Medical Grade Honey as a Promising Treatment to Improve Ovarian Tissue Transplantation.

Ovarian tissue cryopreservation is a female fertility preservation technique that presents major challenges for the maintenance of follicular viability after transplantation. The aim of this study was to evaluate and compare the application of L-Mesitran Soft®, a product containing 40% medical grade honey (MGH), with other strategies to improve ovarian grafts' viability. For this purpose, bovine ovarian tissue was vitrified, warmed and randomly assigned to culture groups: (1) control, (2) MGH 0.2% in vitro, (3) MGH in vivo (direct application in the xenotransplantation), (4) vascular endothelial growth factor (VEGF 50 ng/mL) and (5) vitamin D (100 Nm), during a 48 h period. A sixth group (6) of fragments was thawed on transplantation day and was not cultured. The tissue was xenotransplanted into immunodeficient (Rowett nude homozygous) ovariectomized rats. Grafts were analyzed 48 h after culture, and 7 and 28 days after transplantation. The tissue was subjected to histological and immunohistochemical analysis. Treatments using MGH showed the highest angiogenic and cell proliferation stimulation, with cellular apoptosis, within a healthy cellular turnover pathway. In conclusion, MGH should be considered as a potentially effective and less expensive strategy to improve ovarian tissue transplantation.

Post-transcriptional silencing of Bos taurus prion family genes and its impact on granulosa cell steroidogenesis.

Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a downregulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology.

Molecular basis for the preferential recognition of ß1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum.

Understanding the specific molecular interactions between proteins and ß1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for ß1,3-1,4-mixed-linked over ß1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to ß1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the ß1,3-linkage are important for recognition, and identified the tetrasaccharide Glcß1,4Glcß1,4Glcß1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of ß1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a ß1,3-linkage at the reducing end and at specific positions along the ß1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked ß-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3.

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

Novel insights into the degradation of ß-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase.

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a ß-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ±â€¯0.12 mg/ml and Vmax, 109 ±â€¯1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of ß-1,3-glucan hydrolysed products released initially, showed ß-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Šresolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 ß-strands forming a four-domain structure viz. a ß-sandwich domain I at N-terminal, an α/ß-domain II, an (α/α)6 barrel domain III, and a small 5-stranded ß-sandwich domain IV.

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Cellulosome assembly: paradigms are meant to be broken!

Cohesin-Dockerin interactions are at the core of cellulosomal assembly and organization. They are highly specific and form stable complexes, allowing cellulosomes to adopt distinct conformations. Each cellulosomal system seems to have a particular organizational strategy that can vary in complexity according to the nature of its Cohesin-Dockerin interactions. Hence, several efforts have been undertaken to reveal the mechanisms that govern the specificity, affinity and flexibility of these protein-protein interactions. Here we review the most recent studies that have focused on the structural aspects of Cohesin-Dockerin recognition. They reveal an ever-increasing number of subtle intricacies suggesting that cellulosome assembly is more complex than was initially thought.

Betaine and arginine supplementation of low protein diets improves plasma lipids but does not affect hepatic fatty acid composition and related gene expression profiling in pigs.

BACKGROUND: The individual and combined effects of betaine and arginine supplemented to reduced protein diets were investigated on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid-sensitive factors in commercial pigs. Betaine has previously been shown to reduce carcass fat deposition and arginine improves meat quality of finishing pigs. Forty male crossbred pigs were randomly assigned to one of five diets (n = 8): 160 g kg-1 of crude protein (NPD), 130 g kg-1 of crude protein (RPD), RPD with 3.3 g kg-1 of betaine, RPD with 15 g kg-1 of arginine, and RPD with 3.3 g kg-1 of betaine and 15 g kg-1 of arginine. RESULTS: The restriction of dietary protein increased total lipids (P < 0.001), total cholesterol (P < 0.001), high-density lipoprotein-cholesterol (P < 0.001) and low-density lipoprotein cholesterol (P < 0.001). Betaine and arginine, individually or combined, reduced the majority of plasma lipids (P < 0.05) without affecting total fatty acids in the liver and the overall gene expression pattern. CONCLUSION: These findings suggest a positive effect of betaine and arginine, singly or combined, by reversing plasma lipids increase promoted by dietary protein restriction. © 2017 Society of Chemical Industry.

Arginine supplementation modulates pig plasma lipids, but not hepatic fatty acids, depending on dietary protein level with or without leucine.

BACKGROUND: In the present study, the effect of arginine and leucine supplementation, and dietary protein level, were investigated in commercial crossbred pigs to clarify their individual or combined impact on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid sensitive factors. The experiment was conducted on fifty-four entire male pigs (Duroc × Pietrain × Large White × Landrace crossbred) from 59 to 92 kg of live weight. Each pig was randomly assigned to one of six experimental treatments (n = 9). The treatments followed a 2 × 3 factorial arrangement, providing two levels of arginine supplementation (0 vs. 1%) and three levels of basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet with 2% of leucine, RPDL). RESULTS: Significant interactions between arginine supplementation and protein level were observed across plasma lipids. While dietary arginine increased total lipids, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and triacylglycerols in NPD, the inverse effect was observed in RPD. Overall, dietary treatments had a minor impact on hepatic fatty acid composition. RPD increased 18:1c9 fatty acid while the combination of leucine and RPD reduced 18:0 fatty acid. Arginine supplementation increased the gene expression of FABP1, which contributes for triacylglycerols synthesis without affecting hepatic fatty acids content. RPD, with or without leucine addition, upregulated the lipogenic gene CEBPA but downregulated the fat oxidation gene LPIN1. CONCLUSIONS: Arginine supplementation was responsible for a modulated effect on plasma lipids, which is dependent on dietary protein level. It consistently increased lipaemia in NPD, while reducing the correspondent metabolites in RPD. In contrast, arginine had no major impact, neither on hepatic fatty acids content nor on fatty acid composition. Likewise, leucine supplementation of RPD, regardless the presence of arginine, promoted no changes on total fatty acids in the liver. Ultimately, arginine, leucine and dietary protein reduction seem to be unrelated with fatty liver development.

Correction: L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy.

[This corrects the article DOI: 10.1371/journal.pone.0106974.].

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Diverse specificity of cellulosome attachment to the bacterial cell surface.

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted ß-glucans, ß-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize ß-glucans and ß-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Restriction of dietary protein does not promote hepatic lipogenesis in lean or fatty pigs.

The influence of genotype (lean v. fatty) and dietary protein level (normal v. reduced) on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid-sensitive factors is reported for the first time, using the pig as an experimental model. The experiment was conducted on forty entire male pigs (twenty lean pigs of Large White×Landrace×Pietrain cross-breed and twenty fatty pigs of Alentejana purebreed) from 60 to 93 kg of live weight. Each pig genotype was divided into two subgroups, which were fed the following diets: a normal protein diet (NPD) equilibrated for lysine (17·5 % crude protein and 0·7 % lysine) and a reduced protein diet (RPD) not equilibrated for lysine (13·1 % crude protein and 0·4 % lysine). The majority of plasma metabolites were affected by genotype, with lean pigs having higher contents of lipids, whereas fatty pigs presented higher insulin, leptin and urea levels. RPD increased plasma TAG, free fatty acids and VLDL-cholesterol compared with NPD. Hepatic total lipids were higher in fatty pigs than in the lean genotype. RPD affected hepatic fatty acid composition but had a slight influence on gene expression levels in the liver. Sterol regulatory element-binding factor 1 was down-regulated by RPD, and fatty acid desaturase 1 (FADS1) and fatty acid binding protein 4 (FABP4) were affected by the interaction between genotype and diet. In pigs fed RPD, FADS1 was up-regulated in the lean genotype, whereas FABP4 increased in the fatty genotype. Although there is a genotype-specific effect of dietary protein restriction on hepatic lipid metabolism, lipogenesis is not promoted in the liver of lean or fatty pigs.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

Purification and crystallographic studies of a putative carbohydrate-binding module from the Ruminococcus flavefaciens FD-1 endoglucanase Cel5A.

Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM-Rf2) and a C-terminal dockerin. This novel CBM-Rf2 has been purified and crystallized, and data from cacodylate-derivative crystals were processed to 1.02 and 1.29 Šresolution. The crystals belonged to the orthorhombic space group P212121. The CBM-Rf2 structure was solved by a single-wavelength anomalous dispersion experiment at the As edge.

Adipocyte membrane glycerol permeability is involved in the anti-adipogenic effect of conjugated linoleic acid.

Conjugated linoleic acid (CLA), a group of minor fatty acids from ruminant origin, has long been recognized as a body fat lowering agent. Given the trans(t)10,cis(c)12-CLA well documented interference on lipolysis, we hypothesized for adipocytes altered permeation to glycerol when supplemented with this isomer. 3T3-L1 murine differentiated adipocytes were medium supplemented with linoleic acid (LA) and individual or combined c9,t11 and t10,c12-CLA isomers. Adipocytes treated with the t10,c12-CLA isomer and CLA mixture showed reduced triacylglycerols content (p < 0.001), re-enforcing the t10,c12-CLA as the anti-adipogenic CLA isomer. This finding was supported by decreased Δ9-desaturase index and adipocyte differentiation markers for the t10,c12-CLA group (p < 0.001), which suggest reduced lipogenesis and differentiation, respectively. The glycerol permeability was higher in all CLA treated cells compared to control and LA groups (p < 0.05). The increase in glycerol permeability agrees with both reduced triacylglycerols and non-osmotic cellular volume in the t10,c12-CLA and CLA mixture groups. Taken together, our data suggest that the increased adipocyte plasma membrane glycerol fluxes may be part of the anti-adipogenic response to CLA treatments.

L-ferritin binding to scara5: a new iron traffic pathway potentially implicated in retinopathy.

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.

Is hepatic lipid metabolism of beef cattle influenced by breed and dietary silage level?

BACKGROUND: In ruminants, unsaturated dietary fatty acids are biohydrogenated in the rumen and are further metabolised in various tissues, including liver, which has an important role in lipid and lipoprotein metabolism. Therefore, manipulation of muscle fatty acid composition should take into account liver metabolism. In the present study, the influence of breed and diet on liver lipid composition and gene expression was investigated in order to clarify the role of this organ in the lipid metabolism of ruminants. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã, were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Liver fatty acid composition, mRNA levels of enzymes and transcription factors involved in lipid metabolism, as well as the plasma lipid profile, were assessed. RESULTS: In spite of similar plasma non-esterified fatty acids levels, liver triacylglycerols content was higher in Barrosã than in Alentejana bulls. Moreover, the fatty acid composition of liver was clearly distinct from the remaining tissues involved in fatty acid metabolism of ruminants, as shown by Principal Components Analysis. The hepatic tissue is particularly rich in α-linolenic acid and their products of desaturation and elongation. Results indicate that DGAT1, ELOVL2, FADS1 and FADS2 genes influence the fatty acid composition of the liver the most. Moreover, genes such as DGAT1 and ELOVL2 appear to be more sensitive to genetic background than to dietary manipulation, whereas genes encoding for desaturases, such as FADS1, appear to be modulated by dietary silage level. CONCLUSIONS: Our results indicate that liver plays an important role in the biosynthesis of n-3 LC-PUFA. It is also suggested that dietary silage level influences the hepatic fatty acid metabolism in a breed-dependent manner, through changes in the expression of genes encoding for enzymes associated with the desaturation and elongation pathway. The importance of devising custom-made feeding strategies taking into account the genetic background is, therefore, stressed by the results from this experiment.

Combined effects of dietary arginine, leucine and protein levels on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of crossbred pigs.

The cumulative effects of dietary arginine, leucine and protein levels on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig longissimus lumborum muscle and subcutaneous adipose tissue (SAT) were investigated. The experiment was performed on fifty-four intact male pigs (Duroc × Pietrain × Large White × Landrace crossbred), with a live weight ranging from 59 to 92 kg. The pigs were randomly assigned to one of six experimental treatments (n 9). The treatments followed a 2 × 3 factorial arrangement, with two levels of arginine supplementation (0 v. 1 %) and three levels of a basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet to achieve 2 % of leucine, RPDL). The results showed that dietary arginine supplementation did not affect the intramuscular fat (IMF) content and back fat thickness, but increased the total fat in SAT. This effect was associated with an increase in fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) mRNA levels in SAT, which suggests that arginine might be involved in the differential regulation of some key lipogenic genes in pig muscle and SAT. The increase in IMF content under the RPD, with or without leucine supplementation, was accompanied by increased FASN and SCD mRNA levels. Arginine supplementation did not influence the percentage of main fatty acids, while the RPD had a significant effect on fatty acid composition in both tissues. Leucine supplementation of RPD did not change IMF, total fat of SAT and back fat thickness, but increased 16 : 0 and 18 : 1cis-9 and decreased 18 : 2n-6 in muscle.