Maternal Supplementation with Polyphenols and Omega-3 Fatty Acids during Pregnancy: Prenatal Effects on Fetal Fatty Acid Composition in the Iberian Pig.

Intrauterine Growth Restriction (IUGR) is a major problem in pig production and different strategies, mainly maternal supplementation with different agents, are currently being studied. The combination of hydroxytyrosol and n3-PUFA seems to be a promising treatment to counteract IUGR, since the combination may help improve n3-PUFA composition and lower the inflammatory status of IUGR piglets. The aim of the present study is to determine the effects of a maternal supplementation, from day 35 to day 100 of pregnancy, with linseed oil and hydroxytyrosol on the fetal FA composition. The results showed higher n3 levels, including eicosapentaenoic and docosahexaenoic FA in the offspring from treated gilts, which showed lower n6-PUFA/n3-PUFA (n6/n3) ratios. Saturated and monounsaturated fatty acids were also affected by treatment, especially in the muscle and brain. Thus, a maternal supplementation with linseed oil and hydroxytyrosol affected the fetal FA tissue composition, which could have implications in pig production due to the improvement of the piglets' health status.

Hypothalamic transcriptome analysis reveals male-specific differences in molecular pathways related to oxidative phosphorylation between Iberian pig genotypes.

The hypothalamus is implicated in controlling feeding and adiposity, besides many other physiological functions, and thus can be of great importance in explaining productive differences between lean and fatty pig breeds. The present study aimed to evaluate the hypothalamic transcriptome of pure Iberian (IBxIB) and Large White x Iberian crossbreds (IBxLW) at 60 days-old, produced in a single maternal environment. Results showed the implication of gender and genotype in the hypothalamic transcriptome, with 51 differentially expressed genes (DEGs) between genotypes and 10 DEGs between genders. Fourteen genotype by sex interactions were found, due to a higher genotype effect on transcriptome found in males. In fact, just 31 DEGs were identified when using only females but 158 using only males. A higher expression of genes related to mitochondrial activity in IBxIB male animals (ND3, ND4, ND5, UQCRC2 and ATP6) was found, which was related to a higher oxidative phosphorylation and greater reactive oxygen species and nitric oxide production. IBxLW male animals showed higher expression of SIRT3 regulator, also related to mitochondrial function. When females were analysed, such differences were not found, since only some differences in genes related to the tricarboxylic acid cycle. Thus, the results indicate a significant effect and interaction of the breed and the sex on the hypothalamic transcriptome at this early age.

Changes in Biceps femoris Transcriptome along Growth in Iberian Pigs Fed Different Energy Sources and Comparative Analysis with Duroc Breed.

This experiment was conducted to investigate the effects of developmental stage, breed, and diet energy source on the genome-wide expression, meat quality traits, and tissue composition of biceps femoris muscle in growing pure Iberian and Duroc pigs. The study comprised 59 Iberian (IB) and 19 Duroc (DU) animals, who started the treatment at an average live weight (LW) of 19.9 kg. The animals were kept under identical management conditions and fed two diets with different energy sources (6% high oleic sunflower oil or carbohydrates). Twenty-nine IB animals were slaughtered after seven days of treatment at an average LW of 24.1 kg, and 30 IB animals plus all the DU animals were slaughtered after 47 days at an average LW of 50.7 kg. The main factors affecting the muscle transcriptome were age, with 1832 differentially expressed genes (DEGs), and breed (1055 DEGs), while the effect of diet on the transcriptome was very small. The results indicated transcriptome changes along time in Iberian animals, being especially related to growth and tissue development, extracellular matrix (ECM) composition, and cytoskeleton organization, with DEGs affecting relevant functions and biological pathways, such as myogenesis. The breed also affected functions related to muscle development and cytoskeleton organization, as well as functions related to solute transport and lipid and carbohydrate metabolism. Taking into account the results of the two main comparisons (age and breed effects), we can postulate that the Iberian breed is more precocious than the Duroc breed, regarding myogenesis and muscle development, in the studied growing stage.

Maternal Supplementation with Polyphenols and Omega-3 Fatty Acids during Pregnancy: Prenatal Effects on Growth and Metabolism.

Maternal supplementation with antioxidants and n-3 PUFAs may be a promising strategy to reduce the risk of intrauterine growth restriction and preterm delivery, which may diminish the appearance of low-birth-neonates. A previous studies showed beneficial outcomes of the combination of hydroxytyrosol and linoleic acid, but there is no data of its prenatal effects. The present study aimed to determine the possible prenatal implications of such maternal supplementation at prenatal stages in swine, a model of IUGR pregnancies. Results showed effects on litter size, with treated sows having larger litters and, therefore, smaller fetuses. However, the brain/head weight ratio showed a positive effect of the treatment in development, as well as in some other major organs like lungs, spleen, or kidneys. On the other hand, treated piglets showed better glycemic and lipidemic profiles, which could explain postnatal effects. However, further research on the implications of the treatment on litter size and prenatal and postnatal development must be done before practical recommendation can be given.

Effects of L-Glutamine Supplementation during the Gestation of Gilts and Sows on the Offspring Development in a Traditional Swine Breed.

The use of amino acids during pregnancy, such as glutamine (Gln), seems to be a promising strategy in selected swine breeds to improve the offspring prenatal development. The main goal of the current study was to assess the development of the offspring from parity 1-3 sows of a traditional breed, which were supplemented with 1% glutamine after Day 35 of gestation, under farm conditions. A total of 486 (288 treated) piglets from 78 (46 treated) Iberian sows were used. At birth and slaughterhouse, fatty acid composition, metabolism, and mTOR pathway gene expression were analyzed. At birth, treated newborns showed greater amounts of specific amino acids in plasma, such as glutamine, asparagine, or alanine, and Σn-3 fatty acids in cellular membranes than control newborns. The expression of genes belonging to mTOR Complex 1 was also higher in treated piglets with normal birth-weight. However, these findings did not improve productive traits at birth or following periods in litters from supplemented gilts (parity 1) or sows (parities 2-3). Thus, further research is needed to properly understand the effects of prenatal glutamine supplementation, particularly in traditional swine breeds.

The Role of Offspring Genotype-By-Sex Interactions, Independently of Environmental Cues, on the Phenotype Traits of an Obese Swine Model.

The present study aimed to assess the importance of offspring genotype on postnatal development, independently of confounding factors related to prenatal environment and postnatal lifestyle, using a translational model of obesity and metabolic syndrome (the Iberian pig). Hence, we compared two genotypes (purebred Iberian and crossbreds Iberian × Large White), produced in one single maternal environment (pure Iberian mothers) through artificial insemination of Iberian sows with Iberian and Large White heterospermic semen and maintained in the same conditions during postnatal development. The results indicate that, under same pre- and postnatal environments, the interaction genotype-by-sex has a determinant role on offspring phenotype (i.e., growth and development, metabolic and antioxidant status and fatty acid composition of different tissues). These results may set the basis for future preclinical and clinical research on the differences in the metabolic phenotype among genotypes.

Maternal Supplementation with Polyphenols and Omega-3 Fatty Acids during Pregnancy: Effects on Growth, Metabolism, and Body Composition of the Offspring.

Maternal supplementation with antioxidants and n3 PUFAs may be a promising strategy to reduce the risk of intrauterine growth restriction and preterm delivery, which may diminish the appearance of low-birth-weight neonates. The present study aimed to determine benefits and risks of a dietary supplementation combining hydroxytyrosol, a polyphenol from olive leaves and fruits, and n3 PUFAs, from linseed oil, on developmental patterns and metabolic traits of offspring in swine, a model of IUGR pregnancies. The results obtained indicate that maternal supplementation with hydroxytyrosol and n-3 fatty acids during pregnancy has no deleterious effects on the reproductive traits of the sows (prolificacy, homogeneity of the litter, and percentage of stillborns and low-birth-weight, LBW, piglets) and the postnatal features of the piglets (growth patterns, adiposity, and metabolic traits). Conversely, in spite of a lower mean weight and corpulence at birth, piglets from the supplemented sows showed higher average daily weight gain and fractional growth rate. Thus, at juvenile stages afterwards, the offspring from the treated group reached higher weight and corpulence, with increased muscle development and better lipidemic and fatty acid profiles, in spite of similar adiposity, than offspring in the control group. However, much caution and more research are still needed before practical recommendation and use in human pregnancies.

Short-Term Effects of Early Menopause on Adiposity, Fatty Acids Profile and Insulin Sensitivity of a Swine Model of Female Obesity.

Menopause strongly increases incidence and consequences of obesity and non-communicable diseases in women, with recent research suggesting a very early onset of changes in lipid accumulation, dyslipidemia, and insulin resistance. However, there is a lack of adequate preclinical models for its study. The present trial evaluated the usefulness of an alternative method to surgical ovariectomy, the administration of two doses of a GnRH analogue-protein conjugate (Vacsincel®), for inducing ovarian inactivity in sows used as preclinical models of obesity and menopause. All the sows treated with the compound developed ovarian stoppage after the second dose and, when exposed to obesogenic diets during the following three months, showed changes in the patterns of fat deposition, in the fatty acids profiles at the different tissues and in the plasma concentrations of fructosamine, urea, ß-hydroxibutirate, and haptoglobin when compared to obese fed with the same diet but maintaining ovarian activity. Altogether, these results indicate that menopause early augments the deleterious effects induced by overfeeding and obesity on metabolic traits, paving the way for future research on physiopathology of these conditions and possible therapeutic targets using the swine model.

Differential Effects of Litter Size and Within-Litter Birthweight on Postnatal Traits of Fatty Pigs.

Fatty pigs are characterized by a thrifty genotype, adapted to harsh environments based on changes in metabolism and energy saving. Thus, we hypothesized that feto-maternal energy partitioning in large litters might have postnatal effects that might be independent of intrauterine growth restriction (IUGR) processes. Hence, the current work reported the influence of two effects on postnatal performance and carcass and meat quality of purebred Iberian pigs: (a) the effects of the number of piglets in the litter (high vs. low litter size), and (b) the effects of birthweight (low (LBW) vs. normal (NBW)) in large litters. The results confirmed that NBW piglets born in large litters had differences in developmental patterns of weight, back-fat deposition, and fatty acid (FA) composition when compared to NBW piglets from small litters. These results were different from those found in LBW piglets when compared to their NBW counterparts, which showed an initial asymmetrical growth and altered muscle FA composition at slaughtering. The assessment of FA composition indicated better metabolic status in NBW piglets from large litters than in LBW piglets. These data support the concept that the prenatal environment, even when the individual may cope with it, inescapably affects postnatal life.

Impact of genotype, body weight and sex on the prenatal muscle transcriptome of Iberian pigs.

Growth is dependent on genotype and diet, even at early developmental stages. In this study, we investigated the effects of genotype, sex, and body weight on the fetal muscle transcriptome of purebred Iberian and crossbred Iberian x Large White pigs sharing the same uterine environment. RNA sequencing was performed on 16 purebred and crossbred fetuses with high body weight (340±14g and 415±14g, respectively) and 16 with low body weight (246±14g and 311±14g, respectively), on gestational day 77. Genotype had the greatest effect on gene expression, with 645 genes identified as differentially expressed (DE) between purebred and crossbred animals. Functional analysis showed differential regulation of pathways involved in energy and lipid metabolism, muscle development, and tissue disorders. In purebred animals, fetal body weight was associated with 35 DE genes involved in development, lipid metabolism and adipogenesis. In crossbred animals, fetal body weight was associated with 60 DE genes involved in muscle development, viability, and immunity. Interestingly, the results suggested an interaction genotype*weight for some DE genes. Fetal sex had only a modest effect on gene expression. This study allowed the identification of genes, metabolic pathways, biological functions and regulators related to fetal genotype, weight and sex, in animals sharing the same uterine environment. Our findings contribute to a better understanding of the molecular events that influence prenatal muscle development and highlight the complex interactions affecting transcriptional regulation during development.

The Iberian pig fed with high-fat diet: a model of renal disease in obesity and metabolic syndrome.

BACKGROUND: The pathogenesis of renal disease in the context of overweight/obesity, metabolic syndrome, and insulin resistance is not completely understood. This may be due to the lack of a definitive animal model of disease, which limits our understanding of obesity-induced renal damage. We evaluated the changes in renal histology and lipid deposits induced by obesity in a model of insulin resistance: the Iberian swine fed with fat-enriched food. METHODS: Twenty-eight female sows were randomized to standard (SD) or high-fat diet (HFD: 6.8% of saturated fat) for 100 days. Weight, adiposity, analytics, oral glucose tolerance tests, and measured renal function were determined. Renal histology and lipid deposits in renal tissue were analyzed. RESULTS: Animals on HFD developed obesity, hypertension, high levels of LDL cholesterol, triglycerides, insulin resistance, and glomerular hyperfiltration. No animal developed overt diabetes. Animals on HFD showed "diabetoid changes", including mesangial expansion [21.40% ± 4 vs.13.20% ± 4.0, p < 0.0001], nodular glomerulosclerosis [7.40% ± 7, 0.75 vs. 2.40% ± 4.7, p = 0.02], and glomerulomegaly (18% vs. 10%, p = 0.010) than those on SD. Tubular atrophy, interstitial fibrosis, inflammation, arteriolar hyalinosis, or fibrointimal thickening were mild and similar between groups. Triglyceride content in renal tissue was higher in animals on HFD than in SD (15.4% ± 0.5 vs. 12.7% ± 0.7; p < 0.01). CONCLUSIONS: Iberian pigs fed with fat-enriched food showed diabetoid changes and glomerulomegaly as observed in obese humans making this model suitable to study obesity-induced renal disease.

Polyphenols and IUGR Pregnancies: Effects of Maternal Hydroxytyrosol Supplementation on Postnatal Growth, Metabolism and Body Composition of the Offspring.

Maternal supplementation with the polyphenol hydroxytyrosol in a swine model of intrauterine growth restriction (IUGR) improves the fetal oxidative status, decreases the appearance of low birth-weight neonates and favors growth during early postnatal stages (lactation). The current study aimed to determine whether hydroxytyrosol supplementation can also improve developmental patterns, metabolic traits, and body composition of the offspring during later postnatal stages (from weaning to adulthood). A total of 21 piglets born from control untreated sows and 20 piglets born from sows treated with hydroxytyrosol during the last two-thirds of pregnancy were selected on the basis of similar body weights at weaning, for avoiding any interfering effects occurred during lactation. The pigs in the treated group had higher average daily weight gain (ADWG) and, therefore, reached higher body weight and corpulence, greater muscle development and higher adiposity than their control counterparts. The following were not found: significant effects on metabolism and body composition except changes in the muscular fatty acid composition of the treated pigs coming from the largest litters; those more affected by IUGR processes. These findings suggest that maternal supplementation with hydroxytyrosol may improve juvenile development of offspring in at-risk pregnancies and pave the way for more specific studies aiming to elucidate effects on adiposity, metabolism, and meat organoleptic characteristics.

Breed, Diet, and Interaction Effects on Adipose Tissue Transcriptome in Iberian and Duroc Pigs Fed Different Energy Sources.

In this study, we analyzed the effects of breed, diet energy source, and their interaction on adipose tissue transcriptome in growing Iberian and Duroc pigs. The study comprised 29 Iberian and 19 Duroc males, which were kept under identical management conditions except the nutritional treatment. Two isoenergetic diets were used with 6% high oleic sunflower oil (HO) or carbohydrates (CH) as energy sources. All animals were slaughtered after 47 days of treatment at an average live weight of 51.2 kg. Twelve animals from each breed (six fed each diet) were employed for ham subcutaneous adipose tissue RNA-Seq analysis. The data analysis was performed using two different bioinformatic pipelines. We detected 837 and 1456 differentially expressed genes (DEGs) according to breed, depending on the pipeline. Due to the strong effect of breed on transcriptome, the effect of the diet was separately evaluated in the two breeds. We identified 207 and 57 DEGs depending on diet in Iberian and Duroc pigs, respectively. A joint analysis of both effects allowed the detection of some breed-diet interactions on transcriptome, which were inferred from RNA-Seq and quantitative PCR data. The functional analysis showed the enrichment of functions related to growth and tissue development, inflammatory response, immune cell trafficking, and carbohydrate and lipid metabolism, and allowed the identification of potential regulators. The results indicate different effects of diet on adipose tissue gene expression between breeds, affecting relevant biological pathways.

Polyphenols and IUGR Pregnancies: Effects of Maternal Hydroxytyrosol Supplementation on Hepatic Fat Accretion and Energy and Fatty Acids Profile of Fetal Tissues.

Maternal supplementation with hydroxytyrosol, a polyphenol present in olive leaves and fruits, is a highly promising strategy to improve the oxidative and metabolic status of fetuses at risk of intrauterine growth restriction, which may diminish the appearance of low-birth-weight neonates. The present study aimed to determine whether hydroxytyrosol, by preventing lipid peroxidation, may influence the fat accretion and energy homeostasis in the liver, as well as the fatty acid composition in the liver and muscle. The results indicate that hydroxytyrosol treatment significantly decreased the energy content of the fetal liver, without affecting fat accretion, and caused significant changes in the availability of fatty acids. There were significant increases in the amount of total polyunsaturated fatty acids, omega-3 and omega-6, which are highly important for adequate fetal tissue development. However, there were increases in the omega-6/omega-3 ratio and the desaturation index, which make further studies necessary to determine possible effects on the pro/anti-inflammatory status of the fetuses.

Vegetable Oils Rich in Polyunsaturated Fatty Acids Supplementation of Dairy Cows' Diets: Effects on Productive and Reproductive Performance.

The aim of this study was to determine how polyunsaturated fatty acids (PUFA) supplementation can affect the productive and reproductive performance in dairy cows subjected to a fixed-time artificial insemination (TAI) protocol under farm conditions. One hundred and ninety-eight Holstein non-pregnant cows were used. Treatments consisted of a control diet (CON), without added oil, and two diets supplemented with either 2.3% soybean oil (SOY) or 2.3% linseed oil (LIN) as dry matter. The diets were formulated to be isoenergetic and isoproteic. Dry matter intake and milk yield were similar among treatments (p > 0.05). Both the percentage of fat (p = 0.011) and protein (p = 0.022) were higher in milk from animals not fed with oil (CON). The greatest saturated fatty acid (SFA) concentration (p < 0.0001) was observed in milk from cows fed the control diet, without added oil. The monounsaturated fatty acids (MUFA), PUFA, and the n-3 PUFA content was higher (p < 0.0001) in the milk from animals fed with oil with respect to the control treatment. The C18:2 cis-9, trans-11 in the milk of animals fed with oil supplements was significantly higher (p < 0.0001) than in that of the control group. Animals supplemented with linseed oil tended to show higher plasma progesterone level (p = 0.09) and a higher number of pregnant cows on the first artificial insemination (p = 0.07). These animals tended to reduce the number of TAI (p = 0.08). In brief, results showed that vegetable oils rich in PUFA supplementation considerably improve the nutritional profile of milk. PUFA n-3 supplementation slightly improves some reproductive parameters in dairy cows subjected to the fixed-time artificial insemination (TAI) protocol.

Polyphenols and IUGR Pregnancies: Effects of Maternal Hydroxytyrosol Supplementation on Placental Gene Expression and Fetal Antioxidant Status, DNA-Methylation and Phenotype.

The use of polyphenols is a promising strategy for preventing or alleviating intrauterine growth restriction (IUGR) because polyphenol supplementation increases plasma antioxidant capacity and improves oxidative stress at the feto-placental unit; which are recognized as main issues in IUGR. However, there is a scarcity of experimental data on both realistic benefits and potential hazards of polyphenol supplementation during gestation. Hence, we aimed to use a swine model of IUGR pregnancy to determine possible effects of maternal supplementation with polyphenols (hydroxytyrosol) on placental expression of genes involved in antioxidant homeostasis, vascularization and fetal growth and thus on antioxidant status, DNA-methylation and phenotypic traits (morphology and homeostasis) of the fetus. Hydroxytyrosol improves placental gene expression and fetal antioxidant status and glucose metabolism in a sex-dependent manner, in which males were favored in spite of developmental failures. Concomitantly, hydroxytyrosol prevented hypomethylation of DNA associated with oxidative stress. Finally, no major deleterious effects of hydroxytyrosol supplementation on constriction of the ductus arteriosus, a possible secondary effect of polyphenols during pregnancy, were found.

Characterization of Ageing- and Diet-Related Swine Models of Sarcopenia and Sarcopenic Obesity.

Sarcopenia and sarcopenic obesity are currently considered major global threats for health and well-being. However, there is a lack of adequate preclinical models for their study. The present trial evaluated the suitability of aged swine by determining changes in adiposity, fatty acids composition, antioxidant status and lipid peroxidation, development of metabolic disturbances and structural changes in tissues and organs. Iberian sows with clinical evidence of aging-related sarcopenia were fed a standard diet fulfilling their maintenance requirements or an obesogenic diet for 100 days. Aging and sarcopenia were related to increased lipid accumulation and cellular dysfunction at both adipose tissue and non-adipose ectopic tissues (liver and pancreas). Obesity concomitant to sarcopenia aggravates the condition by increasing visceral adiposity and causing dyslipidemia, insulin resistance and lipotoxicity in non-adipose tissues. These results support that the Iberian swine model represents certain features of sarcopenia and sarcopenic obesity in humans, paving the way for future research on physiopathology of these conditions and possible therapeutic targets.

Modulatory Effects of Breed, Feeding Status, and Diet on Adipogenic, Lipogenic, and Lipolytic Gene Expression in Growing Iberian and Duroc Pigs.

Meat quality depends on tissue composition which is in turn influenced by different factors, such as diet, genotype, age, or sex. We evaluated the effects of breed, 24 h fasting, and dietary energy source (HO: oleic acid versus CH: carbohydrates) on the expression of candidate genes involved in adipogenesis, lipogenesis, and lipolysis in the adipose tissue from Iberian and Duroc growing pigs. The Iberian pigs showed greater feed intake, backfat thickness, and saturated fatty acids (SFA) content in the subcutaneous fat, whereas the Duroc pigs had greater ham weight and polyunsaturated fatty acids (PUFA) content. In both breeds, the diet induced changes in the fatty acid (FA) composition of subcutaneous fat samples. The HO group had higher monounsaturated fatty acids (MUFA) and oleic acid, and lower SFA than the CH group. Regarding gene expression, breed and feeding status (fasting versus postprandial) had significant effects on gene expression, with quantitative interactions between them, while diet showed negligible effects. In general, adipogenic and lipogenic genes were upregulated in the Iberian pigs and in postprandial samples. In contrast, the expression of lipolytic genes showed complex interaction effects. Our results agree with the phenotypic differences between the Iberian and Duroc breeds and with the inhibition of lipogenesis by fasting. Quantitative interactions between breed and feeding status effects were observed, which indicates a different response to fasting of the two breeds, with the obese Iberian breed showing a more stable expression of lipogenic genes. These results highlight the complexity of lipid metabolism regulation, especially in relation to lipolysis processes.

Ontogeny of Sex-Related Differences in Foetal Developmental Features, Lipid Availability and Fatty Acid Composition.

Sex-related differences in lipid availability and fatty acid composition during swine foetal development were investigated. Plasma cholesterol and triglyceride concentrations in the mother were strongly related to the adequacy or inadequacy of foetal development and concomitant activation of protective growth in some organs (brain, heart, liver and spleen). Cholesterol and triglyceride availability was similar in male and female offspring, but female foetuses showed evidence of higher placental transfer of essential fatty acids and synthesis of non-essential fatty acids in muscle and liver. These sex-related differences affected primarily the neutral lipid fraction (triglycerides), which may lead to sex-related postnatal differences in energy partitioning. These results illustrate the strong influence of the maternal lipid profile on foetal development and homeorhesis, and they confirm and extend previous reports that female offspring show better adaptive responses to maternal malnutrition than male offspring. These findings may help guide dietary interventions to ensure adequate fatty acid availability for postnatal development.