Biochemical and physiological bases for utilization of dietary amino acids by young Pigs

Protein is quantitatively the most expensive nutrient in swine diets. Hence it is imperative to understand the physiological roles played by amino acids in growth, development, lactation, reproduction, and health of pigs to improve their protein nutrition and reduce the costs of pork production. Due to incomplete knowledge of amino acid biochemistry and nutrition, it was traditionally assumed that neonatal, post-weaning, growing-finishing, and gestating pigs could synthesize sufficient amounts of all ＂nutritionally nonessential amino acids＂ （NEAA） to support maximum production performance. Therefore, over the past 50 years, much emphasis has been placed on dietary requirements of nutritionally essential amino acids as building blocks for tissue proteins. However, a large body of literature shows that NEAA, particularly glutamine, glutamate, arginine and proline regulate physiological functions via cell signaling pathways, such as mammalian target of rapamycin, AMP-activated protein kinase, extracellular signal-related kinase, Jun kinase, mitogen-activated protein kinase, and NEAA-derived gaseous molecules （e.g., nitric oxide, carbon monoxide, and hydrogen sulfide）. Available evidence shows that under current feeding programs, only 70% and 55% of dietary amino acids are deposited as tissue proteins in 14-day-old sow-reared piglets and in 30-day-old pigs weaned at 21 days of age, respectively. Therefore, there is an urgent need to understand the robes and dietary requirements of NEAA in swine nutrition. This review highlights the basic biochemistry and physiology of absorption and utilization of amino acids in young pigs to enhance the efficacy of utilization of dietary protein and to minimize excretion of nitrogenous wastes from the body.

Fetal and neonatal programming of postnatal growth and feed efficiency in swine

Maternal undernutrition or overnutrition during pregnancy alters organ structure, impairs prenatal and neonatal growth and development, and reduces feed efficiency for lean tissue gains in pigs. These adverse effects may be carried over to the next generation or beyond. This phenomenon of the transgenerational impacts is known as fetal programming, which is mediated by stable and heritable alterations of gene expression through covalent modifications of DNA and histones without changes in DNA sequences(namely, epigenetics). The mechanisms responsible for the epigenetic regulation of protein expression and functions include chromatin remodeling; DNA methylation(occurring at the 5′-position of cytosine residues within CpG dinucleotides); and histone modifications(acetylation, methylation, phosphorylation, and ubiquitination). Like maternal malnutrition, undernutrition during the neonatal period also reduces growth performance and feed efficiency(weight gain:feed intake; also known as weightgain efficiency) in postweaning pigs by 5–10%, thereby increasing the days necessary to reach the market bodyweight. Supplementing functional amino acids(e.g., arginine and glutamine) and vitamins(e.g., folate) play a key role in activating the mammalian target of rapamycin signaling and regulating the provision of methyl donors for DNA and protein methylation. Therefore, these nutrients are beneficial for the dietary treatment of metabolic disorders in offspring with intrauterine growth restriction or neonatal malnutrition. The mechanism-based strategies hold great promise for the improvement of the efficiency of pork production and the sustainability of the global swine industry.

Endoplasmic reticulum stress-induced apoptosis in intestinal epithelial cells:a feed-back regulation by mechanistic target of rapamycin complex 1(mTORC1)

Background: Endoplasmic reticulum(ER) stress is associated with multiple pathological processes of intestinal diseases. Despite a critical role of mechanistic target of rapamycin complex 1(m TORC1) in regulating cellular stress response, the crosstalk between m TORC1 and ER stress signaling and its contribution to the intestinal barrier function is unknown.Results: In the present study, we showed that intestinal epithelial cells(IEC-6) incubated with tunicamycin led to caspase-3-dependent apoptotic cell death. The induction of cell death was accompanied by activation of unfolded protein response as evidenced by increased protein levels for Bi P, p-IRE1α, p-e IF2α, p-JNK, and CHOP. Further study demonstrated that tunicamycin-induced cell death was enhanced by rapamycin, a specific inhibitor of m TORC1.Consistently, tunicamycin decreased transepithelial electrical resistance(TEER) and increased permeability of the cells. These effects of tunicamycin were exacerbated by m TORC1 inhibitor.Conclusions: Taken together, the data presented here identified a previously unknown crosstalk between an unfold protein response and m TORC1 signaling in the intestinal epithelium. This feed-back loop regulation on ER stress signaling by m TORC1 is critical for cell survival and intestinal permeability in epithelial cells.

Dietary L-arginine supplementation reduces lipid accretion by regulating fatty acid metabolism in Nile tilapia(Oreochromis niloticus)

Background: Excessive white fat accumulation in humans and other animals is associated with the development of multiple metabolic diseases. It is unknown whether dietary L-arginine supplementation reduces lipid deposition in high fat diet-fed Nile tilapia(Oreochromis niloticus).Results: In the present study, we found that dietary supplementation with 1% or 2% arginine decreased the deposition and concentration of fats in the liver;the concentrations of triglycerides, low-density lipoprotein, total cholesterol, and high-density lipoprotein in the serum;and the diameter of adipocytes in intraperitoneal adipose tissue. Compared with the un-supplementation control group, the hepatic activities of alanine aminotransferase,aspartate aminotransferase, and lactate dehydrogenase, and hepatic concentration of malondialdehyde were reduced but these for catalase and superoxide dismutase were enhanced by dietary supplementation with 2% arginine. Arginine supplementation reduced the total amounts of monounsaturated fatty acids, while increasing the total amounts of n-3 and n-6 polyunsaturated fatty acids in the liver. These effects of arginine were associated with reductions in mRNA levels for genes related to lipogenesis(sterol regulatory element-binding protein-1, acetyl-CoA carboxylase α, stearoyl-CoA desaturase, and fatty acid synthase) but increases in mRNA levels for genes involved in fatty acid β-oxidation(carnitine palmitoyltransferase 1α and peroxisome proliferator-activated receptor α). In addition, hepatic mRNA levels for Δ4 fatty acyl desaturase 2 and elongase 5 of very long-chain fatty acids were enhanced by arginine supplementation.Conclusion: These results revealed that dietary L-arginine supplementation to tilapia reduced high fat diet-induced fat deposition and fatty acid composition in the liver by regulating the expression of genes for lipid metabolism.

Protein hydrolysates in animal nutrition：Industrial production, bioactive peptides,and functional significance

Recent years have witnessed growing interest in the role of peptides in animal nutrition. Chemical, enzymatic, or microbial hydrolysis of proteins in animal by-products or plant-source feedstuffs before feeding is an attractive means of generating high-quality small or large peptides that have both nutritional and physiological or regulatory functions in livestock, poultry and fish. These peptides may also be formed from ingested proteins in the gastrointestinal tract, but the types of resultant peptides can vary greatly with the physiological conditions of the animals and the composition of the diets. In the small intestine, large peptides are hydrolyzed to small peptides,which are absorbed into enterocytes faster than free amino acids（AAs） to provide a more balanced pattern of AAs in the blood circulation. Some peptides of plant or animal sources also have antimicrobial, antioxidant,antihypertensive, and immunomodulatory activities. Those peptides which confer biological functions beyond their nutritional value are called bioactive peptides. They are usually 2–20 AA residues in length but may consist of 〉20AA residues. Inclusion of some（e.g. 2–8%） animal-protein hydrolysates（e.g., porcine intestine, porcine mucosa,salmon viscera, or poultry tissue hydrolysates） or soybean protein hydrolysates in practical corn-and soybean mealbased diets can ensure desirable rates of growth performance and feed efficiency in weanling pigs, young calves,post-hatching poultry, and fish. Thus, protein hydrolysates hold promise in optimizing the nutrition of domestic and companion animals, as well as their health（particularly gut health） and well-being.

Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Background: Polyamines are essential for cell growth and beneficial for intestinal maturation. To evaluate the effects of putrescine on alleviating intestinal atrophy and underlying molecular mechanisms, both in vivo feeding trial and in vitro cell culture were conducted. Weanling pigs were fed a diet supplemented with 0, 0.1%, 0.2% or0.3% putrescine dihydrochloride, whereas porcine intestinal epithelial cells(IPEC-J2) were challenged with lipopolysaccharide(LPS) in the presence of 200 μmol/L putrescine.Results: Dietary supplementation with 0.2% putrescine dihydrochloride decreased the incidence of diarrhea with an improvement in intestinal integrity. Inhibition of ornithine decarboxylase activity decreased the proliferation and migration of IPEC-J2 cells, and this effect was alleviated by the supplementation with putrescine. The phosphorylation of extracellular signal regulated kinase and focal adhesion kinase was enhanced by putrescine. LPS increased the expression of inflammatory cytokines [tumor necrosis factor α(TNF-α), interleukin 6(IL-6) and IL-8],and inhibited cell proliferation and migration in IPEC-J2 cells. Adding exogenous putrescine suppressed the expression of TNF-α, IL-6 and IL-8, and recovered cell migration and proliferation in LPS-treated IPEC-J2 cells. Dietary putrescine supplementation also reduced the m RNA levels of TNF-α, IL-6 and IL-8 and their upstream regulator nuclear receptor kappa B p65 subunit in the jejunal mucosa of piglets.Conclusions: Dietary supplementation with putrescine mitigated mucosal atrophy in weanling piglets through improving anti-inflammatory function and suppressing inflammatory response. Our results have important implications for nutritional management of intestinal integrity and health in weanling piglets and other neonates.

Glycine represses endoplasmic reticulum stress-related apoptosis and improves intestinal barrier by activating mammalian target of rapamycin complex 1 signaling

Endoplasmic reticulum(ER)stress has been associated with the dysfunction of intestinal barrier in humans and animals.We have previously shown that oral administration of glycine to suckling-piglets improves ER stress-related intestinal mucosal barrier impairment and jejunal epithelial apoptosis.However,the underlying mechanism remains unknown.In this study,the protective effect and the mechanism of glycine on apoptosis and dysfunction in intestinal barrier induced by brefeldin A(BFA),an ER stress inducer,was explored in porcine intestinal epithelial cells(IPEC-1).The results showed that BFA treatment led to enhanced apoptosis and upregulation of proteins involved in ER stress signaling,including inositol-requiring enzyme 1a(IRE1a),activating transcription factor 6a(ATF6a),c-Jun N-terminal kinase(JNK),and C/EBP-homologous protein(CHOP).In addition,BFA induced a dysfunction in intestinal epithelial barrier,as evidenced by the increased paracellular permeability,decreased transepithelial electrical resistance(TEER),and reduced abundance of tight junction proteins(occludin,claudin-1,zonula occludens[ZO]-1,and ZO-2).These alterations triggered by BFA were significantly abolished by glycine treatment(P<0.05),indicating a protective effect of glycine on barrier function impaired by ER stress.Importantly,we found that the regulatory effect of glycine on intestinal permeability,proteins implicated in ER stress and apoptosis,as well as the morphological alterations of the ER were reversed by rapamycin.In summary,our results indicated that glycine alleviates ER stress-induced apoptosis and intestinal barrier dysfunction in IPEC-1 cells in a mammalian target of rapamycin complex 1(mTORC1)-dependent manner.The data provides in vitro evidence and a mechanism for the protective effect of glycine against the disruption of intestinal barrier integrity induced by ER stress.

Hydroxyproline alleviates 4-hydroxy-2-nonenal-induced DNA damage and apoptosis in porcine intestinal epithelial cells

Oxidative stress has been confirmed in relation to intestinal mucosa damage and multiple bowel diseases.Hydroxyproline (Hyp) is an imino acid abundant in sow's milk. Compelling evidence has beengathered showing the potential antioxidative properties of Hyp. However, the role and mechanism ofHyp in porcine intestinal epithelial cells in response to oxidative stress remains unknown. In this study,small intestinal epithelial cell lines of piglets (IPEC-1) were used to evaluate the protective effects of Hypon 4-hydroxy-2-nonenal (4-HNE)-induced oxidative DNA damage and apoptosis. IPEC-1 pretreated with0.5 to 5 mmol/L Hyp were exposed to 4-HNE (40 mmol/L) in the presence or absence of Hyp. Thereafter,the cells were subjected to apoptosis detection by Hoechst staining, flow cytometry, and Western blot orDNA damage analysis by comet assay, immunofluorescence, and reverse-transcription quantitative PCR(RT-qPCR). Cell apoptosis and the upregulation of cleaved-caspase-3 induced by 4-HNE (40 mmol/L) wereinhibited by 5 mmol/L of Hyp. In addition, 5 mmol/L Hyp attenuated 4-HNE-induced reactive oxygenspecies (ROS) accumulation, glutathione (GSH) deprivation and DNA damage. The elevation in transcriptionof GADD45a (growth arrest and DNA-damage-inducible protein 45 alpha) and GADD45b(growth arrest and DNA-damage-inducible protein 45 beta), as well as the phosphorylation of H2AX(H2A histone family, member X), p38 MAPK (mitogen-activated protein kinase), and JNK (c-Jun N-terminalkinase) in cells treated with 4-HNE were alleviated by 5 mmol/L Hyp. Furthermore, Hyp supplementationincreased the protein abundance of Krüppel like factor 4 (KLF4) in cells exposed to 4-HNE.Suppression of KLF4 expression by kenpaulone impeded the resistance of Hyp-treated cells to DNAdamage and apoptosis induced by 4-HNE. Collectively, our results indicated that Hyp serves to protectagainst 4-HNE-induced apoptosis and DNA damage in IPEC-1 cells, which is partially pertinent with theenhanced expression of KLF4. Our data provides an updated explanation for the nutritional values ofHyp-containing animal products.

Flavor supplementation during late gestation and lactation periods increases the reproductive performance and alters fecal microbiota of the sows

This study was conducted to evaluate the effect of flavor on reproductive performance and fecal microbiota of sows during late gestation and lactation.A total of 20 healthy Yorkshire sows were fed a corn-soybean basal diet unsupplemented or supplemented with 0.1%flavor compound from d 90 of gestation to 25 d post-farrowing,and then the piglets were weaned.The reproductive performance and the fecal microbiota of sows were analyzed.Compared with the controls,flavor supplementation in maternal diets increased(P<0.05)weaning litter weight,litter weight gain,weaning body weight,and average daily gain of piglets.There was a trend of increase in the average daily feed intake of sows(P=0.09)by maternal dietary flavor addition.The backfat thickness and litter size were not affected by flavor supplementation(P>0.05).The 16S rRNA analysis showed that flavor supplementation signifi-cantly increased the abundance of Phascolarctobacterium(P<0.05),but significantly decreased genera Terrisporobacter,Alloprevotella,Clostridium_sensu_stricto_1,and Escherichia-shigella(P<0.05).Spearman correlation analysis showed that Phascolarctobacterum was positively correlated with the average daily feed intake of sows(P<0.05),the litter weight gain and average daily gain of piglets(P<0.05).In contrast,Clostridium_sensu_stricto_1 and unclassified_f__Lachnospiraceae were negatively correlated with the litter weight gain and average daily gain of piglets(P<0.05).Taken together,dietary flavor sup-plementation improved the reproductive performance of the sows,which was associated with enhanced beneficial microbiota and decreased potentially pathogenic bacteria in the sows.