High and Low Protein∶ Carbohydrate Dietary Ratios during Gestation Alter Maternal-Fetal Cortisol Regulation in Pigs

Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and 11β-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11β-HSD1 and a reduced expression of c-fos. Additionally, the 11β-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein∶carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.     

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.     

Effects of feeding low protein diets to piglets on plasma urea nitrogen, faecal ammonia nitrogen, the incidence of diarrhoea and performance after weaning

This study evaluated the effects of feeding pigs low protein (LP) diets for different lengths of time after weaning on indices of protein fermentation, the incidence of postweaning diarrhoea (PWD), growth performance, and total-tract apparent digestibility. Sixty weaner pigs weighing 6.1 +/- 0.13 kg (mean +/- SEM) were used in a completely randomised design having five treatments: (i) a high protein diet (HP, 243 g/kg CP) fed for 14 d after weaning (HP14); (ii) a low protein diet (LP, 173 g CP/kg) fed for 5 d after weaning (LP5); (iii) LP diet fed for 7 d after weaning (LP7); (iv) LP diet fed for 10 d after weaning (LP10), and (v) LP diet fed for 14 d after weaning (LP14). All diets were supplemented with lysine, methionine, tryptophan and threonine, with all LP diets additionally fortified with crystalline isoleucine and valine to conform to a proposed ideal amino acid (AA) pattern. A second-stage diet (215 g CP/kg) was fed to pigs at the conclusion of each treatment. None of the diets contained antimicrobial compounds. Feeding a LP diet, regardless of duration of feeding, decreased plasma urea nitrogen (p < 0.001) and faecal ammonia-nitrogen (p < 0.001) contents. Feeding a LP diet, irrespective of feeding duration, decreased the incidence of PWD at day 8 after weaning (p = 0.044), and pigs fed diets LP7, LP010 and LP14 had firmer faeces (p = 0.030, p = 0.047 and p = 0.007, respectively) between days 10 and 12 after weaning. Treatments LP5, LP7, LP10 and LP14 did not reduce (p > 0.05) growth performance up to 106 days after weaning compared to pigs fed the HP diet. Total-tract apparent digestibility of dry matter, energy and crude protein were similar (p > 0.05) between treatments. Our data suggest that feeding a LP diet, supplemented with AA to conform to an ideal AA pattern, for 7-10 days after weaning can reduce PWD in pigs fed antibiotic-free diets without compromising production.     

Gestational low protein diet selectively induces the amino acid response pathway target genes in the liver of offspring rats through transcription factor binding and histone modifications

The amino acid response (AAR) pathway detects a deficiency of dietary amino acid or protein. To investigate the impact of gestational protein restriction on the AAR pathway in offspring, pregnant Sprague-Dawley rats were fed a control (C) or low protein (LP) diet during gestation. Livers of female offspring were collected on postnatal d 38. The mRNA amount of Atf3 in LP offspring increased significantly compared with C offspring, while Asns did not differ between the two groups. ATF4 and p-eIF2α were both induced in LP offspring, whereas p-ERK was significantly decreased. Additionally, amino acid limitation (-AA) in HepG2 cells increased p-ERK and AAR pathway-related genes, while U0126 decreased p-ERK but did not completely reverse the activation of AAR pathway-related genes. Chromatin immunoprecipitation assay demonstrated an increased association of both RNA polymerase II (Pol II) and ATF4 at the Atf3 promoter in LP offspring, while acetylated histone H4, tri-methyl histone H3 at lysine 9, ATF4, ATF3, C/EBPβ, and CHOP, but not Pol II, were all increased at the Asns promoter in LP offspring. In -AA HepG2 cells, C/EBPβ siRNA treatment did not prevent the activation of either ATF3 or ASNS in response to -AA, while ATF4 siRNA prevented the activation of ASNS but not ATF3. Our data demonstrates that a maternal LP diet programs the AAR pathway in the liver of offspring rats. The differential priming of the downstream target genes of the AAR pathway in response to maternal LP diet presents a novel regulatory mechanism related to nutrient-gene interactions.     

Supplementation with branched-chain amino acids to a low-protein diet regulates intestinal expression of amino acid and peptide transporters in weanling pigs

This study determined the effects of dietary branched-chain amino acids (AA) (BCAA) on growth performance, expression of jejunal AA and peptide transporters, and the colonic microflora of weanling piglets fed a low-protein (LP) diet. One hundred and eight Large White × Landrace × Duroc piglets (weaned at 28 days of age) were fed a normal protein diet (NP, 20.9 % crude protein), an LP diet (LP, 17.1 % crude protein), or an LP diet supplemented with BCAA (LP + BCAA, 17.9 % crude protein) for 14 days. Dietary protein restriction reduced piglet growth performance and small-intestinal villous height, which were restored by BCAA supplementation to the LP diet to values for the NP diet. Serum concentrations of BCAA were reduced in piglets fed the LP diet while those in piglets fed the LP + BCAA diet were similar to values for the NP group. mRNA levels for Na⁺-neutral AA exchanger-2, cationic AA transporter-1, b^0,+ AA transporter, and 4F2 heavy chain were more abundant in piglets fed the LP + BCAA diet than the LP diet. However, mRNA and protein levels for peptide transporter-1 were lower in piglets fed the LP + BCAA diet as compared to the LP diet. The colonic microflora did not differ among the three groups of pigs. In conclusion, growth performance, intestinal development, and intestinal expression of AA transporters in weanling piglets are enhanced by BCAA supplementation to LP diets. Our findings provide a new molecular basis for further understanding of BCAA as functional AA in animal nutrition.     

Effects of feed restriction during pregnancy on maternal reproductive outcome,foetal hepatic IGF gene expression and offspring performance in the rabbit

Primiparous female rabbits have high nutritional requirements and, while it is recommended that they are subjected to an extensive reproductive rhythm, this could lead to overweight, affecting reproductive outcomes. We hypothesised that restricting food intake during the less energetic period of gestation could improve reproductive outcome without impairing offspring viability. This study compares two groups of primiparous rabbit does in an extensive reproductive programme, one in which feed was restricted from Day 0 to Day 21 of gestation (R021), and another in which does were fed ad libitum (control) throughout pregnancy. The mother and offspring variables compared were (1) mother reproductive outcomes at the time points pre-implantation (Day 3 postartificial insemination [AI]), preterm (Day 28 post-AI) and birth; and (2) the prenatal offspring characteristic IGF system gene expression in foetal liver, liver fibrosis and foetus sex ratio, and postnatal factor viability and growth at birth, and survival and growth until weaning. Feed restriction did not affect the conception rate, embryo survival, or the number of morulae and blastocysts recovered at Day 3 post-AI. Preterm placenta size and efficiency were similar in the two groups. However, both implantation rate (P < 0.001) and the number of foetuses (P = 0.05) were higher in the R021 mothers than controls, while there was no difference in foetal viability. Foetal size and weight, the weights of most organs, organ weight/BW ratios and sex ratio were unaffected by feed restriction; these variables were only affected by uterine position (P < 0.05). Conversely, in the R021 does, foetal liver IGBP1 and IGF2 gene expression were dysregulated despite no liver fibrosis and a normal liver structure. No effects of restricted feed intake were produced on maternal fertility, prolificacy, or offspring birth weight, but control females weaned more kits. Litter weight and mortality rate during the lactation period were also unaffected. In conclusion, pre-implantation events and foetal development were unaffected by feed restriction. While some genes of the foetal hepatic IGF system were dysregulated during pregnancy, liver morphology appeared normal, and the growth of foetuses and kits until weaning was unmodified. This strategy of feed restriction in extensive reproductive rhythms seems to have no significant adverse effects on dam reproductive outcome or offspring growth and viability until weaning.     

Influence of benzoic acid and dietary protein level on performance, nitrogen metabolism and urinary pH in growing-finishing pigs

An experiment was conducted to examine the effect of benzoic acid and two dietary protein levels on pig performance, nitrogen balance and urinary pH. A total of 24 crossbred barrows (26 kg to 106 kg BW) received one of four diets: low protein level with and without 1% benzoic acid (LP- and LP+, respectively) and high protein level with and without 1% benzoic acid (HP- and HP+, respectively). The animals were fed restrictively grower and finisher diets and were kept in metabolic cages in weeks 3, 6, 9, and 12 of the experiment. The addition of benzoic acid did not improve weight gain and feed conversion ratio. N-intake and digested N were only influenced by dietary protein level (p< 0.01), while N-balance was similar in all four diets. Dietary benzoic acid improved N-digestibility in the grower period (p<0.01) but not in the finisher period. The addition of benzoic acid reduced urinary pH by about one pH-unit in both feeding periods independent of the protein level of the diet (p< 0.01) and increased the concentration of urinary hippuric acid markedly (p<0.01). The results of this study indicate a positive influence of dietary benzoic acid on pigs especially in case of feeding a low protein diet in the grower period.     

Birth weight and postnatal dietary protein level affect performance, muscle metabolism and meat quality in pigs

Intrauterine growth restriction (IUGR), resulting in low birth body weight (LBW) occurs naturally in pigs. However, IUGR may also cause persistent changes in physiology and metabolism resulting in poorer performance, organogenesis and meat quality. As IUGR pigs have a lower daily gain from birth to slaughter they may differ in utilization of nutrients and requirements for dietary protein compared with their larger littermates. Thus, the objective in this study was to examine the interaction between birth body weight (BW) and the postnatal dietary protein level, in relation to postnatal performance, organogenesis, muscle metabolism and meat quality. The experiment was carried out with offspring from 16 purebred Danish Landrace gilts mated to Danish Landrace boars. The female and entire male pigs with LBW that survived at weaning were compared with the female and male pigs with the highest/high birth body weight (HBW) within each litter. The offspring were reared individually from weaning and were fed ad libitum a diet containing either a normal level of protein (NP) for optimal growth or an isocaloric diet containing a 30% lower protein content (LP) from 3 weeks to 150 days of age. At slaughter, we found no interactions between birth weight group and dietary protein level for any of the measured traits. The relative crown-rump length (cm/kg) at birth indicates that LBW pigs were thinner than HBW pigs. Daily gain and feed intake were reduced by 14% and 10%, respectively, while the kg feed/kg gain was slightly increased by 3% in LBW pigs compared with HBW pigs. The LP diet reduced daily gain by 27% due to reduced feed intake and increased kg feed/kg gain by 12% and 21%, respectively compared with the NP diet. LBW male pigs produced meat with a higher shear force than male HBW pigs and also LP pigs produced meat with higher shear force than NP pigs. The activity of lactate dehydrogenase in the Longissimus dorsi muscle (LD) was reduced in pigs fed the LP diet. Calpastatin was increased in LD of LBW pigs and decreased in pigs fed the NP diet. In conclusion, these results suggest a rejection of our hypothesis that low birth weight littermates have a lower requirement for dietary protein compared with heavy weight littermates. Furthermore, LBW male pigs and LP fed pigs of both genders produced less tender meat than HBW pigs or NP fed pigs, respectively.     

High-protein diet during gestation and lactation affects mammary gland mRNA abundance, milk composition and pre-weaning litter growth in mice

We evaluated the effect of a high-protein diet (HP) on pregnancy, lactational and rearing success in mice. At the time of mating, females were randomly assigned to isoenergetic diets with HP (40% w/w) or control protein levels (C; 20%). After parturition, half of the dams were fed the other diet throughout lactation resulting in four dietary groups: CC (C diet during gestation and lactation), CHP (C diet during gestation and HP diet during lactation), HPC (HP diet during gestation and C diet during lactation) and HPHP (HP diet during gestation and lactation). Maternal and offspring body mass was monitored. Measurements of maternal mammary gland (MG), kidney and abdominal fat pad masses, MG histology and MG mRNA abundance, as well as milk composition were taken at selected time points. HP diet decreased abdominal fat and increased kidney mass of lactating dams. Litter mass at birth was lower in HP than in C dams (14.8 v. 16.8 g). Dams fed an HP diet during lactation showed 5% less food intake (10.4 v. 10.9 g/day) and lower body and MG mass. On day 14 of lactation, the proportion of MG parenchyma was lower in dams fed an HP diet during gestation as compared to dams fed a C diet (64.8% v. 75.8%). Abundance of MG α-lactalbumin, β-casein, whey acidic protein, xanthine oxidoreductase mRNA at mid-lactation was decreased in all groups receiving an HP diet either during gestation and/or lactation. Milk lactose content was lower in dams fed an HP diet during lactation compared to dams fed a C diet (1.6% v. 2.0%). On days 14, 18 and 21 of lactation total litter mass was lower in litters of dams fed an HP diet during lactation, and the pups' relative kidney mass was greater than in litters suckled by dams receiving a C diet. These findings indicate that excess protein intake in reproducing mice has adverse effects on offspring early in their postnatal growth as a consequence of impaired lactational function.     

The importance of catch-up growth after early malnutrition for the programming of obesity in male rat

Objective: To investigate whether catch‐up growth after maternal malnutrition would favor the development of obesity in adulthood. Research Methods and Procedures: Pregnant rats were submitted to protein or calorie restriction during the course of gestation. During lactation, pups were protein‐restricted, normally fed, or overfed [reduced litter size, control (C) diet]. At weaning, rats were transferred to chow or to a hypercaloric diet (HCD) known to induce obesity. Body weight, food intake, blood parameters, glucose tolerance, adipocyte cellularity, and adipose factors contributing to cardiovascular disease development were measured. Results: Protein and calorie restriction during gestation led to growth retardation at birth. If malnutrition was prolonged throughout lactation, adult body weight was permanently reduced. However, growth‐retarded offspring overfed during the suckling period underwent a rapid catch‐up growth and became heavier than the normally fed Cs. Offspring of calorie‐restricted rats gained more weight than those of dams fed protein‐restricted diet. Feeding an HCD postnatally amplified the effect of calorie restriction, and offspring that underwent catch‐up growth became more obese than Cs. The HCD was associated with hyperphagia, hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, and adipocyte hypertrophy. The magnitude of effects varied depending on the type and the timing of early malnutrition. The expression of genes encoding factors implicated in cardiovascular disease was also modulated differently by early malnutrition and adult obesity. Discussion: Catch‐up growth immediately after early malnutrition should be a key point for the programming of obesity.     

Perinatal exposure of rats to a maternal diet with varying protein quantity and quality affects the risk of overweight in female adult offspring

The maternal protein diet during the perinatal period can program the health of adult offspring. This study in rats evaluated the effects of protein quantity and quality in the maternal diet during gestation and lactation on weight and adiposity in female offspring. Six groups of dams were fed a high-protein (HP; 47% protein) or normal-protein (NP; 19% protein) isocaloric diet during gestation (_G) using either cow's milk (M), pea (P) or turkey (T) proteins. During lactation, all dams received the NP diet (protein source unchanged). From postnatal day (PND) 28 until PND70, female pups (n=8) from the dam milk groups were exposed to either an NP milk diet (NPM_W) or to dietary self-selection (DSS). All other pups were only exposed to DSS. The DSS design was a choice between five food cups containing HPM, HPP, HPT, carbohydrates or lipids. The weights and food intakes of the animals were recorded throughout the study, and samples from offspring were collected on PND70. During the lactation and postweaning periods, body weight was lower in the pea and turkey groups (NP_G and HP_G) versus the milk group (P<.0001). DSS groups increased their total energy and fat intakes compared to the NPM_W group (P<.0001). In all HP_G groups, total adipose tissue was increased (P=.03) associated with higher fasting plasma leptin (P<.05). These results suggest that the maternal protein source impacted offspring body weight and that protein excess during gestation, irrespective of its source, increased the risk of adiposity development in female adult offspring.     

Foraging behaviour,nutrient intake from pasture and performance of free-range growing pigs in relation to feed CP level in two organic cropping systems

In organic pig production one of the major challenges is to be able to fulfil amino acid requirements based on organic and locally grown protein feed crops. The pig is an opportunistic omnivore with a unique capacity for foraging above and below the soil surface. It is hypothesized that direct foraging in the range area can pose an important contribution in terms of fulfilling nutrient requirements of growing pigs. Foraging activity, lucerne nutrient intake and pig performance were investigated in 36 growing pigs, foraging on lucerne or grass and fed either a standard organic pelleted feed mixture (HP: high protein) or a grain mixture containing 48% less CP (LP: low protein) compared with the high protein feed mixture, from an average live weight of 58 kg to 90 kg in a complete block design in three replicates. The pigs were fed 80% of energy recommendations and had access to 4 m² of pasture/pig per day during the 40 days experimental period from September to October 2013. Behavioural observations were carried out 12 times over the entire experimental period. For both crops, LP pigs rooted significantly more compared with HP pigs but the effect of CP level was more pronounced in grass (44% v. 19% of all observations) compared with lucerne (28% v. 16% of all observations). Feed protein level turned out not to have any significant effect on grazing behaviour but pigs foraging on lucerne grazed significantly more than pigs foraging on grass (10% v. 4% of all observations). Daily weight gain and feed conversion ratio were significantly affected by feed protein and forage crop interactions. Compared to HP pigs, LP treated pigs had 33% lower daily weight gain (589 v. 878 g) and 31% poorer feed conversion ratio (3.75 v. 2.59 kg feed/kg weight gain) in grass paddocks, whereas in lucerne paddocks LP pigs only had 18% lower daily weight gain (741 v. 900 g) and a 14% poorer feed conversion ratio (2.95 v. 2.54 kg feed/kg weight gain) compared with HP pigs. LP pigs foraging on lucerne used 169 g less concentrate CP/kg weight gain, compared with HP pigs, indicating the nitrogen efficiency of the system. The results indicate that direct foraging of lucerne may be a valuable strategy in terms of accommodating CP and lysine requirements of organic growing pigs.     

Does Early Mismatched Nutrition Predispose to Hypertension and Atherosclerosis,in Male Mice?

Background

A link between early mismatched nutritional environment and development of components of the metabolic syndrome later in life has been shown in epidemiological and animal data. The aim of this study was to investigate whether an early mismatched nutrition produced by catch-up growth after fetal protein restriction could induce the appearance of hypertension and/or atherosclerosis in adult male mice.

Methodology/Principal Findings

Wild-type C57BL6/J or LDLr−/− dams were fed a low protein (LP) or a control (C) diet during gestation. Catch-up growth was induced in LP offspring by feeding dams with a control diet and by culling the litter to 4 pups against 8 in controls. At weaning, male mice were fed either standard chow or an obesogenic diet (OB), leading to 4 experimental groups. Blood pressure (BP) and heart rate (HR) were assessed in conscious unrestrained wild-type mice by telemetry. Atherosclerosis plaque area was measured in aortic root sections of LDLr−/− mice. We found that: (1) postnatal OB diet increased significantly BP (P<0.0001) and HR (P<0.008) in 3-month old OB-C and OB-LP offspring, respectively; (2) that maternal LP diet induced a significant higher BP (P<0.009) and HR (P<0.004) and (3) an altered circadian rhythm in addition to higher plasma corticosterone concentration in 9 months-old LP offspring; (4) that, although LP offspring showed higher plasma total cholesterol than control offspring, atherosclerosis assessed in aortic roots of 6-mo old mice featured increased plaque area due to OB feeding but not due to early mismatched nutrition.

Conclusions/Significance

These results indicate a long-term effect of early mismatched nutrition on the appearance of hypertension independently of obesity, while no effect on atherosclerosis was noticed at this age.     

Maternal protein restriction leads to hyperinsulinemia and reduced insulin-signaling protein expression in 21-mo-old female rat offspring

Human adult diseases such as cardiovascular disease, hypertension, and type 2 diabetes have been epidemiologically linked to poor fetal growth and development. Male offspring of rat dams fed a low-protein (LP) diet during pregnancy and lactation develop diabetes with concomitant alterations in their insulin-signaling mechanisms. Such associations have not been studied in female offspring. The aim of this study was to determine whether female LP offspring develop diabetes in later life. Control and LP female offspring groups were obtained from rat dams fed a control (20% protein) or an isocaloric (8% protein) diet, respectively, throughout pregnancy and lactation. Both groups were weaned and maintained on 20% normal laboratory chow until 21 mo of age when they underwent intravenous glucose tolerance testing (IVGTT). Fasting glucose was comparable between the two groups; however, LP fasting insulin was approximately twofold that of controls (P < 0.02). Glucose tolerance during IVGTT was comparable between the two groups; however, LP peak plasma insulin at 4 min was approximately threefold higher than in controls (P < 0.001). LP plasma insulin area under the curve was 1.9-fold higher than controls (P < 0.02). In Western blots, both muscle protein kinase C-zeta expression and p110beta-associated p85alpha in abdominal fat were reduced (P < 0.05) in LPs. Hyperinsulinemia in response to glucose challenge coupled with attenuation of certain insulin-signaling molecules imply the development of insulin resistance in LP muscle and fat. These observations suggest that intrauterine protein restriction leads to insulin resistance in females in old age and, hence, an increased risk of type 2 diabetes.