Review: Methods and biomarkers to investigate intestinal function and health in pigs.

Society is becoming increasingly critical of animal husbandry due to its environmental impact and issues involving animal health and welfare including scientific experiments conducted on farm animals. This opens up two new fields of scientific research, the development of non- or minimally invasive (1) methods and techniques using faeces, urine, breath or saliva sampling to replace existing invasive models, and (2) biomarkers reflecting a disease or malfunction of an organ that may predict the future outcome of a pig's health, performance or sustainability. To date, there is a paucity of non- or minimally invasive methods and biomarkers investigating gastrointestinal function and health in pigs. This review describes recent literature pertaining to parameters that assess gastrointestinal functionality and health, tools currently used to investigate them, and the development or the potential to develop new non- and minimally invasive methods and/or biomarkers in pigs. Methods described within this review are those that characterise gastrointestinal mass such as the citrulline generation test, intestinal protein synthesis rate, first pass splanchnic nutrient uptake and techniques describing intestinal proliferation, barrier function and transit rate, and microbial composition and metabolism. An important consideration is gut health, and several molecules with the potential to act as biomarkers of compromised gut health in pigs are reported. Many of these methods to investigate gut functionality and health are considered 'gold standards' but are invasive. Thus, in pigs, there is a need to develop and validate non-invasive methods and biomarkers that meet the principles of the 3 R guidelines, which aim to reduce and refine animal experimentation and replace animals where possible.

Invited review: impact of specific nutrient interventions during mid-to-late gestation on physiological traits important for survival of multiple-born lambs.

To improve production efficiency, the sheep meat industry has increased flock prolificacy. However, multiple-born lambs have lower birth weights, increased mortality and reduced growth rate compared with single-born lambs. Lamb mortality is a major issue for livestock farming globally and solutions are required to increase survival to realise the value of increased flock fecundity. Nutrition during gestation can influence maternal-foetal placental nutrient transfer and thus foetal growth and organ/tissue development, as well as improve postnatal productivity. This review covers the challenges and opportunities associated with increased prolificacy, highlights gaps in our knowledge and identifies some opportunities for how targeted intervention with specific nutrients during mid-to-late pregnancy may influence lamb survival and productivity with a specific focus on pasture-based systems. This time frame was selected as intervention strategies in short-time windows post-pregnancy scanning and before lambing to improve lamb survival in high-risk groups (e.g. triplets) are likely to be the most practical and economically feasible options for pasture-based extensive farming systems.

Intravenous maternal -arginine administration to twin-bearing ewes, during late pregnancy, is associated with increased fetal muscle mTOR abundance and postnatal growth in twin female lambs.

The aims of this study were to determine whether parenteral Arg administered to well-fed twin-bearing ewes from 100 to 140 d of pregnancy influences fetal skeletal muscle growth, the abundance and activation of mechanistic target of rapamycin (mTOR) protein, and postnatal muscle growth of the offspring. Ewes fed 100% of NRC-recommended nutrient requirements for twin-bearing ewes were administered an intravenous bolus of either 345 µmol Arg HCl/kg BW or saline solution (Control) 3 times per day. At 140 d of pregnancy (P140), a group of 11 Control and 9 Arg-treated ewes were euthanized and hind leg muscles and longissimus dorsi (LD) were excised and weighed. A sample of LD was snap frozen in liquid nitrogen for later analysis of free AA (FAA) concentration, mTOR abundance and phosphorylation, and biochemical indices (DNA, RNA, and protein content). For the remaining 25 ewes (Arg, = 13, and Control, = 12), Arg administration was continued until the initiation of parturition and ewes were allowed to lamb. Lambs were weaned at postnatal Day 82 and grazed on pasture until postnatal day 153 (PN153), when a subset of 20 lambs ( = 10 per group) was euthanized. At P140, only the psoas major was heavier in the Arg-administered group compared with the Control group. Female lambs from ewes supplemented with Arg (Arg-F) had increased abundance of total mTOR, RNA concentration, and RNA:DNA ratio in LD compared with female lambs from Control ewes (Con-F), whereas males did not differ. At PN153, Arg-F were heavier than Con-F and had heavier LD and plantaris and a trend for heavier psoas major muscles compared with Con-F. In contrast, BW and individual muscle weights did not differ in male lambs. Lambs from Arg-treated ewes had heavier semimembranosus and tended to have heavier biceps femoris compared with Control lambs. The RNA concentration in LD was greater in Arg-F compared with Con-F, and DNA concentration was greater in the Arg group compared with the Control group. In conclusion, Arg administration to the ewe during gestation increases female lamb weight and muscle weight after birth and these changes are associated with altered mTOR protein abundance and have potential implications for sheep production.

Mammary transcriptome analysis of lactating dairy cows following administration of bovine growth hormone.

The galactopoietic effect of growth hormone (GH) in lactating ruminants is well established; however the mechanisms that mediate these effects are not well understood. The first objective of this study was to determine the effect of GH on the synthesis of the major casein and whey proteins. The second objective was to identify the genes and pathways that may be involved in mediating the effect of GH on milk synthesis. A single subcutaneous injection of a commercially available slow release formulation of GH (Lactatropin®), or physiological saline solution (control) was administered to non-pregnant dairy cows (n=4/group) in mid-late lactation. Milk samples were collected for composition analysis and mammary lobulo-alveolar tissue was collected postmortem 6 days post injection. Gene expression profiles were evaluated using either a 22 000 bovine complementary DNA microarray or quantitative PCR (qPCR), and microarrays were validated by qPCR. The yield of all the major casein and whey proteins was increased 32% to 41% in GH-treated cows, with the exception of α-lactalbumin yield which was elevated by 70% relative to controls. Treatment with GH treatment tended to increase the concentration of α-lactalbumin but had no effect on the concentration of any of the major milk proteins. Messenger RNA (mRNA) abundance of the major whey and casein genes, with the exception of α-s2-casein, was increased in response to GH compared with controls, which is consistent with the positive effect of GH on milk production. Treatment with GH treatment influenced the mRNA abundance of genes involved in cell growth and proliferation, transcriptional and translational regulation, actin cytoskeleton signalling, lipid metabolism and cell death. This study has provided new insights into the cell signalling that may be involved in mediating the effect of GH on milk production in the mammary gland of lactating dairy cows.

Intravenous maternal -arginine administration to twin-bearing ewes during late pregnancy enhances placental growth and development.

This study aimed to investigate if intravenous maternal Arg administration to well-fed twin-bearing ewes, from 100 to 140 d of gestation or birth, could enhance placental development and placental nutrient transport. Ewes received intravenous infusions of saline (control) or 345 µmol Arg HCl/kg of BW 3 times daily from d 100 of pregnancy (P100) to d 140 of pregnancy (P140; cohort 1) or from P100 to birth (cohort 2). At P140, ewes in cohort 1 were euthanized and individual placentae per fetus were dissected and placentomes were classed per type (A to D) and size (light to heavy). Placentome number and individual weight were recorded. As an indicator of placental nutrient transport, blood plasma was collected from the uterine ovarian vein (UOV), uterine artery (UA), and umbilical vein and artery at the time of euthanasia and analyzed for metabolites and free AA concentrations. The ewes in cohort 2 were allowed to lamb and lambs were weighed at birth. The expelled placenta was dissected and number of cotyledons and weights of total cotyledons, remaining fetal membranes, and total placenta were recorded. At P140, Arg-infused ewes had a 63% ( = 0.03) greater number of unoccupied caruncles than control ewes. No differences were observed for placental weight at P140. At birth, lambs from Arg-infused ewes tended to have 11% ( = 0.09) greater placental weight and 34% ( = 0.03) greater total cotyledon weight compared with control lambs. Arginine-infused ewes (Arg-infused) had increased concentrations of Arg ( = 0.0001) and ornithine (Orn; = 0.004) but decreased concentrations of Met ( = 0.01) and His ( = 0.02 and = 0.09, respectively) compared with control ewes in plasma UOV and UA. Fetuses from Arg-infused ewes had increased concentrations of Orn ( = 0.005) and decreased concentrations of His ( = 0.006), Met ( = 0.003), and Lys ( = 0.01) but no differences in Arg ( > 0.10) concentrations were found compared with control fetuses in umbilical artery and vein plasma. This study showed that maternal Arg administration of well-fed twin-bearing ewes during late pregnancy tended to improve placental growth and development.

Nutritional plane of twin-bearing ewes alters fetal mammary gland biochemical composition and mTOR/MAPK pathway signaling.

Identifying the biochemical changes and molecular pathways that regulate fetal mammary development in response to maternal nutrition is important for understanding the link between fetal programming of mammary development and future lactation performance. Although there are published studies regarding biochemical changes in the developing mammary gland, there are currently no data on molecular pathway involvement in regulating ruminant fetal mammary development. This study investigated changes in fetal mammary biochemical indices and mechanistic target of rapamycin (mTOR)/mitogen activated protein kinase (MAPK) signaling at d 100 and 140 of gestation in an ovine model of restricted maternal nutrition. Ewes were randomly allocated to ad libitum (A) or maintenance (M) nutritional regimens, under New Zealand pastoral grazing conditions, from d 21 to 140 of pregnancy. At d 100 and 140 of pregnancy, a subgroup of twin-bearing dams was euthanized, and whole fetal mammary glands (fiber, skin, fat, and ducts) were collected. Mammary glands of fetuses carried by M-fed dams were heavier at d 100 than those of fetuses carried by A-fed dams ( = 0.03), with no difference in the abundance of mTOR/MAPK signaling proteins observed. At d 140, mammary glands of fetuses carried by M-fed dams were lighter ( = 0.07) than fetuses carried by A-fed dams because of decreased hyperplasia ( = 0.04) and hypertrophy ( = 0.09) but had increased protein synthetic capacity ( = 0.02). Increased protein synthetic capacity was associated with increased abundance of MAPK pathway signaling proteins eukaryotic intiation factor 4E (eIF4E)/eIF4E and mTOR pathway signaling proteins eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/4E-BP1 and ribosomal protein S6 (RPS6)/RPS6 ( ≤ 0.05). Increased abundance of MAPK/mTOR pathway proteins is proposed to mediate increased protein synthetic capacity via ribosome biogenesis and the availability of factors required to initiate protein translation. The primary regulator of 4E-BP1 phosphorylation at Ser65 and RPS6 at Ser235/236 is the activated form of mTOR: mTOR. To study potential tissue-specific mTOR, mTOR abundance mammary glands, separated into parenchyma and fat pad, were collected from d 140 fetuses carried by dams fed a lucerne-based pellet diet formulated to meet 100% of the NRC-recommended maintenance requirements. Results showed that the abundance of mTOR was primarily localized to the fat pad, indicating that the fat pad plays a potential role in regulating development of the fetal mammary gland.

Increased milk protein synthesis in response to exogenous growth hormone is associated with changes in mechanistic (mammalian) target of rapamycin (mTOR)C1-dependent and independent cell signaling.

The objective of this study was to determine if increased milk protein synthesis observed in lactating dairy cows treated with growth hormone (GH) was associated with mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) regulation of downstream factors controlling nucleocytoplasmic export and translation of mRNA. To address this objective, biochemical indices of mammary growth and secretory activity and the abundance and phosphorylation status of mTORC1 pathway factors were measured in mammary tissues harvested from nonpregnant lactating dairy cows 6 d after treatment with a slow-release formulation of GH or saline (n=4/group). Treatment with GH increased mammary parenchymal weight and total protein content and tended to increase ribosome number and cell size, whereas protein synthetic efficiency, capacity, and cell number were unchanged. Cellular abundance of the mTORC1 components mTOR and (phosphorylated) mTOR(Ser2448) increased, as did complex eukaryotic initiation factor 4E:eukaryotic initiation factor 4E binding protein 1 (eIF4E:4EBP1), whereas no change was observed for mTORC1-downstream targets 4EBP1, 4EBP1(Ser65), p70/p85(S6K) and p70(S6K)Thre389/p85(S6K)Thre412. Changes in activation were not observed for any of the targets measured. These results indicate that GH treatment influences signaling to mTORC1 but not downstream targets involved in the nucleocytoplasmic export and translation of mRNA. Increased eIF4E:4EBP1 complex formation indicates involvement of the mitogen-activated protein kinase (MAPK) pathway. Abundance of MAPK pathway components eIF4E, eIF4E(Ser209), eIF4E:eIF4G complex, MAP kinase-interacting serine/threonine-protein kinase 1 (MKNK1), MKNK1(Thr197202), and ribosomal protein S6 kinase, 90kDa, polypeptide 1 (RPS6KA1) increased significantly in response to GH, whereas relative activation of the proteins was unchanged. Expression of IGFBP3 and IGFBP5 increased, that of IGF1R decreased, and that of IGF1 remained unchanged in response to GH. PatSearch analysis of the milk caseins αS1-casein, αS2-casein, and ß-casein, MAPK signaling target RPS6KA1, and proliferation gene IGFBP3 mRNA indicated that all contained putative eIF4E-sensitivity elements. In response to GH, these genes were all upregulated, suggesting that increased abundance of eIF4E and eIF4E(Ser209) plays a role in mediating their nucleocytoplasmic export. We propose that, in response to GH, the IGF1-IGF1R-MAPK signaling cascade regulates eIF4E-mediated nucleocytoplasmic export and translation of mRNA, whereas mTOR controls cell renewal, cell turnover, and rRNA transcription through an alternative signaling cascade.

Placental nutrient transport is affected by pregnancy rank in sheep.

Understanding the link between placental function and fetal growth is critical to comprehend the mechanisms underlying altered fetal growth. This study investigated the relationship between fetal weight and placentome type and size in placentae of singleton and twin fetuses and fetuses within a twin pair from ad libitum-fed ewes at d 140 of pregnancy. In addition, insulin, IGF-I, metabolites, and free AA profiles in fetal, umbilical artery, and vein plasma of singleton and twin fetuses were investigated and used as an indicator of placental nutrient transport. Individual placentae per fetus were dissected, placentomes were classed per type (A to D) and size (light to heavy), and placentome number and individual weight were recorded. Twin fetuses were 16% lighter (P = 0.01) than singletons and had a smaller placenta, with 28% decreased placentome weight (P = 0.03) and 35% fewer placentomes (P = 0.001). Twins also had a different distribution of placentome type and size compared with placentae of singletons, such that twins showed a greater proportion of type B and light placentomes compared with singletons. In twins, umbilical artery plasma had less Glu (P < 0.05) and greater Gln (P < 0.05) concentrations than fetal plasma or umbilical vein plasma, but no differences in AA concentrations were observed between these pools in singletons. Glutamate is a major oxidation energy source for the placenta, and the fetal liver is the net producer of Glu using Gln as its main precursor, indicating that the functionality of the fetoplacental unit may be different between singletons and twins. Twin fetuses had 13% less insulin (P = 0.04) concentrations in umbilical artery plasma than singletons. plasma of twin fetuses had 39% less IGF-I (P = 0.003), 33% less His (P = 0.03), and 22% less Gln (P = 0.02) concentrations and tended to have 44% less Arg (P = 0.07) and 20% less Leu (P = 0.06) concentrations than singletons. Arginine, His, and Leu are examples of AA that can promote insulin secretion, and in turn, insulin can increase fetal IGF-I concentrations. In addition, insulin and IGF-I are important fetal growth factors by stimulating and regulating AA transport across the placenta. Collectively, these results indicate that the functionality of the fetoplacental unit may be different between singletons and twins and that AA transport may be reduced in twin placentae.