Affinity chromatography of ovine casein

Abstract Sheep milk casein was separated into two fractions: one containing α s1 -plus β- and the other α s2 plus κ-caseins by affinity chromatography on activated thiol-Sepharose 4B. Milk samples were from the Leccese breed with the most common electrophoretic pattern. Electrophoresis of the chromatographic fractions on SDS-PAGE and on starch urea gel at pH 8.6 and 1.7 clarified the electrophoretic pattern of whole casein. Acidic pH electrophoresis of the two fractions obtained by affinity chromatography may be useful for investigations on the polymorphism of the casein fractions

The Effects of Milk Protein Polymorphisms on Milk Components and Cheese-Producing Ability

Abstract A Total of 2005 first lactation Holstein-Friesian cows with known 305-d lactation yield for milk, fat, and protein were available. For each cow, genotypes for α s1 -casein, β-casein, κ-casein, and β-lactoglobulin were known. It appears that the milk protein variants α s1 -casein, β-casein, and κ-casein may not be segregating independently. Effects of genetic variants of milk proteins on estimated individual Parmesan cheese yields were investigated. The relationships of the genetic variants of milk proteins to total lactation milk yield, fat yield, protein yield, fat percentage, and protein percentage were also investigated. Least squares analysis of the data indicated that α s1 -casein genotype significantly influenced milk yield, fat yield, and protein yield with the highest yields obtained for the genotype BB . Cheese yield on a fixed amount of milk and fat percentage were significantly related to β-lactoglobulin genotype with the highest estimates obtained for BB . Protein percentage was influenced by α s1 -casein and κ-casein, with the genotypes BC and BB , respectively, having the highest percentages. Significantly higher lactation cheese yields were estimated with α s1 -casein genotype BB . Using the prediction equation to estimate cheese yield (on data from another study), it was found that differences in Parmesan cheese yield from milk of either κ-casein genotype AA or BB were greater than expected based on composition. Differences in salted curd yield from another study using milk of either β-lactoglobulin genotype AA or BB were also greater than expected

Molecular Pathways and Key Genes Associated With Breast Width and Protein Content in White Striping and Wooden Breast Chicken Pectoral Muscle

Growth-related abnormalities affecting modern chickens, known as White Striping (WS) and Wooden Breast (WB), have been deeply investigated in the last decade. Nevertheless, their precise etiology remains unclear. The present study aimed at providing new insights into the molecular mechanisms involved in their onset by identifying clusters of co-expressed genes (i.e., modules) and key loci associated with phenotypes highly related to the occurrence of these muscular disorders. The data obtained by a Weighted Gene Co-expression Network Analysis (WGCNA) were investigated to identify hub genes associated with the parameters breast width (W) and total crude protein content (PC) of Pectoralis major muscles (PM) previously harvested from 12 fast-growing broilers (6 normal vs. 6 affected by WS/WB). W and PC can be considered markers of the high breast yield of modern broilers and the impaired composition of abnormal fillets, respectively. Among the identified modules, the turquoise (r = -0.90, p < 0.0001) and yellow2 (r = 0.91, p < 0.0001) were those most significantly related to PC and W, and therefore respectively named “protein content” and “width” modules. Functional analysis of the width module evidenced genes involved in the ubiquitin-mediated proteolysis and inflammatory response. GTPase activator activity, PI3K-Akt signaling pathway, collagen catabolic process, and blood vessel development have been detected among the most significant functional categories of the protein content module. The most interconnected hub genes detected for the width module encode for proteins implicated in the adaptive responses to oxidative stress (i.e., THRAP3 and PRPF40A), and a member of the inhibitor of apoptosis family (i.e., BIRC2) involved in contrasting apoptotic events related to the endoplasmic reticulum (ER)-stress. The protein content module showed hub genes coding for different types of collagens (such as COL6A3 and COL5A2), along with MMP2 and SPARC, which are implicated inCollagen type IV catabolism and biosynthesis. Taken together, the present findings suggested that an ER stress condition may underly the inflammatory responses and apoptotic events taking place within affected PM muscles. Moreover, these results support the hypothesis of a role of the Collagen type IV in the cascade of events leading to the occurrence of WS/WB and identify novel actors probably involved in their onset

Detection of differentially expressed genes in broiler pectoralis major muscle affected by White Striping – Wooden Breast myopathies

Abstract White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broiler fillets present an impaired visual appearance that negatively affects consumers' acceptability. Previous studies have highlighted in affected fillets a severely damaged muscle, showing profound inflammation, fibrosis, and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation, and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further defining the transcription patterns associated with these myopathies

Functional analysis finds differences on the muscle transcriptome of pigs fed an n-3 PUFA-enriched diet with or without antioxidant supplementations

Supplementing pig diets with n-3 polyunsaturated fatty acids (n-3 PUFA) may produce meat products with an increased n-3 fatty acid content, and the combined antioxidants addition could prevent lipid oxidation in the feed. However, to date, the effects of these bioactive compounds at the molecular level in porcine skeletal muscle are mostly unknown. This study aimed to analyse changes in the Longissimus thoracis transcriptome of 35 pigs fed three diets supplemented with: linseed (L); linseed, vitamin E and Selenium (LES) or linseed and plant-derived polyphenols (LPE). Pigs were reared from 80.8 \ub1 5.6 kg to 151.8 \ub1 9.9 kg. After slaughter, RNA-Seq was performed and 1182 differentially expressed genes (DEGs) were submitted to functional analysis. The L vs LES comparison did not show differences, while L vs LPE showed 1102 DEGs and LES vs LPE 80 DEGs. LPE compared to the other groups showed the highest number of up-regulated genes involved in preserving muscle metabolism and structure. Results enlighten that the combined supplementation of bioactive lipids (n-3 PUFA from linseed) with plant extracts as a source of polyphenols increases, compared to the only addition of linseed, the expression of genes involved in mRNA metabolic processes and transcriptional regulation, glucose uptake and, finally, in supporting muscle development and physiology. These results improve the knowledge of the biological effect of bioactive compounds in Longissimus thoracis muscle, and sustain the growing interest over their use in pig production

A molecular insight into the lipid changes of pig Longissimus thoracis muscle following dietary supplementation with functional ingredients

: In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning

Effect of dietary polyunsaturated fatty acid and antioxidant supplementation on the transcriptional level of genes involved in lipid and energy metabolism in swine

Porcine fat traits depend mostly on the interaction between nutritional and genetic factors. However, the pathways and biological processes influenced by this interaction are still poorly known in pigs, although they can have a huge impact on meat quality traits. The present research provides new knowledge insight into the effect of four diets (D1 = standard diet; D2 = linseed supplementation; D3 = linseed, vitamin E and selenium supplementation; D4 = linseed and plant-derived polyphenols supplementation) on the expression of 24 candidate genes selected for their role in lipid and energy metabolism. The data indicated that 10 out of 24 genes were differentially expressed among diets, namely ACACA, ADIPOQ, ADIPOR1, CHREBP (MLXPL), ELOVL6, FASN, G6PD, PLIN2, RXRA and SCD. Results from the univar- iate analysis displayed an increased expression of ACACA, ADIPOQ, ADIPOR1, CHREBP, ELOVL6, FASN, PLIN2, RXRA and SCD in D4 compared to D2. Similarly, ACACA, ADIPOQ, ADIPOR1, ELOVL6 and SCD were highly expressed in D4 compared to D3, while no differ- ences were observed in D2-D3 comparison. Moreover, an increased expression of G6PD and ELOVL6 genes in D4 compared to D1 was observed. Results from the multivariate analysis confirmed that D2 was not different from D3 and that ACACA, SCD and FASN expression made D4 different from D2 and D3. Comparing D4 and D1, the expression levels of ELOVL6 and ACACA were the most influenced. This research provides evidence that the addition of both n-3 PUFA and polyphenols, derived from linseed, grape-skin and oregano supplementa- tion in the diets, stimulates the expression of genes involved in lipogenesis and in oxidative processes. Results evidenced a greater effect on gene expression of the diet added with both plant extracts and n-3 PUFA, resulting in an increased expression of genes coding for fatty acid synthesis, desaturation and elongation in pig Longissimus thoracis muscle

The evolution of vimentin and desmin in Pectoralis major muscles of broiler chickens supports their essential role in muscle regeneration

Vimentin (VIM) and desmin (DES) are muscle-specific proteins having crucial roles in maintaining the lateral organization and alignment of the sarcomeric structure during myofibrils’ regeneration. The present experiment was designed to ascertain the evolution of VIM and DES in Pectoralis major muscles (PM) of fast-growing (FG) and medium-growing (MG) meat-type chickens both at the protein and gene levels. MG broilers were considered as a control group whereas the evolution of VIM and DES over the growth period was evaluated in FG by collecting samples at different developmental stages (7, 14, 21, 28, 35, and 42 days). After performing a preliminary classification of the samples based on their histological features, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. Overall, the findings obtained at the protein level mirrored those related to their encoding genes, although a potential time lag required to observe the consequences of gene expression was evident. The two- and 3-fold higher level of the VIM-based heterodimer observed in FG at d 21 and d 28 in comparison with MG of the same age might be ascribed to the beginning and progressive development of the regenerative processes. This hypothesis is supported by IHC highlighting the presence of fibers to coexpressing VIM and DES. In addition, gene expression analyses suggested that, unlike VIM common sequence, VIM long isoform may not be directly implicated in muscle regeneration. As for DES content, the fluctuating trends observed for both the native protein and its heterodimer in FG might be ascribed to its importance for maintaining the structural organization of the regenerating fibers. Furthermore, the higher expression level of the DES gene in FG in comparison with MG further supported its potential application as a marker of muscle fibers’ regeneration. In conclusion, the findings of the present research seem to support the existence of a relationship between the occurrence of muscle regeneration and the growth rate of meat-type chickens and corroborate the potential use of VIM and DES as molecular markers of these cellular processes

Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age