Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

University of Guelph

Ruminants absorb little glucose and rely on hepatic gluconeogenesis and ketogenesis in the fed state to convert short-chain fatty acids produced during digestion into glucose and ketone bodies, respectively. In contrast to the non-ruminant response, fluxes through gluconeogenic and ketogenic pathways decrease during food restriction. Transcriptional regulation responsible for these unique food restriction responses has not been established. To determine the hepatic transcriptional response of ruminants to an acute drop in dietary nutrient supply, 102 yearling heifers were assigned to either ad libitum feeding or 24 h of food withdrawal in a randomized block design. Liver biopsies were obtained for microarray and quantitative real-time PCR analyses of gene expression. Plasma concentrations of non-esterified fatty acids were higher in food restricted heifers, while levels of β-hydroxybutyrate, triacylglycerol, and glucose were decreased. Despite a decline in substrate supply and a lower hepatic production of glucose, expression of the key gluconeogenic enzymes pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase was upregulated as in non-ruminants. Downregulation of cholesterolgenic genes and upregulation of fatty acid oxidative genes were consistent with SREBP-2 and PPARα control, respectively. Ketogenesis from short-chain fatty acids was downregulated, contrary to the non-ruminant response to food restriction. Short-chain fatty acids may exert transcriptional control in the ruminant liver similar to that demonstrated in the large intestine of non-ruminants.

Transcript profiling of the ruminant liver indicates a unique program of transcriptional regulation of ketogenic enzymes during food restriction.

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.

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

The objectives of this study were to investigate the effects of branched-chain AA (BCAA) supplementation when glucose is infused postruminally into lactating dairy cows consuming a diet low in crude protein (CP) and to test the hypothesis that low BCAA concentrations are responsible for the poor stimulation of milk protein yield by glucose. Twelve early-lactation Holstein cows were randomly assigned to 15% and 12% CP diets in a switchback design of 6-wk periods. Cows consuming the 12% CP diet received 96-h continuous jugular infusions of saline and 1 kg/d of glucose with 0, 75, or 150 g/d of BCAA in a Latin square sequence of treatments. Compared with saline, glucose infusion did not affect dry matter intake but increased milk yield by 2.2 kg/d and milk protein and lactose yields by 63 and 151 g/d, respectively. Mammary plasma flow increased 36% during glucose infusion compared with saline infusion, possibly because of a 31% decrease in total acetate plus β-hydroxybutyrate concentrations. Circulating concentrations of total essential AA and BCAA decreased 19 and 31%, respectively, during infusion of glucose, yet net mammary uptakes of AA remained unchanged compared with saline infusion. The addition of 75 and 150 g/d of BCAA to glucose infusions increased arterial concentrations of BCAA to 106 and 149%, respectively, of the concentrations in saline-infused cows, but caused a decrease in concentrations of non-branched-chain essential AA in plasma, as well as their mammary uptakes and milk protein yields. Plasma urea concentration was not affected by BCAA infusion, indicating no change in catabolism of AA. The lack of mammary and catabolic effects leads us to suggest that BCAA exerted their effects on plasma concentrations of the other essential AA by stimulating utilization in skeletal muscle for protein accretion. Results indicate that the glucose effect on milk protein yield was not limited by low BCAA concentrations, and that a stimulation of extra-mammary use of non-branched-chain essential amino acids by BCAA led to a decrease in milk protein yield.

Maintenance of plasma branched-chain amino acid concentrations during glucose infusion directs essential amino acids to extra-mammary tissues in lactating dairy cows.

Milk protein yield responses to changes in the profile of essential amino acids absorbed by the gastrointestinal tract or circulating in blood plasma do not follow the classic limiting amino acid response, in part because of an ability of the mammary glands to modify their blood flow rate and net clearance of amino acids out of plasma. The hypothesis that mammary blood flow is locally regulated to maintain ATP balance accounts for observed changes in flow due to postruminal glucose, insulin, and essential amino acid (EAA) infusions. An additional hypothesis that net mammary uptakes of metabolites from blood are affected by perturbations in their respective arterial concentrations and the rate of mammary blood flow also appears to hold for the energy metabolites glucose, acetate, β-hydroxybutyrate, and fatty acids. However, net EAA uptakes by the mammary glands are poorly predicted by models considering arterial concentrations and blood flow rates only. Evidence points to intramammary protein synthesis and secretion as the determinant of net EAA uptake. The intracellular signaling network anchored by the mechanistic target of rapamycin complex 1 stands as an excellent candidate to explain nutritional effects on milk protein synthesis because it integrates information on physiological and nutritional state to affect protein synthesis and cell metabolism, growth, proliferation, and differentiation in many cell types. In mammary cells in vitro and in vivo, the mechanistic target of rapamycin complex 1, integrated stress response, and glycogen synthase kinase-3 networks that contribute to regulation of initiation of mRNA translation are responsive to acute changes in nutrient supply and EAA profile. However, after several days of postruminal infusion of balanced and imbalanced EAA profiles, these signaling networks do not appear to continue to account for changes in milk protein yields. Gene expression evidence suggests that regulation of components of the unfolded protein response that control biogenesis of the endoplasmic reticulum and differentiation of a secretory phenotype may contribute to effects of nutrition on milk protein yield. Connections between early signaling events and their long-term consequences should be sought.

John Doelman

Department of Animal Biosciences

University of Guelph

Bovine Mammary Gland Biopsy Techniques

John Doelman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Animal Biosciences

University of Guelph

Bovine Mammary Gland Biopsy Techniques

Department of Animal Biosciences