RESUMEN
To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF.
Asunto(s)
Lactancia , Glándulas Mamarias Animales/irrigación sanguínea , Óxido Nítrico/metabolismo , Adenosina/farmacología , Animales , Circulación Sanguínea , Glucemia/metabolismo , Vasos Sanguíneos/metabolismo , Bovinos , Epoprostenol/metabolismo , Femenino , Glucosa/farmacología , Gliburida/farmacología , Insulina/sangre , Insulina/farmacología , Leche/química , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Triglicéridos/sangre , Ácido p-Aminohipúrico/sangreRESUMEN
Removing His from a postruminal AA infusion decreases milk protein and increases milk fat content. Feather meal is an inexpensive protein source, high in rumen undegradable protein but low in His. The objective of our study was to investigate dietary feather meal as a method for creating a His deficiency or imbalance to alter milk composition. Four dietary treatments were fed for 4 wk each to 8 multiparous mid-lactation Holstein cows in a replicated 4 × 4 Latin square design. A standard-protein control diet (SP-C) was formulated to provide 3,100g/d of metabolizable protein (MP). Feather meal was added to the control diet either to replace the MP isonitrogenously (SP-FM) or to increase the MP supply to 3,484 g/d (HP-FM). As an isonitrogenous control for HP-FM, a high-protein diet (HP-C) was formulated with His-adequate protein sources to provide the same MP content as HP-FM. Dry matter intake tended to decrease when feather meal was fed. Predicted flows of digestible His, Met, and Lys, and plasma concentrations of these AA were reduced on both feather meal diets. Predicted flows of total digestible essential AA were not different between HP-FM and SP-C. We concluded that the DMI depression on HP-FM prevented an imbalance of excess AA over His, and created a deficiency of His, Met, and Lys compared with SP-C. Milk production decreased on the 2 feather meal treatments, partly explained by a tendency for DMI to decrease. Milk yield was lowest on SP-FM at 30.3 kg/d and highest on HP-C at 37.9 kg/d. Milk fat yield was not affected by diet but protein and lactose yields were both lower with feather meal. Protein yields were 860 and 998 g/d, whereas lactose yields were 1,384 and 1,561 g/d for SP-FM and HP-FM, respectively. This resulted in a higher fat content and lower protein percentage on FM diets. The ratio of solids-not-fat:fat in milk was lowest on SP-FM at 2.11 compared with 2.56 on SP-C. Adding feather meal to the diet by replacing MP isonitrogenously was more effective at lowering the solids-not-fat:fat ratio than increasing the MP content with an imbalanced protein source.
Asunto(s)
Alimentación Animal/análisis , Dieta/veterinaria , Plumas/química , Histidina/sangre , Leche/química , Animales , Glucemia/metabolismo , Peso Corporal , Bovinos , Proteínas en la Dieta/administración & dosificación , Femenino , Histidina/deficiencia , Lactancia , Lactosa/metabolismo , Proteínas de la Leche/metabolismo , Rumen/metabolismoRESUMEN
The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 µM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Estrés Oxidativo/fisiología , Selenometionina/farmacología , Animales , Apoptosis/efectos de los fármacos , Bovinos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/citología , Femenino , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Glándulas Mamarias Animales/citología , Modelos BiológicosRESUMEN
This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.
Asunto(s)
Suplementos Dietéticos , Células Epiteliales/citología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Selenometionina/administración & dosificación , Selenometionina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Regulación Enzimológica de la Expresión Génica/fisiología , Inmunohistoquímica , Glándulas Mamarias Animales/citología , Glutatión Peroxidasa GPX1RESUMEN
An experiment was conducted to test the hypothesis that a sufficient proportion of histidine (His) included in the drinking water of lactating cows bypasses the rumen to have an effect on milk synthesis. Eight dairy cows (45 +/- 15 d in milk) were given either 0 or 2.5 g/L of His in the drinking water in a crossover design of two 7-d periods. Cows were offered a corn and alfalfa silage-based total mixed ration for ad libitum intake. Water was provided ad libitum to each cow in an individual automatic drinking vessel with a flow meter attached. Water intake tended to increase from 85.1 to 92.1 L/d when His was added. Concentrations of His in plasma samples collected on the last day of each period tended to increase from 14.6 to 21.6 muM, corresponding to an estimated 0.4% bypass of the imbibed histidine. Other amino acid concentrations in plasma were not affected by His supplementation. Milk yield increased by 1.7 L/d with His treatment, lactose yield increased by 90 g/d, and there were tendencies for protein yield to increase, fat percentage to decrease, and protein to fat ratio to increase. An improvement in postruminal histidine flow can influence milk production and composition but the proportion of imbibed water that bypasses the rumen will have to be increased to take advantage of drinking water as a vehicle to transfer His postruminally.
Asunto(s)
Suplementos Dietéticos , Agua Dulce , Histidina/farmacología , Lactancia/efectos de los fármacos , Aminoácidos/sangre , Animales , Bovinos , Ingestión de Líquidos/efectos de los fármacos , Femenino , Agua Dulce/química , Histidina/administración & dosificación , Leche/química , Leche/metabolismoRESUMEN
Low concentrations of the essential amino acid histidine in circulation have been shown to increase mammary blood flow and it has been suggested that this effect is mediated by histamine. The hypotheses tested in this experiment were that interstitial histamine concentrations in the mammary gland are related to arterial His concentrations and that mammary blood flow is reduced by extracellular histamine via H(1) receptors. The hypotheses were tested by infusing saline or chlorpheniramine, a blocker of the H(1) histamine receptor, into the arterial supply of the mammary glands of lactating cows infused with 44 g/h of amino acid mixtures with or without His for 10 h. Infusates were administered in a 2 x 2 factorial arrangement within a 4 x 4 Latin square to 4 multiparous Holstein cows in mid lactation. Exclusion of His from the infusate decreased protein content in milk from the infused udder half from 3.98 to 3.77%, and increased arterial alpha-aminonitrogen concentration from 3.2 to 3.4 mM. Neither the decreased arterial His concentration nor the H(1) blocker affected plasma flow to the infused udder half. We conclude that histamine is not involved in the regulation of mammary blood flow. The H(1) blocker decreased milk production in the infused udder half from 4.6 to 3.5 kg without affecting protein, fat, and lactose percentages, suggesting an inhibition of milk ejection. Cows on chlorpheniramine ate less feed during the infusion than saline-infused cows, which resulted in lower arterial concentrations and mammary uptakes of acetate. The efficiency of plasma triacylglycerol uptake across the mammary glands was decreased by chlorpheniramine but net uptake of long-chain fatty acids was not affected. The mechanism by which an amino acid deficiency influences mammary blood flow does not involve histamine signaling through the H(1) receptor and remains unidentified.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/farmacología , Histamina/metabolismo , Glándulas Mamarias Animales/irrigación sanguínea , Leche/metabolismo , Receptores Histamínicos H1/fisiología , Acetatos/farmacología , Aminoácidos/deficiencia , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Bovinos , Clorfeniramina/farmacología , Femenino , Histamina/sangre , Histidina/metabolismo , Lactancia , Glándulas Mamarias Animales/efectos de los fármacos , Leche/química , Eyección Láctea/efectos de los fármacosRESUMEN
Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10 h with 0 or 40 g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0 g/h, 30 g/h in the profile of rumen microbes, or 30 g/h in the profile of milk proteins, in a 3 x 2 factorial arrangement with the 2 acetate treatments of 0 and 40 g/h, all in a 6 x 6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and beta-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields of both protein and fat in milk showed a low sensitivity to the concentration of their precursors in circulation. It was concluded that the Km in Michaelis-Menten-type equations describing milk synthesis should be assigned a low value, and that the Vmax is regulated to bring about changes in milk yield and composition.
Asunto(s)
Acetatos/administración & dosificación , Aminoácidos/administración & dosificación , Bovinos/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Acetatos/sangre , Acetatos/metabolismo , Aminoácidos/sangre , Aminoácidos/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Glucemia/metabolismo , Ingestión de Alimentos/fisiología , Ácidos Grasos/metabolismo , Femenino , Insulina/sangre , Cinética , Lactancia , Leche/química , Proteínas de la Leche/metabolismoRESUMEN
Concentrations of glucose in the external iliac artery feeding one udder half of 14 midlactation Holstein cows were increased by infusion to test the following three hypotheses of mammary function: 1) that mammary glands control their blood supply to maintain intracellular energy balance, 2) that milk precursors are taken out of capillary blood according to mass action kinetics, and 3) that the rate of milk component synthesis is dependent on its precursor's uptake from blood. The first seven cows received 20 g/h glucose during 10 h of infusion. Arterial concentrations of glucose were locally increased by only 10%, and the iliac plasma flow was not affected by glucose infusion, so the next seven cows were given 90 g/h glucose. Quantitative predictions resulting from the hypotheses were that arterial plasma flow would decrease by 32% with 90 g/h glucose infusion, glucose uptakes would increase and acetate, fatty acid, and amino acid uptakes decrease, and milk protein and fat yields and percentages would decrease. Iliac plasma flow decreased 16%, half of what was predicted, which suggests that other regulatory processes besides blood flow control took part in the response. Acetate and fatty acid uptakes by the mammary glands were reduced as predicted because of the lower blood flow, but an unexpected depression in extraction of plasma triacylglycerol also contributed to the reduced fatty acid uptake. Milk fat and protein yields were not affected by the exogenous glucose, falsifying the third hypothesis that milk component secretion is a function of uptake of its precursor. Milk fat and protein percentages declined with glucose infusion because of increased lactose synthesis and secretion of water into milk.