Acute and persistent effects of oral glutamine supplementation on growth, cellular proliferation, and tight junction protein transcript abundance in jejunal tissue of low and normal birthweight pre-weaning piglets.

Breeding for higher fertility has resulted in a higher number of low birthweight (LBW) piglets. It has been shown that LBW piglets grow slower than normal birthweight (NBW) littermates. Differences in growth performance have been associated with impaired small intestinal development. In suckling and weaning piglets, glutamine (Gln) supplementation has been associated with improved growth and intestinal development. This study was designed to examine the effects of oral Gln supplementation on growth and small intestinal parameters in LBW and NBW suckling piglets. At birth (day 0), a total of 72 LBW (1.10 ± 0.06 kg) and 72 NBW (1.51 ± 0.06) male piglets were selected. At day 1, litters were standardized to 12 piglets, and experimental piglets supplemented daily with either Gln (1 g/kg BW) or isonitrogenous amounts of Alanine (Ala) as control (1.22 g/kg BW) until day 12. Creep feed was offered from day 14 onward. Subgroups of piglets were euthanized at days 5, 12, and 26 for the analyses of jejunal morphometry, cellular proliferation, glutathione concentration and transcript abundance of tight junction proteins. From age day 11 to 21, Gln supplemented LBW (LBW-Gln) piglets were heavier than Ala supplemented LBW (LBW-Ala) littermates (P = 0.034), while NBW piglets were heavier until age day 26 compared to LBW littermates. Villus height was higher in LBW-Gln compared to LBW-Ala on age day 12 (P = 0.031). Sporadic differences among supplementation and birthweight groups were detected for jejunal cellular proliferation, cellular population and glutathione concentration, whereas age was the most dominant factor. These results show that Gln supplementation improved the growth of LBW piglets compared to LBW-Ala beyond the termination of Gln supplementation, but this was not associated with consistent effects on selected parameters of jejunal development.

Identification and Quantification of Proliferating Cells in Skeletal Muscle of Glutamine Supplemented Low- and Normal-Birth-Weight Piglets.

One way to improve the growth of low-birth-weight (LBW) piglets can be stimulation of the cellular development of muscle by optimized amino acid supply. In the current study, it was investigated how glutamine (Gln) supplementation affects muscle tissue of LBW and normal-birth-weight (NBW) piglets. Longissimus and semitendinosus muscles of 96 male piglets, which were supplemented with 1 g Gln/kg body weight or alanine, were collected at slaughter on day 5 or 26 post natum (dpn), one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Immunohistochemistry was applied to detect proliferating, BrdU-positive cells in muscle cross-sections. Serial stainings with cell type specific antibodies enabled detection and subsequent quantification of proliferating satellite cells and identification of further proliferating cell types, e.g., preadipocytes and immune cells. The results indicated that satellite cells and macrophages comprise the largest fractions of proliferating cells in skeletal muscle of piglets early after birth. The Gln supplementation somewhat stimulated satellite cells. We observed differences between the two muscles, but no influence of the piglets' birth weight was observed. Thus, Gln supplements may not be considered as effective treatment in piglets with low birth weight for improvement of muscle growth.

Effects of Oral Glutamine Supplementation, Birthweight and Age on Colonic Morphology and Microbiome Development in Male Suckling Piglets.

Mortality, impaired development and metabolic dysfunctions of suckling low-birthweight piglets may be influenced by modulating the intestinal microbiome through glutamine supplementation. Therefore, this study examined whether glutamine supplementation may affect the colonic development and microbiome composition of male low- and normal-birthweight piglets at 5 and 12 days of age. Suckling piglets were supplemented orally with glutamine or alanine. Colonic digesta samples were obtained for 16S rDNA sequencing, determination of bacterial metabolites and histomorphological tissue analyses. Glutamine-supplemented piglets had lower concentrations of cadaverine and spermidine in the colonic digesta (p < 0.05) and a higher number of CD3+ colonic intraepithelial lymphocytes compared to alanine-supplemented piglets (p < 0.05). Low-birthweight piglets were characterised by a lower relative abundance of Firmicutes, the genera Negativibacillus and Faecalibacterium and a higher abundance of Alistipes (p < 0.05). Concentrations of cadaverine and total biogenic amines (p < 0.05) and CD3+ intraepithelial lymphocytes (p < 0.05) were lower in low- compared with normal-birthweight piglets. In comparison to the factor age, glutamine supplementation and birthweight were associated with minor changes in microbial and histological characteristics of the colon, indicating that ontogenetic factors play a more important role in intestinal development.

Effects of oral glutamine supplementation on jejunal morphology, development, and amino acid profiles in male low birth weight suckling piglets.

BACKGROUND: It has been shown that small intestine development in low birth weight (LBW) piglets is impaired. Glutamine (Gln) has been reported to improve piglet health and intestinal function in weaned piglets, but data is scarce in suckling piglets. This study was conducted to investigate the effects of oral Gln supplementation compared to Alanine (Ala) on jejunal development and function in 5 and 12 d old male LBW and normal birth weight (NBW) suckling piglets. RESULTS: Gln had no effect on the jejunal morphology, development, tissue and digesta amino acid profiles and mRNA abundance of genes involved in amino acid transport, metabolism, glutathione synthesis in LBW piglets when compared to Ala supplementation and birth weight controls at 5 and 12 d. Only the concentration of Gln in jejunal tissue was higher in NBW piglets supplemented with Gln compared to Ala at 5 d (P < 0.05). A comparison of the birth weight groups showed no differences between LBW and NBW piglets at 5 and 12 d in any parameter. Jejunal crypt depth, villus height / width, tunica muscularis thickness, number of goblet and IgA positive cells, the ratio of jejunal RNA to DNA and the concentration of DNA, protein and RNA changed (P < 0.05) from 5 compared to 12 d. The concentrations of several free, and protein bound amino acids as well as amino metabolites differed between age groups in jejunal tissue but the digesta concentrations were affected to a lesser extent. CONCLUSIONS: Oral Gln supplementation to suckling male piglets over the first 12 d of life was not associated with changes in jejunal parameters measured in this study. The absence of effects may indicate that Gln is absorbed as well as metabolized in the upper intestinal tract and thus could benefit intestinal development at a more proximal location.

Glutamine supplementation moderately affects growth, plasma metabolite and free amino acid patterns in neonatal low birth weight piglets.

Low birth weight (LBW) neonates show impaired growth compared with normal birth weight (NBW) neonates. Glutamine (Gln) supplementation benefits growth of weaning piglets, while the effect on neonates is not sufficiently clear. We examined the effect of neonatal Gln supplementation on piglet growth, milk intake and metabolic parameters. Sow-reared pairs of newborn LBW (0·8-1·2 kg) and NBW (1·4-1·8 kg) male piglets received Gln (1 g/kg body mass (BM)/d; Gln-LBW, Gln-NBW; n 24/group) or isonitrogenous alanine (1·22 g/kg BM/d; Ala-LBW; Ala-NBW; n 24/group) supplementation at 1-5 or 1-12 d of age (daily in three equal portions at 07:00, 12:00 and 17:00 by syringe feeding). We measured piglet BM, milk intake (1, 11-12 d), plasma metabolite, insulin, amino acid (AA) and liver TAG concentrations (5, 12 d). The Gln-LBW group had higher BM (+7·5%, 10 d, P = 0·066; 11-12 d, P < 0·05) and milk intake (+14·7%, P = 0·015) than Ala-LBW. At 5 d, Ala-LBW group had higher plasma TAG (+34·7%, P < 0·1) and lower carnosine (-22·5%, P < 0·05) than Ala-NBW and Gln-LBW, and higher liver TAG (+66·9%, P = 0·029) than Ala-NBW. At 12 d, plasma urea was higher (+37·5%, P < 0·05) with Gln than Ala supplementation. Several proteinogenic AA in plasma were lower (P < 0·05) in Ala-NBW v. Gln-NBW. Plasma arginine was higher (P < 0·05) in Gln-NBW v Ala-NBW piglets (5, 12 d). Supplemental Gln moderately improved growth and milk intake and affected lipid metabolism in LBW piglets and AA metabolism in NBW piglets, suggesting effects on intestinal and liver function.

The Effect of Dietary Protein Imbalance during Pregnancy on the Growth, Metabolism and Circulatory Metabolome of Neonatal and Weaned Juvenile Porcine Offspring.

Protein imbalance during pregnancy affects women in underdeveloped and developing countries and is associated with compromised offspring growth and an increased risk of metabolic diseases in later life. We studied in a porcine model the glucose and urea metabolism, and circulatory hormone and metabolite profile of offspring exposed during gestation, to maternal isoenergetic low-high (LP-HC), high-low (HP-LC) or adequate (AP) protein-carbohydrate ratio diets. At birth, LP-HC were lighter and the plasma acetylcarnitine to free carnitine ratios at 1 day of life was lower compared to AP offspring. Plasma urea concentrations were lower in 1 day old LP-HC offspring than HP-LC. In the juvenile period, increased insulin concentrations were observed in LP-HC and HP-LC offspring compared to AP, as was body weight from HP-LC compared to LP-HC. Plasma triglyceride concentrations were lower in 80 than 1 day old HP-LC offspring, and glucagon concentrations lower in 80 than 1 day old AP and HP-LC offspring. Plasma urea and the ratio of glucagon to insulin were lower in all 80 than 1 day old offspring. Aminoacyl-tRNA, arginine and phenylalanine, tyrosine and tryptophan metabolism, histidine and beta-alanine metabolism differed between 1 and 80 day old AP and HP-LC offspring. Maternal protein imbalance throughout pregnancy did not result in significant consequences in offspring metabolism compared to AP, indicating enormous plasticity by the placenta and developing offspring.

Distinct Roles of Perilipins in the Intramuscular Deposition of Lipids in Glutamine-Supplemented, Low-, and Normal-Birth-Weight Piglets.

Piglets with low birth weight (LBW) usually have reduced muscle mass and increased lipid deposition compared with their normal-birth-weight (NBW) littermates. Supplementation of piglets with amino acids during the first days of life may improve muscle growth and simultaneously alter the intramuscular lipid deposition. The aim of the current study was to investigate the influence of glutamine (Gln) supplementation during the early suckling period on lipid deposition in the longissimus muscle (MLD) and the role of different perilipin (PLIN) family members in this process. Four groups were generated consisting of 72 male LBW piglets and 72 NBW littermates. Piglets were supplemented with either 1 g Gln/kg body weight or an isonitrogenous amount of alanine (Ala) between days post natum (dpn) 1 and 12. Twelve piglets per group were slaughtered at 5, 12, and 26 dpn, and muscle tissue was collected. Perilipins were localized by immunohistochemistry in muscle sections. The mRNA and protein abundances of PLIN family members and related lipases were quantified by quantitative RT-PCR (qPCR) and western blots, respectively. While PLIN1 was localized around lipid droplets in mature and developing adipocytes, PLIN2 was localized at intramyocellular lipid droplets, PLIN3 and 4 at cell membranes of muscle fibers and adipocytes, and PLIN5 in the cytoplasm of undefined cells. The western blot results indicated higher protein abundances of PLIN2, 3, 4, and 5 in LBW piglets (p < 0.05) at 5 dpn compared with their NBW littermates independent of supplementation, while not directly reflecting the mRNA expression levels. The mRNA abundance of PLIN2 was lower while PLIN4 was higher in piglets at 26 dpn in comparison with piglets at 5 dpn (p < 0.01). Relative mRNA expression of LPL and CGI-58 was lowest in piglets at 5 dpn (p < 0.001). However, ATGL mRNA was not influenced by birth weight or supplementation, but the Spearman correlation coefficient analysis revealed close correlations with PLIN2, 4, and 5 mRNA at 5 and 26 dpn (r > 0.5, p < 0.001). The results indicated the importance of birth weight and age for intramuscular lipid deposition and different roles of PLIN family members in this process, but no clear modulating effect of Gln supplementation.

Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets.

Muscle growth of low birth weight (LBW) piglets may be improved with adapted nutrition. This study elucidated effects of glutamine (Gln) supplementation on the cellular muscle development of LBW and normal birth weight (NBW) piglets. Male piglets (n = 144) were either supplemented with 1 g Gln/kg body weight or an isonitrogeneous amount of alanine (Ala) between postnatal day 1 and 12 (dpn). Twelve piglets per group were slaughtered at 5, 12 and 26 dpn, one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Muscle samples were collected and myogenic cells were isolated and cultivated. Expression of muscle growth related genes was quantified with qPCR. Proliferating, BrdU-positive cells in muscle sections were detected with immunohistochemistry indicating different cell types and decreasing proliferation with age. More proliferation was observed in muscle tissue of LBW-GLN than LBW-ALA piglets at 5 dpn, but there was no clear effect of supplementation on related gene expression. Cell culture experiments indicated that Gln could promote cell proliferation in a dose dependent manner, but expression of myogenesis regulatory genes was not altered. Overall, Gln supplementation stimulated cell proliferation in muscle tissue and in vitro in myogenic cell culture, whereas muscle growth regulatory genes were barely altered.

Effects of Oral Glutamine Supplementation on Early Postnatal Muscle Morphology in Low and Normal Birth Weight Piglets.

Adapted nutrition can improve the growth of low birth weight (LBW) piglets. Since maternal milk is thought to provide insufficient glutamine (Gln) for LBW piglets, the current study investigated the influence of Gln supplementation during the early suckling period on development and lipid deposition in skeletal muscle. The weight differences between LBW and normal birth weight (NBW) littermates persisted from birth to slaughter (p < 0.001). However, intramuscular Gln and Ala concentrations were altered in piglets according to the supplementation (p < 0.01). There were larger muscle fibers (p = 0.048) in Gln-supplemented piglets. Capillarization or nuclei number per muscle fiber was not influenced by birth weight (BiW) or Gln supplementation. Abundance of myosin heavy chain (MYH) isoforms was slightly altered by Gln supplementation. LBW piglets had more lipid droplets than NBW piglets at day 5 of life in both muscles (p < 0.01). The differences decreased with age. Adipocyte development increased with age, but was not influenced by BiW or supplementation. The results indicate that BiW differences were accompanied by differences in lipid deposition and muscle fiber structure, suggesting a delayed development in LBW piglets. Supplementation with Gln may support piglets to overcome those disadvantages.

Transcript profile of skeletal muscle lipid metabolism genes affected by diet in a piglet model of low birth weight.

Dysregulated skeletal muscle metabolism (DSMM) is associated with increased inter- and intramuscular fat deposition in low birth weight (L) individuals. The mechanisms behind DSMM in L individuals are not completely understood but decreased muscle mass and shifts in lipid and carbohydrate utilisation may contribute. Previously, we observed lower fat oxidation in a porcine model of low birth weight. To elucidate the biological activities underpinning this difference microfluidic arrays were used to assess mRNA associated with lipid metabolism in longissimus dorsi (LD) and semitendinosus (ST) skeletal muscle samples from thirty-six female L and normal birth weight (N) pigs. Plasma samples were collected from a sub-population to measure metabolite concentrations. Following overnight fasting, skeletal muscle and plasma samples were collected and the association with birth weight, diet and age was assessed. Reduced dietary fat was associated with decreased LD intermuscular fat deposition and beta-oxidation associated mRNA, in both birth weight groups. Lipid uptake and intramuscular fat deposition associated mRNA was reduced in only L pigs. Abundance of ST mRNA associated with lipolysis, lipid synthesis and transport increased in both birth weight groups. Lipid uptake associated mRNA reduced in only L pigs. These changes were associated with decreased plasma L glucose and N triacylglycerol. Post-dietary fat reduction, LD mRNA associated with lipid synthesis and inter- and intramuscular fat deposition increased in L, whilst beta-oxidation associated mRNA remains elevated for longer in N. In the ST, mRNA associated with lipolysis and intramuscular fat deposition increased in both birth weight groups, however this increase was more significant in L pigs and associated with reduced beta-oxidation. Analysis of muscle lipid metabolism associated mRNA revealed that profile shifts are a consequence of birth weight. Whilst, many of the adaptions to diet and age appear to be similar in birth weight groups, the magnitude of response and individual changes underpin the previously observed lower fat oxidation in L pigs.

Parenteral administration of l-arginine to twin-bearing Romney ewes during late pregnancy is associated with reduced milk somatic cell count during early lactation.

Maternal milk is the primary source of nutrition for suckling mammals, and its yield and composition are important determinants of survival during the early neonatal period. The objective of this study was to examine whether parenteral administration of l-Arg to twin-bearing ewes, during mid to late pregnancy, influenced prepartum maternal mammary gland development and subsequent lactation performance in the early postpartum period (14 d). At 80 d of pregnancy, multiparous Romney ewes were housed indoors in group pens, split into 2 cohorts, and fed a lucerne-based pellet diet, formulated to meet 100% of National Research Council-recommended requirements for twin-bearing pregnant ewes, once a day. Cohort 1 was administered l-Arg (72.7 mg/kg of live weight via i.v, 3 times a day) from d 100 of pregnancy until d 140. At d 140, ewes were euthanized and maternal mammary tissues were collected for analysis of the biochemical indices total DNA, RNA, protein, protein synthetic efficiency (protein:RNA), cell size (protein:DNA), transcriptional efficiency (RNA:DNA), and the abundance of mammalian target of rapamycin (mTOR) and mTORSer2448 protein. Cohort 2 was administered an identical l-Arg regimen as cohort 1, but from d 100 until parturition. Milk was collected over a 14-d period (d 1, 4, 7, 10, and 14) to assess milk yield and composition. In cohort 1, total mammary DNA (cell number) tended to be higher in l-Arg ewes, with no change in total mammary RNA or protein content, biochemical indices of protein synthetic efficiency, cell size or transcriptional efficiency, or mTOR protein abundance or phosphorylation. In cohort 2, milk composition analysis from l-Arg ewes showed lower (d 7-14) milk somatic cell counts, greater crude protein percentage from d 7 to 10 but lower at d 14, and altered absolute concentrations of some free AA (d 7 and 14) compared with controls. We propose that parenteral administration of l-Arg during late pregnancy is associated with increased mammary gland cellular content and decreased somatic cell counts during early lactation.

Amino acids in sheep production.

Increasing production efficiency with a high standard of animal welfare and respect for the environment is a goal of sheep farming systems. Substantial gains in productivity have been achieved through improved genetics, nutrition and management changes; however the survival and growth performance of multiple-born lambs still remains a problem. This is a significant production efficiency and animal well-being issue. There is a growing body of evidence that some amino acids have a role in regulating growth, reproduction and immunity through modulation of metabolic and cell signaling pathways. The purpose of this review is to provide an overview of what is currently known about the role of amino acids in sheep production and the potential for supplementation strategies to influence on-farm survival and growth of lambs.

Administration of Exogenous Growth Hormone Is Associated with Changes in Plasma and Intracellular Mammary Amino Acid Profiles and Abundance of the Mammary Gland Amino Acid Transporter SLC3A2 in Mid-Lactation Dairy Cows.

The objectives of this study were to (1) identify changes in plasma and mammary intracellular amino acid (AA) profiles in dairy cows treated with growth hormone (GH), and (2) evaluate the expression of mammary gland genes involved in the transport of AA identified in (1). Eight non-pregnant (n = 4 per group) lactating dairy cows were treated with a single subcutaneous injection of either a slow-release formulation of commercially available GH (Lactotropin 500 mg) or physiological saline solution. Six days after treatment, cows were milked and blood collected from the jugular vein for the analysis of free AA in the plasma. Cows were euthanized and mammary tissue harvested. Treatment with GH increased milk, protein, fat and lactose yields, with no effect on dry matter intake. Plasma concentrations of lysine and group I AA decreased significantly, and arginine, methionine, tyrosine and arginine-family AA tended to decrease in GH-treated cows. Concentrations of intracellular glycine, serine and glutamate increased significantly, with a trend for decreased arginine observed in the mammary gland of GH-treated cows. A trend for increased concentrations of intracellular total AA, NEAA and arginine-family AA were observed in the mammary gland of GH-treated cows. Variance in the concentration of plasma methionine, tyrosine, valine, alanine, ornithine, BCAA, EAA was significantly different between treatments. Variance in the concentration of intracellular lysine, valine, glutamine, EAA and group II was significantly different between treatments. AA changes were associated with increased mRNA abundance of the mammary gland AA transporter SLC3A2. We propose that these changes occur to support increased milk protein and fatty acid production in the mammary gland of GH-treated cows via potential mTOR pathway signaling.

Identification of the dialysable serum inducer of germ-tube formation in Candida albicans.

Yeast cells of Candida albicans are induced by serum at 37 degrees C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be d-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that d-glucose was the only active component in these fractions. Induction of germ-tube formation by d-glucose required a temperature of 37 degrees C and the pH optimum was between pH 7.0 and 8.0. d-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although d-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1.4 % (v/v) serum or an equivalent concentration of d-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8.5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.

Deletion of the Candida albicans G-protein-coupled receptor, encoded by orf19.1944 and its allele orf19.9499, produces mutants defective in filamentous growth.

Filamentous growth of Candida albicans occurs in response to a variety of environmental signals. The C. albicans gene orf19.1944 and its allele orf19.9499 are identical and are predicted to encode an 823-residue, 7-transmembrane-domain protein that has all the expected features of a G-protein-coupled receptor. The protein is 20.9% identical to the Saccharomyces cerevisiae Gpr1p receptor that signals both glucose availability and nitrogen limitation. Deletion of both copies of the gene in C. albicans abolished filamentation by colonies embedded in rich media (YPS, YPGal, and YPGlu), whereas mutants carrying a single copy of the gene were indistinguishable from the parental strain under these conditions. On medium containing low concentrations of ammonia (SLAD and SLAM media), surface colonies of both the homozygous deletion mutants and the mutants carrying a single copy of the gene were defective in filamentation. Serum-induced germ tube formation was unaffected by deletion of this gene, as was filamentation of the mutants growing on the surface of solid Spider medium at 37 degrees C or embedded in solid Spider medium at 25 degrees C. The protein encoded by orf19.1944 and orf19.9499 has a role in filamentation by both surface and embedded colonies, presumably as a sensor of environmental cues.