myopathies.

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as "non-defective" (n = 4), "medium-WS" (n = 6), "heavy-WS" (n = 7) and "heavy-WS+WB" (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm2. Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated

Isolation and characterization of mannanase-producing bacteria for potential synbiotic application in shrimp farming

SIMPLE SUMMARY: Mannooligosaccharides (MOS) can promote growth and immunity in aquatic animals. Although most commercial MOS are derived from yeast, the use of MOS from copra meal, a waste product of the coconut industry, is of interest. MOS from copra meal, when given as a dietary supplement to shrimp, could help improve resistance to pathogenic Vibrio and could also act as an immunostimulant. Our objective was to identify bacterial isolates that could be used as probiotics for shrimp and together with MOS as synbiotics to synergistically improve shrimp performance and feed utilization. In this study, two bacterial candidates, Man26 and Man122, were isolated from shrimp intestines and screened for mannanase, the enzyme for mannan digestion. The crude enzymes were evaluated for their biological properties and potential application in the digestion of feedstuffs in vitro. Both strains were able to produce and secrete mannanase to digest MOS and other mannan-rich materials and could tolerate a wide pH range. The addition of crude enzymes significantly increased the reducing sugars of copra meal, palm kernel cake, and soybean meal (p 70% relative activity). The thermostability range of Man26 was 20–40 °C for 20 min (>50%), while that of Man122 was 20–60 °C for 30 min (>50%). The V(max) of Man122 against locust bean gum substrate was 41.15 ± 12.33 U·mg(−1), six times higher than that of Man26. The K(m) of Man26 and Man122 were 18.92 ± 4.36 mg·mL(−1) and 34.53 ± 14.46 mg·mL(−1), respectively. With the addition of crude enzymes, reducing sugars of copra meal, palm kernel cake, and soybean meal were significantly increased (p < 0.05), as well as protein release. The results suggest that Man26 and Man122 could potentially be used in animal feeds and synbiotically with copra meal to improve absorption and utilization of feedstuffs

Lysine Depletion during Different Feeding Phases: Effects on Growth Performances and Meat Quality of Broiler Chickens

The present study aimed at assessing the impact of lysine restriction performed during different feeding phases on growth performances, meat quality traits and technological properties as well as on the incidence and severity of breast muscle abnormalities. For this purpose, a total of 945 one-day-old Ross 308 male chicks was randomly divided into three experimental groups: CONT, fed a four feeding phases commercial diet, GRW I, and GRW I + II fed CONT diet with the depletion of synthetic lysine during grower I and grower I and II feeding phases, respectively. Productive performances were recorded throughout the whole rearing cycle and the incidence of breast muscle growth-related abnormalities assessed at slaughter (49 d) on 280 breasts/group. Quality traits and technological properties of breast meat were measured on a total of 54 Pectoralis major muscles. Lysine restriction only marginally affected the productive performances and the quality parameters of breast meat. The increased (p &lt; 0.05) solubility of the protein fraction along with the remarkably higher (p &lt; 0.05) anserine content found in GRW I + II suggests an increased energy requirement in the pectoral muscles belonging to lysine-restricted birds and supports the hypothesis of a reduced protein synthesis taking place within these muscles

Monitoring of white striping and wooden breast cases and impacts on quality of breast meat collected from commercial broilers (Gallus gallus)

ObjectiveThis study aimed at investigating white striping (WS) and wooden breast (WB) cases in breast meat collected from commercial broilers.MethodsA total of 183 breast samples were collected from male Ross 308 broilers slaughtered at the age of 6 weeks (n = 100) and 7 weeks (n = 83). The breasts were subjected to meat defect inspection, meat quality determination and histology evaluation.ResultsOf 183, 4 breasts from 6-week-old broilers were classified as non-defective while the others exhibited the WS lesion. Among the 6-week-old birds, the defective samples from the medium size birds (carcass weight ≤2.5 kg) showed mild to moderate WS degree with no altered meat quality. Some of the breasts from the 6-week-old birds with carcass weight above 2.5 kg exhibited WB in accompanied with the WS condition. Besides of a reduction of protein content, increases in collagen matter and pH values in the defective samples (p&lt;0.05), no other impaired quality indices were detected within this group. All 7-week-old broilers yielded carcasses weighing above 2.5 kg and showed abnormal characteristics with progressive severity. The breasts affected with severe WS and WB showed the greatest cook loss, hardness, springiness and chewiness (p&lt;0.05). Development of WB induced significantly increased drip loss in the samples (p&lt;0.05). Histology indicated necrotic events in the defective myofibers. Based on logistic regression, increasing percent breast weight by one unit enhanced the chance of WS and WB development with advanced severity by 50.9% and 61.0%, respectively. Delayed slaughter age from 6 to 7 weeks increased the likelihood of obtaining increased WS severity by 56.3%.ConclusionCases of WS and WB defects in Southeast Asia have been revealed. Despite few cases of the severe WS and WB, such abnormal conditions significantly impaired technological properties and nutritional quality of broiler breasts