The effect of pregnancy on nitrogen retention, maternal insulin sensitivity, and mRNA abundance of genes involved in energy and amino acid metabolism in gilts.

Twenty-one of each pregnant (P) and nonserviced, nonpregnant (NP) sister-pairs of gilts were selected to investigate the effect of pregnancy on protein deposition (Pd; whole body and maternal), insulin sensitivity, and mRNA abundance of genes involved in energy and AA metabolism. Between breeding (study day 0) and day 111, P and NP gilts received 2.16 kg of the experimental diet (3.34 Mcal ME/kg, 17.6% crude protein, 0.78% standardized ileal digestible lysine) that was formulated to meet the estimated ME requirements of pregnant gilts (and meet or exceed AA requirements). Nitrogen balances were conducted on day 63 and 102 ± 0.2 of the study during 4-d periods. Blood samples were collected on day 43, 56, 71, 85, 98, and 108 ± 0.3 of the study to determine plasma concentrations of fasted IGF-1, estradiol (E2), and estrone sulfate (E1S). Frequently sampled intravenous glucose tolerance tests (FSIGTT) were conducted on day 75 ± 0.7 in 6 P and 5 NP gilts and on day 107 ± 0.4 in 17 P and 17 NP gilts and the MINMOD approach was applied to evaluate whole body insulin sensitivity and pancreatic responsiveness. Longissimus muscle (LM) and s.c. adipose tissue (AD) samples were excised from 12 P and 12 NP gilts at day 111 ± 0.4 of the study after euthanasia to determine mRNA abundance of key genes. Whole body Pd was greater (P < 0.001) at day 102 and maternal Pd was lower (P < 0.002) at day 63 and 102 for P compared to NP gilts. Plasma concentrations of E1S and E2 increased (P < 0.05) with study day for P gilts and remained constant for NP gilts, which coincided with reduced plasma concentrations of IGF-1 and increased estrogen receptor alpha (ESR1) mRNA abundance in LM of P gilts. Glucose effectiveness was not different between P and NP gilts, but whole body insulin sensitivity was lower (P = 0.004) in P compared to NP gilts on day 75 and 107, which corresponded with reduced mRNA abundances of SLC2A4, HK2, SREBF1, and FASN, and increased abundances of PDK4 and PPARGC1A in LM and AD. When fed identically, P gilts had greater whole body Pd at day 102, which reflects Pd in the pregnancy-associated tissues (at the expense of maternal Pd), likely driven by estrogen-stimulated insulin resistance in peripheral tissue and subsequent modulation of gene expression relating to glucose metabolism.

The effect of graded concentrations of dietary tryptophan on canine behavior in response to the approach of a familiar or unfamiliar individual.

Tryptophan (Trp) is an indispensable dietary amino acid that supports the modulation of mood and behavior in mammalian species through its role in the serotonergic pathway. This study assessed the behavior patterns of 36 healthy, female adult mixed-breed hound dogs fed a control diet (tryptophan = 0.18% as-fed) or 1 of several experimental diets with graded concentrations of tryptophan (0.05%, 0.1%, and 0.15% of the total diet) supplemented on top of the 0.18% basal level. At baseline, and every 8 weeks throughout the 24-week period, behavioral parameters were evaluated for each dog in response to the approach of an individual familiar to the dogs and another individual who was unfamiliar to the dogs. Differences in behavior scores (activity, P = 0.0197; distance, P = 0.0358; confidence, P < 0.0001; and ear position, P < 0.0001) between the unfamiliar and familiar individuals supported the efficacy of the behavioral ethogram used. No consistent and significant differences in behavior were observed, however, among dogs fed the control diet and those fed an experimental diet with any level of tryptophan supplementation. Future research should consider the tryptophan-to-large-neutral-amino-acid ratio and not just tryptophan concentrations, seek to understand the variation in tryptophan requirements among breeds, and look to utilize additional markers of serotonin status.

Le tryptophane (Trp) est un acide aminé alimentaire indispensable qui supporte la modulation de l'humeur et le comportement chez des espèces animales via son rôle dans le cycle sérotonergique. La présente étude a évalué les patrons de comportement de 36 chiennes adultes en santé de race mélangée de type chien de chasse nourries avec une diète contrôlée (tryptophane = 0,18 %, tel que nourri) ou 1 de plusieurs diètes expérimentales avec des concentrations graduées de tryptophane (0,05 %, 0,1 %, et 0,15 % de la diète totale) ajoutées en plus du niveau de base de 0,18 % de la diète. Au temps 0, et à toutes les 8 semaines pendant la période de 24 semaines de l'étude, les paramètres de comportement furent évalués pour chaque chien en réponse à l'approche d'un individu familier aux chiens et un autre individu qui n'était pas familier aux chiens. Les différences dans les pointages de comportement (activité, P = 0,0197; distance, P = 0,0358; confiance, P < 0,0001; et position des oreilles P < 0,0001) entre l'individu familier et le non-familier supporte l'efficacité de l'éthogramme de comportement utilisé. Aucune différence constante ou significative dans le comportement ne fut observée, toutefois, parmi les chiens nourris la diète témoin et ceux nourris avec une diète expérimentale quelque soit le niveau de Trp ajouté. Des recherches ultérieures devraient considérer le ratio tryptophan/gros acide aminé neutre et pas seulement les concentrations de tryptophane, essayer de comprendre la variation dans les besoins de tryptophane parmi les races, et voir à utiliser des marqueurs additionnels du statut de la sérotonine.(Traduit par Docteur Serge Messier).

Effects of a wax organogel and alginate gel complex on holy basil (Ocimum sanctum) in vitro ruminal dry matter disappearance and gas production.

BACKGROUND: The objectives of this study were to: (a) select an ideal organogel for the oil phase of a novel gel encapsulation technology, (b) optimize the formulation of an organogel and sodium alginate-based gel complex, and (c) examine the rumen protective ability of the gel by measuring 48-h in vitro ruminal dry matter disappearance and gas production from encapsulated dried and ground holy basil leaves. RESULTS: A rice-bran wax and canola oil organogel was selected for the oil phase of the gel complex as this combination had a 48-h dry matter disappearance of 6%, the lowest of all organogels analyzed. The gel complex was formulated by homogenizing the organogel with a sodium alginate solution to create a low-viscosity oil-in-water emulsion. Average dry matter disappearance of gel-encapsulated holy basil was 19%, compared to 42% for the free, unprotected holy basil. However, gel encapsulation of holy basil stimulated gas production. Specifically, gas production of encapsulated holy basil was four times higher than the treatment with holy basil added on top of the gel prior to incubation rather than encapsulated within the gel. CONCLUSION: Although the gel itself was highly degradable, it is speculated encapsulation thwarted holy basil's antimicrobial activity. © 2018 Society of Chemical Industry.

Lessons from animal nutritionists: dietary amino acid requirement studies and considerations for healthy aging studies.

Dietary restriction (DR) increases median life span and protects against age-related disease. Improved longevity can be achieved by restriction of dietary energy, protein, or amino acids (AAs), such as methionine (Met). Met requirements have been defined using methodologies that measure the dose response to Met when all other dietary variables are held constant and with outcomes focused on protein turnover. Here, we cover protein and sulfur AA requirements and discuss the terms "deficient," "optimal," and "excess" and how these need to be considered. We additionally discuss the effect of methyl-donating compounds on sulfur AA metabolism and outcomes. We will discuss how the mechanistic target of rapamycin complex 1 (mTORC1) signaling network regulates protein turnover, lipogenesis and cell growth, proliferation, differentiation, and metabolism in response to hormones, AAs, and cellular energy status. Inhibition of mTORC1 signaling with rapamycin or genetic mutation increases median life span in model organisms, and mTORC1 inhibition may be responsible for some of the life span-extending effects of DR. Finally, we discuss how the sulfur AAs may regulate aspects of reactive oxygen species (ROS) mitigation. Overall, we suggest that approaches evaluating AA intake need to consider whole-body protein synthesis and measures related to tissue-specific and whole-body metabolism that have been associated with longevity.

Bovine Mammary Gland Biopsy Techniques.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

Supranutritional selenium intake from enriched milk casein impairs hepatic insulin sensitivity via attenuated IRS/PI3K/AKT signaling and decreased PGC-1α expression in male Sprague-Dawley rats.

Selenium (Se)-enriched milk provides antioxidant benefits and has therapeutic potential against cancer. However, both antidiabetic and prodiabetic effects have been attributed to Se. Our objective was to evaluate the effect of Se-enriched milk casein on insulin sensitivity in rats when given at the requirement of 0.25 ppm Se and supranutritionally on both low- and high-fat diets. Two hundred sixteen male Sprague-Dawley rats were fed low- or high-fat diets containing one, two or eight times the Se requirement in a randomized block design. After 7 weeks, 72 rats were subjected to the hyperinsulinemic-euglycemic clamp with [3-3H]glucose infusion to estimate glucose fluxes. Tissues were collected from the remaining 144 rats 8 min after ip saline or insulin injection. During hyperinsulinemic-euglycemic clamps, glucose infusion rate was 22% lower (P=.058), and endogenous glucose production was 76% higher (P=.054) when Se content increased from one to eight times the requirement on low-fat diets, indicating impaired hepatic insulin sensitivity. Se also decreased the ability for insulin to stimulate Akt phosphorylation at Thr308. Hepatic oxidation state and expression of selenoprotein P and glutathione peroxidase-1 were unaffected while expression of insulin receptor substrate (IRS)-1 and-2 and PPARγ coactivator-1α (PGC-1α) decreased with supranutritional Se and high-fat intake. In addition, hepatic expression of regulatory and catalytic subunits of phosphatidylinositol 3-kinase (PI3K) decreased with supranutritional intake of Se. Se intake from enriched casein up to eight times the requirement impairs hepatic insulin sensitivity in a mechanism similar to fat feeding, via attenuated IRS/PI3K/Akt signaling and decreased PGC-1α expression.

Branched-chain amino acid and lysine deficiencies exert different effects on mammary translational regulation.

Deficiencies and imbalances of specific group II essential amino acids (EAA) were created in lactating cows by an infusion subtraction protocol to explore effects on milk production and abundance and phosphorylation state of regulators of mRNA translation in the mammary glands. Five lactating cows on a diet of 11.2% crude protein were infused abomasally for 5d with saline, 563 g/d of a complete EAA mix, or EAA mixes without the branched-chain amino acids (BCAA), Leu, or Lys in a 5 × 5 Latin square design. Milk protein yield was stimulated by EAA infusion and returned to saline levels upon subtraction of BCAA, Leu, or Lys. Mammary abundance of phosphorylated S6K1 was measured as an indicator of mammalian target of rapamycin complex 1 (mTORC1) activity and was found not to be affected by the complete EAA mix but was increased by the mixture lacking Lys. Total S6K1 abundances in mammary tissue were elevated by complete and BCAA-lacking infusions. All of the EAA treatments except the one lacking BCAA upregulated mammary eIF2Bε and eIF2α abundances, which is stimulatory to global mRNA translation. Phosphorylation state of eIF2Bε tended to decrease when complete or Lys-lacking EAA mixtures were infused. Phosphorylation state of eIF2α was not affected by treatment. We detected a correlation of 0.62 between phosphorylation state of S6K1 and total eIF2Bε abundance, and a correlation of 0.58 between phosphorylation state of S6K1 and total eIF2α abundance, suggesting that mTORC1 activation may have upregulated eIF2Bε and eIF2α expression. Despite maintenance of mammary eIF2Bε and eIF2α abundances during Leu and Lys deficiencies, milk protein yield declined, suggesting that other factors are responsible for mediating effects of Lys and Leu. A deficiency of all 3 BCAA may impair milk protein yield through deactivation of mTORC1-mediated upregulation of eIF2Bε and eIF2α abundances.

Selenized milk casein in the diet of BALB/c nude mice reduces growth of intramammary MCF-7 tumors.

BACKGROUND: Dietary selenium has the potential to reduce growth of mammary tumors. Increasing the Se content of cows' milk proteins is a potentially effective means to increase Se intake in humans. We investigate the effects of selenized milk protein on human mammary tumor progression in immunodeficient BALB/c nude mice. METHODS: Four isonitrogenous diets with selenium levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk casein isolates with a rodent premix. MCF-7 cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 ß-estradiol pellets. Mice with palpable tumors were randomly assigned to one of the four diets for 10 weeks, during which time weekly tumor caliper measurements were conducted. Individual growth curves were fit with the Gompertz equation. Apoptotic cells and Bcl-2, Bax, and Cyclin D1 protein levels in tumors were determined. RESULTS: There was a linear decrease in mean tumor volume at 70 days with increasing Se intake (P < 0.05), where final tumor volume decreased 35% between 0.16 and 1.15 ppm Se. There was a linear decrease in mean predicted tumor volume at 56, 63 and 70 days, and the number of tumors with a final volume above 500 mm3, with increasing Se intake (P < 0.05). This tumor volume effect was associated with a decrease in the proportion of tumors with a maximum growth rate above 0.03 day-1. The predicted maximum volume of tumors (Vmax) and the number of tumors with a large Vmax, were not affected by Se-casein. Final tumor mass, Bcl-2, Bax, and Cyclin D1 protein levels in tumors were not significantly affected by Se-casein. There was a significantly higher number of apoptotic cells in high-Se tumors as compared to low-Se tumors. CONCLUSIONS: Taken together, these results suggest that turnover of cells in the tumor, but not its nutrient supply, were affected by dairy Se. We have shown that 1.1 ppm dietary Se from selenized casein can effectively reduce tumor progression in an MCF-7 xenograft breast cancer model. These results show promise for selenized milk protein as an effective supplement during chemotherapy.

Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model.

Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-d-[1-(3)H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37 degrees C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 +/- 5.7% and 67.4 +/- 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.

Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation.

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3-O-methyl-D-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37 degrees C under zero-trans, equilibrium-exchange, high-cis, and high-trans concentrations of 3-O-methyl-D-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol.min-1.mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.