Effects of Pig Dietary n-6/n-3 Polyunsaturated Fatty Acids Ratio and Gender on Carcass Traits, Fatty Acid Profiles, Nutritional Indices of Lipid Depots and Oxidative Stability of Meat in Medium-Heavy Pigs.

The effects of different dietary n-6/n-3 polyunsaturated fatty acids (PUFA) ratios and gender on key carcass traits, as well as the nutritional and technological quality of lipids in medium-heavy pig tissues have been poorly studied. To investigate the subject, 24 Large White, barrows and gilts, evenly divided into two groups of 12, were fed from 80 kg of live-weight (LW) until slaughter at 150 kg LW, either a high (9.7:1) (HPR) or low (1.4:1) (LPR) dietary n-6/n-3 PUFA ratio. On individual samples of longissimus thoracis muscle (LTM), subcutaneous (SF) and perirenal (PF) adipose tissues (ATs), the fatty acid (FA) composition was determined by gas chromatography, and lipid nutritional indices (LNIs) were calculated. The oxidative stability of meat was evaluated by determining the malondialdehyde content on raw and cooked (24 h postmortem) and refrigerated (8 days postmortem) LTM samples. The carcass traits did not vary between genders and diets. The LPR group showed a higher n-3 PUFA level and a lower n-6/n-3 PUFA ratio in all the tissues examined and better LNI, especially in the ATs. Diet did not affect the oxidative stability of meat. Gender did not influence the n-6/n-3 PUFA ratio, while barrows showed improvements in some LNI in ATs. Reducing the n-6/n-3 ratio in the diet of growing-finishing medium-heavy pigs improved the FA profile in all tissues and most LNI in ATs without impairing the oxidative stability of meat.

Genetic parameters and analysis of factors affecting variations between backfat and Semimembranosus muscle fatty acid composition in heavy pigs.

This study was conceived to evaluate the variations between backfat (BF) and Semimembranosus (SM) muscle fatty acid (FA) composition in 789 heavy pigs and to estimate the genetic and phenotypic correlations and the heritability values of these variations. Most FAs showed a common genetic basis controlling their proportion in SM muscle and BF, while the n-6/n-3 ratio, α-linolenic and erucic acids displayed a genetic control more oriented towards tissue-specific molecular pathways. All variations between the FA composition of BF and SM muscle showed low-to-medium heritability values, suggesting that there are also genetic mechanisms capable of differentiating the deposition of FAs in BF from those in SM muscle. This result implies that a better knowledge of the genes differentiating the FA composition of BF and SM muscle could provide new tools allowing to select, in a partially independent manner, the FA composition of muscle and subcutaneous fat.

Describing backfat and Semimembranosus muscle fatty acid variability in heavy pigs: Analysis of non-genetic factors.

This study aimed to describe the multivariate structure of Semimembranosus muscle and backfat fatty acid (FA) composition in 798 Italian Large White heavy pigs and to investigate the effects of environmental factors and carcass characteristics on FA variations. The total FA variability in muscle and backfat was characterized by a negative correlation between saturated and polyunsaturated FAs, which strongly depended on the carcass adiposity. Slaughtering season was also relevant, with pigs slaughtered in autumn having more n-6 FAs and eicosadienoic acid in backfat, while pigs slaughtered in winter displayed more saturated FAs. Regarding Semimembranosus muscle, pigs with heavier belly cuts and slaughtered in autumn had higher proportions of cis-vaccenic and palmitoleic acids, while those slaughtered in summer had more saturated FAs. Slaughtering season emerged as a relevant factor shaping both backfat and muscle FA composition, indicating that more studies and attention should be paid to environmental factors, which may have effects on FA metabolism and deposition in finishing pigs.

Relationships between EUROP carcass grading and backfat fatty acid composition in Italian Large White heavy pigs.

The amount and quality of covering adipose tissue affect the suitability of hind legs for the production of high-quality seasoned hams. To date, no studies exist on the correlation between EUROP carcass classification and backfat fatty acid (FA) composition in heavy pigs used for dry-cured hams. A sample of 898 Italian Large White heavy pigs was used to verify the relationship between carcass classification based on lean meat percentage and backfat FA composition. A Canonical Discriminant Analysis (CDA) was used to verify the power of individual FA and FA categories in discriminating among EUROP classes. The results proved that saturated FAs (i.e. palmitic, stearic and arachidic acids) and the n-6 polyunsaturated FAs have the highest discriminating power, thus permitting to differentiate among E, U, R, O carcass classes. For the first time, this work demonstrates the relationship between EUROP pig carcass grading, which is only based on an estimate of the percentage of lean meat, and backfat FA composition.

Influence of linseed and antioxidant-rich diets in pig nutrition on lipid oxidation during cooking and in vitro digestion of pork.

Enrichment of pig diets with polyunsaturated fatty acids (PUFA) is considered an emerging strategy to increase their intake in the human diet. However, PUFA are particularly vulnerable to oxidative reactions leading to the generation of toxic compounds. The aim of this study was to evaluate the effect of supplementation of pig diets with extruded linseed (L), either or not in combination with synthetic antioxidants (E, tocopheryl-acetate and selenium) or natural extracts (P, grape-skin and oregano), and basal diet (C, without linseed) on the oxidative stability in raw, grilled and in vitro digested pork. The diet supplementation with antioxidant-rich ingredients resulted in the accumulation of specific metabolites in meat. Actually, 11 different phenolic- and 6 tocopherol-derived metabolites were identified by UHPLC/HR-MS. These metabolites were potentially correlated with the reduction in the oxidative phenomena occurring during meat cooking and digestion. Specifically, 16% and 35% reduction in the amounts of lipid hydroperoxides and TBA-RS were assessed after cooking of meat from P diet, respect to the L diet. Diet supplementations with α-tocopheryl acetate and selenium reduced the oxidative reactions only during meat cooking. A significant reduction was attended at the end of in vitro digestion, showing about 24% and 34% hydroperoxides and TBA-RS concentration reductions, respectively, in P diet samples respect to the L ones. Thus, our study suggests that the appearance of phenolic metabolites in meat could be associated to a reduction in the oxidative phenomena during meat cooking and digestion.

Genetic parameters of muscle fatty acid profile in a purebred Large White heavy pig population.

Semimembranosus muscle samples from 795 Large White heavy pigs were used to determine their intramuscular fatty acid composition and to estimate the heritability and the genetic correlations of these traits. Muscle fatty acids showed heritability estimates of low-to-moderate magnitude, ranging from 0.157 for total fatty acids to 0.237 for docosahexaenoic acid. Only small differences in heritability appeared among fatty acids based on their chain length, saturation and double bond position. Omega-6 polyunsaturated fatty acids showed positive genetic correlations with carcass lean % (0.563 ± 0.005) and loin thickness (0.438 ± 0.005) while being negatively related to backfat thickness measured both by calibre (-0.225 ± 0.008) and Fat-O-Meat'er (FOM) apparatus (-0.603 ± 0.004). Interestingly, the monounsaturated fatty acid class was not correlated with carcass measures and presented only a weak positive genetic correlation with intramuscular fat (0.145 ± 0.002). This result suggests that in heavy pig breeds monounsaturated fatty acids in muscle could be selected for without interfering with carcass traits.

Is the protein profile of pig Longissimus dorsi affected by gender and diet?

The impact of gender and diet on the proteome of Longissimus dorsi was addressed by 2D-PAGE analysis of male and female pigs, fed with a barley-based control diet and a diet enriched with extruded linseed and plant extracts. No statistically significant difference in protein number between female and male samples was found. Furthermore, PCA excluded gender-dependent protein clusters. For both the control and enriched diet, several spots exhibited at least a 1.5-fold intensity difference, but none showed a statistically relevant variation. Protein profiles PCA for both diets indicated that the first two principal components account up to 47% of total variance, with two diet-dependent separated clusters. Among 176 common spots, 29 exhibited >1.5 fold change, mostly more abundant in the control diet. PMF identified 14 distinct proteins, including myofibrillar proteins, glycolytic enzymes and myoglobin, thus suggesting a diet-dependent meat quality. A statistically significant increase in carbonylated proteins of enriched diet samples was detected using the 2,4-dinitrophenylhydrazine method but not using fluorescein-5-thiosemicarbazide-labeled bands. ROS induction and DNA oxidative damage, detected in a human cell line exposed to digested meat from both diets, further support the notion that the enriched diet does not protect against oxidative stress. SIGNIFICANCE: The comparison of the protein profile of female and male Longissimus dorsi from pigs fed by a control diet and a diet enriched with polyphenols, indicate no gender effect, whereas diet affects the abundance of several proteins, possibly linked to meat quality. Protein carbonylation was statistically higher in meat from the enriched diet, suggesting that polyphenols at the concentration present in the diet did not exert a protective effect against oxidation.

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 ± 5.6 kg to 151.8 ± 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.

Effect of diets supplemented with linseed alone or combined with vitamin E and selenium or with plant extracts, on Longissimus thoracis transcriptome in growing-finishing Italian Large White pigs.

BACKGROUND: Supplementing farm animals diet with functional ingredients may improve the nutritional quality of meat products. Diet composition has been also demonstrated to influence the gene expression with effect on biological processes and pathways. However, the knowledge on the effect of nutrients at the molecular level is scant. In particular, studies on the effects of antioxidants and polyphenols dietary supplementation have been investigated mainly in rodents, and only scarcely in farm animals so far. RNA-Seq with next-generation sequencing is increasingly the method of choice for studying changes in the transcriptome and it has been recently employed also in pig nutrigenomics studies to identify diet-induced changes in gene expression. The present study aimed to investigate the effect of diets enriched with functional ingredients (linseed, vitamin E and plant extracts) on the transcriptome of pig Longissimus thoracis to elucidate the role of these compounds in influencing genes involved in muscle physiology and metabolism compared to a standard diet. RESULTS: Eight hundred ninety-three significant differentially expressed genes (DEGs) (FDR adjusted P-value ≤ 0.05) were detected by RNA-Seq analysis in the three diet comparisons (D2-D1, D3-D1, D4-D1). The functional analysis of DEGs showed that the diet enriched with n-3 PUFA from linseed (D2) mostly downregulated genes in pathways and biological processes (BPs) related to muscle development, contraction, and glycogen metabolism compared to the standard diet. The diet supplemented with linseed and vitamin E/Selenium (D3) showed to mostly downregulate genes linked to oxidative phosphorylation. Only few genes involved in extracellular matrix (ECM) organization were upregulated by the D3. Finally, the comparison D4-D1 showed that the diet supplemented with linseed and plant extracts (D4) upregulated the majority of genes compared to D1 that were involved in a complex network of pathways and BPs all connected by hub genes. In particular, IGF2 was a hub gene connecting protein metabolism, ECM organization, immune system and lipid biosynthesis pathways. CONCLUSION: The supplementation of pig diet with n-3 PUFA from linseed, antioxidants and plant-derived polyphenols can influence the expression of a relevant number of genes in Longissimus thoracis muscle that are involved in a variety of biochemical pathways linked to muscle function and metabolism.

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 univariate 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 differences 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 supplementation 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.

Single step extraction and derivatization of intramuscular lipids for fatty acid Ultra Fast GC analysis: application on pig thigh.

Two different methods for single step transesterification from pig meat without fat extraction have been tested. Freeze-drying of the meat with and without anhydrous salt, followed by a base-catalyzed transmethylation (KOH/MeOH) was carried out. Both methods were compared with the standard Folch procedure of fat extraction followed by transmethylation. The methods were tested on a complete sample set of biceps femoris of pig thigh, used for the production of dry-cured ham. The set was divided in three subgroups according to total fat content. Both derivatization protocols on freeze-dried pork muscle were proven to be a valid alternative to the Folch procedure for FAME analysis. Freeze-drying method offered several advantages in comparison with the Folch procedure, including a lower solvent requirement, and process temperature, as well as considerable saving of time. In freeze-drying, the addition of an anhydrous salt (Na2SO4) gave more friable samples which resulted in higher yields for some fatty acids, particularly evident in the case of tissues with high lipid content.

Calculating the iodine value for Italian heavy pig subcutaneous adipose tissue from fatty acid methyl ester profiles.

In this work, different equations were compared as for their effectiveness in predicting the iodine value (IV), based on fatty acid (FA) composition of subcutaneous adipose tissue of Italian heavy pigs. In particular, six equations were tested: AOCS (1); modified AOCS (2), including all unsaturated FA (UFA); regression models obtained using the stepwise regression procedure as variable selection method, calculated considering only UFA (3) or all the FA (4); regression models obtained using the backward elimination procedure, calculated considering only UFA (5) or all the FA (6). The comparison of the equations performance, estimated using an external test set, showed that the use of regression models led to significant enhancements of prediction accuracy with respect to the AOCS equations. Using both equations 4 and 6, the average paired differences between experimental and predicted IV values were not statistically significant. Therefore, it is possible to use these equations for IV estimation of the subcutaneous adipose tissue of Italian heavy pigs.

Confirmed association between a single nucleotide polymorphism in the FTO gene and obesity-related traits in heavy pigs.

We recently showed that a polymorphism in the fat mass and obesity associated (FTO) gene (AM931150: g.276T > G) is associated with fat deposition traits in pigs. To confirm this result, we genotyped this polymorphism in an Italian Duroc population made up by 313 performance tested pigs with known estimated breeding values (EBVs) for average daily gain, back fat thickness (BFT), feed:gain ratio, lean cuts (LC), and visible intermuscular fat (VIF, a measure of intermuscular fat in the hams). In addition, we genotyped 148 commercial heavy pigs for which several fat deposition traits and lean meat percentage were measured. The results of the association analyses confirmed the effect of the FTO mutation on obesity-related traits (VIF, BFT and LC) in the Italian Duroc pigs (P < 0.01) and in the commercial pigs (intramuscular fat content of different muscles, P < 0.05 or P < 0.10; lean meat content, P < 0.05; BFT, P < 0.05; intermuscular fat content in the hams, P < 0.05).