The trend towards high ovulation rates in mature commercial sows has resulted in intra-uterine crowding in the immediate post-implantation period, with negative impacts on placental development and later fetal development (Town et al. 2004).Factors that improve placental angiogenesis could offset the effects of intra-uterine crowding by supporting placental development at critical times in gestation.Feeding of L-arginine has been shown to have beneficial effects on placental vascularization in gilts (Hazeleger et al. 2007) and on litter size born in gilts (Mateo et al. 2007) and sows (Ramaekers et a/. 2006).In the present study, we investigated the effects of L-arginine supplementation to commercial sows on global placental gene expression, and on temporal changes in the expression of a panel of eight candidate genes known to be involved in angiogenesis, in early pregnancy.Multiparous sows (n -48) were either non-supplemented Controls or were fed an L-arginine supplement (20g.d)from d 15 through 29 of gestation.A representative number of sows were euthanized on days 16 through 49 of gestation and embryonic and placental tissues were collected from two average-sized conceptuses from each uterine horn and placed in RNAlater for later analysis.To obtain temporal expression profiles for specific genes involved in placental angiogenesis, total placental RNA was extracted from all Control samples collected, reverse transcribed and real-time PCR used to determine the transcript abundance of: vascular endothelial growth factor (VEGF) -A; the two VEGF receptors, fms-related tyrosine kinase 1 (FLT1) and fetal liver kinase-1(flk-1/KDR); hypoxia-inducible factor (HIF)1A; the Angiopoietins (ANGPT) -1 and -2 and their receptor, TEK tyrosine kinase; and finally Angiogenin (ANG) -1.The delta ACt values were calculated using 185 as an internal control, and data were analyzed using regression analysis (SAS Institute Inc., Cary, NC).To determine the cumulative effect of L-arginine treatment, real-time PCR for these same candidate genes was also performed on the d 30 placental samples from both Control and L-arginine sows.The relative ACt values for d 30 samples were again calculated using 185 as an internal control and data were analyzed using MIXED models (SAS Institute Inc., Cary, NC).Effects of L-arginine on global placental gene expression (n =4 representative sows per treatment) were also analyzed using PigOligoArray slides and placental tissues collected at d 30 of gestation.Total RNA was extracted, purified using mRNA mini kits (lnvitrogen), amplified with aminoallyl mRNA amplification kit (Ambion), and labeled with Cy3 or Cy5 in a random block dye-swap design.The hybridized slide images were captured with Genepix software and an Axon Scanner set for optimized PMT for each dye.Median spot intensities underwent Loess and quantile normalization and were analyzed using linear models, all in limma (Smyth, 2004).
The supplementation of total parenteral nutrition (TPN) formulas with short-chain fatty acids (SCFAs) increases glucose uptake and the expression of glucose transporters in parenterally fed animals. Several signals may be involved in intestinal adaptation; however, increased messenger RNA (mRNA) levels for proglucagon and several early-response genes, including c-myc and c-fos, are seen in animals receiving SCFA-supplemented TPN. Although the effects of a mixture of SCFAs are well documented, the relative contribution of individual SCFAs is unknown. Butyrate is a preferred fuel of colonocytes, with documented effects on cellular proliferation and gene expression. Accordingly, this study was undertaken to determine the relative role of butyrate in initiating an adaptive response in nonresected rats receiving TPN.Animals received standard TPN for 66 hours, followed by 6 hours of either standard TPN, TPN supplemented with a mixture of SCFAs (acetate, propionate, and butyrate, 60 mmol/L total), or TPN supplemented with butyrate alone (9 mmol/L). An oral control group was fed an elemental diet, similar in macronutrient content to the TPN, so that all animals received the same amount of energy daily.SCFAs increased ileal glucose transporter 2 (GLUT2) mRNA expression compared with the orally fed group. SCFAs also increased proglucagon mRNA expression compared with the TPN group. No changes in Na+K(+)-adenosine triphosphatase or early-response gene expression were found in this study.In a rat model of TPN, the use of 9 mmol/L butyrate did not have the same effect on GLUT2 and proglucagon expression as a 60-mmol/L mixture of SCFAs. This suggests that the effect of a mixture of SCFAs on intestinal gene expression is not butyrate specific.
The objectives of the current experiment were to determine whether boars heterozygous for the mutation in skeletal ryanodine receptors (sRyR), known to cause porcine stress syndrome, differed from wild-type boars in hypothalamic-pituitary-adrenal axis (HPA) function. We have examined basal plasma ACTH, cortisol, and corticosteroid-binding globulin (CBG) concentrations; plasma ACTH and cortisol responses to a nose-snare stressor and at slaughter; dexamethasone suppression of plasma ACTH and cortisol concentrations; and glucocorticoid receptor (GR) density in the pituitary gland, hippocampus, hypothalamus, and frontal cortex. We have also examined carcass yields, composition, and meat quality to determine whether differences in HPA activity were accompanied by an increased incidence of meat quality characteristics associated with pale, soft, exudative (PSE) meat. Thirty boars either heterozygous or wild-type (n = 15 per genotype) for mutated sRyR were tested for HPA function at 7 mo of age. Heterozygous boars had lower basal plasma ACTH (P < .05) and cortisol (P < .04) concentrations. Integrated basal plasma ACTH and cortisol levels were also lower (P < .05 and P < .005, respectively). Genotype had no significant effect on basal CBG, stressor-induced (nose snare or slaughter) or dexamethasone suppression of plasma ACTH or cortisol concentrations. No differences in immunoreactive GR levels were found in the pituitary gland or any brain region examined. We did find a significant, negative correlation (r = −.62, P < .02) between peak (0800) basal plasma ACTH concentrations and hippocampal GR levels. The alterations in basal HPA function in heterozygous boars were accompanied by lighter body weights (P < .03), decreased carcass fat depth (P < .04), and increased carcass lean yields (P < .02). There was a higher incidence of meat quality characteristics associated with PSE meat in heterozygous boars indicated by higher carcass temperatures (P < .04) and meat brightness (P < .0001) with lower carcass pH at slaughter (P < .03) and after chilling (P < .003). In conclusion, we have found differences in basal and not stressor-induced HPA function between boars heterozygous and wild-type for mutated sRyR. This altered basal HPA activity was accompanied by an increased incidence of meat quality aspects associated with PSE meat in heterozygous boars.
Neonatal handling permanently alters hypothalamic- pituitary-adrenal axis (HPA) function in rats. In the rat, this treatment increases hippocampal glucocorticoid receptors (GR) and dampens plasma ACTH and corticosterone responses to stressors. The objectives of this study were to determine whether neonatal handling of pigs would effect permanent changes in plasma corticosteroid binding capacity (CBG), basal or stressor-induced plasma cortisol and ACTH concentrations, brain or pituitary GR levels, dexamethasone suppression of plasma cortisol and ACTH concentrations, behaviour in an open field-test pen, and body weights. Twelve litters of pigs were randomly assigned to either neonatal handling or no disturbance. Handled litters were removed from the farrowing crate for 10 min per day for the first 14 days of life. Male pigs were kept for the study and the boars were weighed monthly. At 7 months of age, boars were tested for locomotory behaviour in an open field-test pen. The boars were implanted with indwelling ear-vein catheters and blood samples were obtained basally, during and after application of a nose snare, and after 0.04 mg/kg dexamethasone. Boars were killed and blood samples were obtained and the brain and pituitary glands collected. Handled boars had greater (P<0.05) plasma CBG binding and lower basal total (P<0.05) and calculated free (P<0.03) plasma cortisol concentrations. No significant differences between treatments were found in plasma ACTH or cortisol responses to a nose-snare stressor; however, when killed, handled boars had greater (P<0.02) plasma ACTH concentrations. Handled and non-handled boars did not differ in plasma ACTH or cortisol responses to dexamethasone. There was no treatment effect on GR expression in the pituitary gland, frontal cortex, hippocampus, or hypothalamus. Behaviourally, the handled boars had higher (P<0.03) locomotor scores over inner squares and a lower (P<0.05) ratio of outer:inner squares entered in open field-tests. During the first 7 months of life, body weights were lower (P<0.004) for handled boars. In conclusion, neonatal handling permanently altered HPA function in pigs, but in a manner dissimilar to that found in the rat. These changes induced in the pig were not beneficial for commercial production with respect to body weight.
Adipogenesis is of significant relevance from an agricultural perspective. Traits such as subcutaneous fat thickness, marbling and waste fat are of substantial economic importance in animal production. In order to discover more about the genetic basis of this process, a study was undertaken to examine the changes that occur daily in global gene expression as 3T3-L1 cells differentiate from preadipocyte to adipocyte. Duplicate RNA samples were collected daily during the differentiation process and probed with the Affymetrix U74Av2 GeneChip ® microarray to allow the time-course analysis of the gene expression profile in these differentiating cells. Self-organizing maps (SOM) clustering was performed to extract patterns of expression over the course of the experiment (day 0 to day 6). The clustering generated nine distinct expression patterns containing between 74 and 420 genes/ESTs. Functional clusters and important chronological changes in the expression of key genes and gene groups were identified. The pattern of expression observed for many genes not only confirmed what has been shown previously for the early stages of differentiation, but also expanded this pattern to cover the whole differentiation process thus giving a very comprehensive overview of patterns and changes in gene expression over the time course of adipocyte differentiation. Key words: Adipocyte differentiation, gene expression, SOM clustering
It is considered that the rate of glucose uptake by the mammary gland is the main regulator of lactose synthesis (Threadgold and Kuhn, 1984) and hence the main factor determining milk production (Faulkner and Peaker, 1987). GLUT1 is the only glucose transporter that has been found in lactating mammary tissue (Burnol et al, 1990). The aim of this experiment was to determine whether GLUT1 mRNA could be detected in sow mammary tissue, to investigate how abundance of GLUT 1 mRNA in mammary tissue changed between late gestation, early lactation and established lactation, and to establish whether this was correlated with milk output and/or feed intake.
Activation of β adrenergic receptors increased heat production in sheep as much as 40 %, whereas α-2 receptor activation reduced heat production by up to 20 %. Expression profiles for genes related to cellular efficiency (Uncoupling protein-2,-3), whole animal orectic (Neuropeptide-Y) and satiety signaling (Leptin) as well as cell surface receptors involved in mediating metabolic activity of cells (adrenergic receptors, leptin receptors and neuropeptide-Y receptors) were examined in ruminant tissues (biceps femoris (BF) muscle, heart, liver, rumen, abomasum, duodenum, subcutaneous adipose (SA), peri-renal adipose (PA) and mesenteric adipose (MA)). Comparison of the gene expression profiles was made with measurements of heat production, metabolizable energy intake, weight gain and retained energy values. We found greater variation in heat production between animals within a breed than between breeds, and this was mirrored by variability in gene expression between individual animals. There was a positive correlation between expression of uncoupling protein -2 (UCP-2) mRNA in skeletal muscle, sub-cutaneous adipose and mesenteric adipose as compared to average daily gain (ADG). We documented expression of uncoupling protein-3 (UCP-3) in bovine cardiac muscle and found that this showed modest negative correlation with retained energy. It was also of note that cardiac muscle expression of neuropeptide-Y receptor message was negatively correlated with both efficiency of energy retention and maintenance ME requirement across all breeds and feeding levels. Expression patterns for leptin receptor mRNA in mesenteric and subcutaneous adipose are positively correlated with ADG. Acute cold exposure increased UCP-2 expression specifically in peripheral tissue (skeletal muscle and sub-cutaneous adipose) (P < 0.01), whereas, winter acclimatization enhanced expression of UCP-3 in muscle (P < 0.05). UCP-2, UCP-3, leptin receptor and NPY receptor genes appear to be good potential candidates as useful markers of energetic efficiency but future research is required to establish accuracy and reliability.
Thirty-six steers were used to determine the effects of acute cold (-20°C) and acute feed restriction (0.4× maintenance) on mRNA abundance of five candidate genes. To investigate adaptational physiology in cattle we evaluated gene expression patterns for uncoupling protein 2 (UCP-2), uncoupling protein 3 (UCP-3), leptin, leptin receptor and neuropeptide Y receptor (NPY receptor) in specific tissues. We measured relative mRNA abundance using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) on mRNA extracted from duodenum, iver, skeletal muscle, mesenteric adipose tissue, subcutaneous adipose tissue, and peri-renal adipose tissue. We obtained carcass grades for comparison to mRNA abundance profiles. Leptin receptor mRNA abundance was increased by acute feed restriction in the liver (P = 0.02) and trended to increase in the duodenum (P = 0.06) of acute feed-restricted steers. NPY receptor RNA abundance decreased (P = 0.02) in the liver of feed-restricted steers, and acute cold-stress-treated steers (P = 0.03), but increased (P = 0.03) in mesenteric adipose tissue of acute-cold-treated steers. Leptin mRNA abundance was decreased in subcutaneous adipose tissue (P = 0.04) by acute cold, but trended to increase in the peri-renal adipose tissue (P = 0.09). UCP-3 mRNA abundance was not significantly altered by our acute treatments. UCP-2 mRNA abundance increased in subcutaneous adipose tissue of both acute cold (P = 0.004) and acute feed-restricted steers (P = 0.03) and in biceps femoris, after acute cold (P = 0.003) and acute feed restriction (P = 0.002). There was an inverse relationship between the leptin mRNA abundance in subcutaneous adipose and the carcass grade, where the AAA , AA and A grade carcasses had mean values of 501.6 ± 30.0, 535.5 ± 14.0 and 653.4 ± 61.0. Our results show that tissue-specific mRNA abundance of these candidate genes may be altered by acute stressors, and that may relate to tissue-specific adaptational strategies in cattle. Furthermore, leptin mRNA abundance in subcutaneous adipose tissue may inversely reflect the maturity or finishing characteristics of beef cattle such that it is inversely related to the final carcass grade. Key words: Acute cold, acute feed restriction, UCP-2, UCP-3, leptin, leptin receptor, NPY receptor, cattle
