Changes in mammary blood flow during the lactation period in goats measured by the ultrasound Doppler principle

1. Mammary blood flow was measured during the lactation period in two low- and two high-yielding dairy goats (peak milk yields approx. 1.5 and 3.6 kg/day respectively), using the ultrasound Doppler principle for determination of blood velocity in both milk veins (subcutaneous abdominal veins) of the animals, and ultrasound scanning for measurement of cross-sectional area of the veins. 2. Milk vein cross-sectional area ranged from 0.11 to 0.38 cm2 in the four goats, with a close to significant (P = 0.06) difference between the veins in the two sides of the animals. Cross-sectional area remained constant during the lactation period. Changes in mammary blood flow was therefore caused by changes in blood velocity. 3. Milk vein blood velocities ranged from 4.4 to 34.7 cm/sec independently of the time of the day, and were of the same magnitude in the two sides of the animals. Except in one goat (P = 0.1), blood velocity decreased significantly (P less than 0.01) with progressing lactation, during which period also milk yield was declining. 4. In the two low-yielding goats, a positive linear relationship (R2 = 0.20) was found between milk yield and milk vein blood velocity, whereas a diminishing exponential relationship (R2 = 0.97) was found in the two high-yielding goats. At a given milk vein blood velocity, high-yielding goats obtained a higher milk yield and also responded to changes in blood velocity (up to approx. 15 cm/sec) with greater increases in milk yield than low-yielding goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in mammary uptake of free fatty acids,triglyceride, cholesterol and phospholipid in relation to milk synthesis during lactation in goats

Uptake of free fatty acids (FFA), triglycerides (TG), cholesterol (CHOL) and phospholipids (PL) was measured in both mammary glands of dairy goats during lactation. Arterial concentrations of TG, CHOL and PL as well as arterio-venous difference (AV) and extraction rate (E) for TG were highest in goats with the highest dietary feed intake. AV were linearly related to arterial concentrations for the four lipid classes, and arterial concentrations of CHOL were linearly related to output of lactose, protein and fat in milk. Arterial supply, and not mammary synthetic activity, is the main determinant of mammary FFA, TG and CHOL uptake.     

Evaluation of mammary blood flow measurements in lactating goats using the ultrasound Doppler principle

1. Non-invasive methods were developed for measuring mammary blood flow in lactating goats. 2. A Doppler principle ultrasound device was equipped with an external detector measuring maximal blood velocity (Vmax) and average blood velocity (Vav) was calculated as Vmax/2. Volume flow then depended on determination of the angle of insonation and the cross-sectional area of the milk vein (the caudal superficial epigastric or subcutaneous abdominal vein). 3. Blood velocities were measured on the milk vein of either side of the animal while clamping the pudendal veins manually. Blood velocities ranged from 7-34 cm/sec. 4. The milk vein diameter was measured by means of a slide gauge which, for clearly protruding veins, gave similar results to that measured by ultrasound scanning. In protruding veins the cross-section was circular. In non-protruding veins the cross-section was elliptical and the slide gauge significantly (P less than 0.01) overestimated the cross-sectional area. The milk vein diameter of either side measured in 10 lactating goats was 8.8 +/- 1.1 mm (means +/- SD). 5. Blood flow ranged from 90-675 ml/min in a dry and a high-yielding (3.4 l milk daily) goat, respectively. The reproducibility of the blood flow measurements was 12-16%. 6. It is concluded that the present method may be used for quantitative measurements of mammary blood flow in goats.     

Acetate metabolism in the mammary gland of the lactating ewe

Acetate metabolism in the mammary gland of lactating ewes was studied by continuous infusion of radioisotopic [U-14C]sodium acetate and measurement of mammary gland arteriovenous difference and blood flow. Entry rate of acetate into the whole body averaged 75 +/- 7 mumol min-1 kg-1 liveweight and 22.1 +/- 2.7% of total CO2 production was derived from acetate. Acetate was both utilized and produced by the mammary gland. Acetate uptake was related linearly (r2 = 0.94) to arterial concentration and gross utilization of acetate accounted for 16.2 +/- 2.6% of whole-body entry rate. Endogenous acetate production by the mammary gland increased linearly (r2 = 0.90) as milk yield rose, and accounted for 25.6 +/- 2.7% of the gross mammary utilization of acetate. The proportion of mammary CO2 derived from acetate (22.5 +/- 3.9%) was similar to that of the whole body. The uptake of acetate, 3-hydroxybutyrate, esterified fatty acids and plasma free fatty acids accounted for about 25, 13, 60 and 4% of milk fatty acid carbon respectively, after correction for the oxidation of acetate, but not of the other substrates. Metabolism of acetate in the mammary glands of lactating ewes appears quantitatively more important than that in cows, but similar to that in goats.     

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland

The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats.     

The effects of colchicine on milk secretion, mammary metabolism and blood flow in the goat

Mammary function in the conscious goat was studied during colchicine-induced depression of milk secretion in one mammary gland. Milk yield of the treated gland was reduced to approximately a quarter of previous, while there were significant increases in afternoon milk yield from the untreated glands on the 2nd and 3rd days after treatment in goats in late lactation. Milk composition in the untreated glands was not significantly affected. In the treated gland, milk [Na+], [Cl-], [citrate] and [protein] increased while [K+] and [lactose] decreased, although the time course of these changes differed; milk [fat] was unaffected. Mammary extractions ((A-V)/A) of glucose, acetate and most amino acids were significantly decreased during the period of maximal inhibition of secretion. There were no significant changes in arterial plasma concentrations of glucose, acetate or any essential amino acids. In another series of experiments, mammary blood flow increased and then returned to normal after colchicine treatment even though milk yield and mammary glucose uptake decreased markedly; oxygen uptake was not significantly affected. The results are discussed in relation to the actions of colchicine on the mammary secretory cell, to the normal control of mammary blood flow and to the mechanism of compensation by the untreated gland.     

Insulin regulation of amino-acid metabolism in the mammary gland of sheep in early lactation and fed fresh forage

Insulin plays an important role in regulating the partitioning of nutrients to the mammary gland, particularly in lactating ruminants fed concentrate-based diets. There is evidence that the nutritional status of the animals might also affect their response to insulin. This is largely untested in early lactating ruminants fed fresh forage. To investigate nutritional effects on insulin response, 12 lactating sheep, housed indoors, were allocated to one of two treatment groups (hyperinsulinaemic euglycaemic clamp (HEC) or control) in a randomised block design and fed perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. Mammary amino acid (AA) net uptake from plasma and utilisation for milk protein synthesis was measured during the 4th day of the HEC using arterio-venous concentration differences, and 1-13C-leucine was used to estimate whole body and mammary gland leucine kinetics. There was no change in feed intake, milk protein output and mammary blood flow during the HEC (P > 0.1). The HEC decreased (P < 0.1) the arterial concentrations of all essential AA (EAA) except histidine. The mammary net uptake of some EAA (isoleucine, leucine, methionine and phenylalanine) was reduced by the HEC (P < 0.1). Leucine oxidation in the mammary gland was not altered during the HEC (P > 0.1) but mammary protein synthesis was reduced by the HEC (P < 0.05). These results show that sheep mammary gland can adapt to changing AA precursor supply to maintain milk protein production during early lactation, when fed fresh forage. How this occurs remains unclear, and this area deserves further study.     

High ambient temperature directly decreases milk synthesis in the mammary gland in Saanen goats

The mammary gland is a privileged organ for mammals. Because of their high capacity for milk synthesis, dairy ruminants have been distributed throughout the world. In tropical areas, dairy animals face high ambient temperatures (HTa). The indirect effect of HTa on milk synthesis is mediated in part by a reduction in feed intake. The current experiment focused on the direct natural effect of HTa on mammary function. Multiparous Saanen goats were used in this study. The physiological responses for HTa were evaluated from the control period during the winter and from the natural HTa during the summer. Milk samples were collected for isolation of the goat milk cells to study the expression of the β-1,4 galactosyltransferase (β-GALT1), Akt, and heat shock protein 70 (HSP70) genes. Although goats in the summer maintained rectal temperature and plasma cortisol levels similar to those observed in the winter, the higher respiratory rate and lower feed intake and milk yield (MY) from the goats in the summer indicated that the goats in the summer were exposed to a higher degree of HTa. This was supported by the significantly higher level of plasma glutathione peroxidase (GPX) activity. Moreover, the relative expression levels of β-GALT1 and Akt were not different. The relative expression of HSP70 during the summer was significantly higher than what was observed in cells isolated in the winter. In conclusion, the HTa effect on MY during the summer was related to its indirect effect on feed intake. The direct HTa effect might be related to HSP70 gene expression in goat milk cells and to plasma GPX activity. However, the natural HTa did not affect the expression of Akt or β-GALT1.     

Changes in mammary glucose and protein uptake in relation to milk synthesis during lactation in high- and low-yielding goats

• 1.1. Glucose and protein uptake were measured in both mammary glands of two low- and two high-yielding dairy goats during lactation.
• 2.2. Low-yielding goats tended to have higher arterial glucose concentrations, but approximately 40% lower arterio-venous differences (AV) and extraction rates (E) for glucose than high-yielding goats.
• 3.3. AV and E for glucose (but not protein) were linearly related to yields of both lactose, milk protein and fat.
• 4.4. Mammary uptake of glucose is determined primarily by mammary glucose metabolism, not glucose supply; lower intracellular glucose concentration in mammary cells of genetically superior animals thus explains the more efficient mammary uptake.

     

Use of magnetic resonance imaging in the study of goat mammary glands in vivo

A technique is described for the in-vivo determination of mammary gland size and gross composition in goats by using nuclear magnetic resonance imaging (MRI). The volume of test objects determined with MRI had an error of +0.4 +/- 1.6% of the actual volume. In lactating goats the in-vivo MRI estimate of mammary parenchymal volume was significantly greater than, but highly significantly correlated with, the weight of parenchyma determined post mortem (for the whole udder, r = 0.88, P less than 0.001; for individual glands, r = 0.85, P less than 0.001). MRI-determined estimates of the volume of fluid within the mammary gland were within 1.2% of the volume of milk removed from the udders after imaging. The spin-lattice (T1) relaxation time of the whole udder correlated closely with the volume of fluid within the udder. The T1 relaxation time of parenchymal tissue measured in vivo did not differ significantly from that determined immediately after post-mortem excision.     

The excretion of prostacyclin (PGI2) in milk and its possible role as a vasodilator in the mammary gland of goats

1. Prostacyclin production in mammary gland of two lactating goats measured as the excretion in milk of 6-ketoprostaglandin F1 alpha (6-KPGF1 alpha) was followed for 16 days before, during and after exogenous administration of recombinant bovine growth hormone (GH). 2. 6-KPGF1 alpha was detected in all milk samples in concentrations ranging from 32-99 pg/ml milk independently of the time of sampling. 3. GH-treatment significantly increased milk yield, the concentration and excretion of 6-KPGF1 alpha in milk. 4. The concentration of milk 6-KPGF1 alpha was positively correlated with milk yield in the high (R2 = 0.35), but not in the low yielding goat (R2 = 0.003). 5. The possible role of prostacyclin as a local vasodilator in the mammary gland of goats is discussed.     

Adaptive shifts in the activity of amino acid transport into mammary secretory cells in changed nutritional conditions

On the basis of analysis of published data, direct (using 13C) and indirect methods of estimating the amino acid transfer into mammary secretory cells in vivo were compared and the modified indirect method was used to determine quantitatively the shifts in transport activity in lactating cows and goats in trials with amino acid deficit or excess and in investigations that used a hyperinsulemic-euglycemic clamp. The analysis suggests that inadequacy of traditional use of extraction efficiency as a measure of tissue affinity to substrate is associated with two shortcomings: 1) if the changes in mammary blood flow are more expressed compared to transport activity, the arteriovenous difference and extraction efficiency may change in opposite direction to the shifts in transport activity; 2) due to the effect of nonlinearity, in situations characterized by small ratio of blood flow: the transport activity extraction efficiency is insensitive to shifts on activity of transport. The re-analysis of published data using the modified inbdirect method indicated that the deficit of individual amino acid caused a rise in activity of their transport and the excess decreased a net transfer into cell. The insulineuglycemic clamp treatments increased the activity of amino acid transport into the mammary cell and milk protein yield. The results obtained suggest that net transmembrane transfer of amino acid into mammary secretory cell can be controlled by the cell itself according to metabolic demand.     

Changes in mammary uptake and metabolic fate of glucose with once-daily milking and feed restriction in dairy cows

The aim of this review is to better understand the regulation of milk yield in response to once-daily milking and feed restriction. Glucose is the principal precursor for the synthesis of lactose (a major osmotic agent in milk), and participates in determining the milk volume produced. When applying these two breeding factors, reductions in milk yield are associated with a reduction in milk lactose yield and in the arterial flow of glucose, due to a decrease in the mammary blood flow. The ability of the udder to extract glucose is altered with once-daily milking but not necessarily with feed restriction. Lactose synthesis is down-regulated in response to once-daily milking and feed restriction but the percentage of the extracted glucose which is converted into lactose is differently affected in response to treatments. No marked change is observed with once daily milking whereas this would be increased with feed restriction and in contrast, depressed with fasting.     

Effects of exogenous growth hormone on mammary function in lactating goats

The galactopoietic effect of daily injections, for five day periods, of growth hormone was examined by measuring milk yield, mammary blood flow and arteriovenous differences of glucose and amino acids on 12 occasions in four goats. In 10 periods there were marked increases (mean 18.1%) in mammary blood flow (8 statistically significant) and less-marked increases (mean 8.0%) in milk yield (6 statistically significant). On 8 of the occasions on which it was measured the maximum blood plasma growth hormone concentration was increased to more than 8 ng/ml. There were no statistically significant changes in mammary arteriovenous concentration differences of glucose or amino acids in response to growth hormone injections. It is suggested that, contrary to the usual situation in which the rate of mammary blood flow appears to be regulated by the metabolic activity of the gland, the galactopoietic response to growth hormone may be a consequence of elevated blood flow, which increases the supply to the gland of rate-limiting metabolic substrates.     

The conjugate regulation of mammary blood flow and secretory cell metabolism: analysis of problem

In the review the essential features of system regulation of mammary blood flow, substrate supply, uptake and utilization by cellular metabolism and for milk component synthesis are considered, with emphasis on productive ruminants (cows and goats). The conception of local control of regional vascular resistance in described. in which the criterion of maintenance of energetic balance in secretory cells is used. The some steps of substrates utilization and their regulation are described (glucose, acetate, free amino acids, triacylglycerols, free fatty acids). The secretory cell is considered as control unit equilibrating the extraction of substrates from blood with the rate of milk components synthesis. The role of hormonal factors in these processes is discussed.     

Studies on the mode of uptake of blood triglycerides by the mammary gland of the lactating goat. The uptake and incorporation into milk fat and mammary lymph of labelled glycerol, fatty acids and triglycerides

1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with (3)H and (14)C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the (3)H/(14)C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of (3)H and (14)C into milk and lymph were closely similar after the infusion of [2-(3)H]glycerol tri[1-(14)C]oleate or of a mixture of [2-(3)H]glycerol and [1-(14)C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.     

Cyclic nucleotides in goats' milk: changes with physiological state

Concentrations of cAMP and cGMP were measured in mammary secretions of goats. [cAMP] and [cGMP] increased during late pregnancy. At the onset of copious milk secretion, [cGMP] decreased while [cAMP] increased further. After the cessation of milking in late lactation, [cGMP] increased and [cAMP] tended to decrease. During withdrawal of food for 48 hr, the rate of milk secretion fell, [cGMP] increased and [cAMP] decreased; the changes were reversed on re-feeding. During temporary inhibition of milk secretion by intramammary treatment with colchicine, [cGMP] increased while [cAMP] decreased. It is concluded that cyclic nucleotide concentrations in milk change with physiological state.     

In-vivo studies of mammary development in the goat using magnetic resonance imaging (MRI)

Mammary development and regression were measured in goats in vivo using magnetic resonance imaging (MRI). Measurements were made during the first and second cycles of pregnancy, lactation and involution. In primiparous goats, and exponential pattern of growth was evident during gestation and for the first 2 weeks of lactation. Parenchyma volume correlated significantly with milk yield across goats during early lactation, and across stage of lactation within goats. Milking was discontinued in Week 26 of the first lactation. Involution was characterized by an initial accumulation of fluid (over 2 days) followed by reabsorption; parenchyma volume did not decrease significantly until the 3rd week of involution, which was also the time at which these goats were mated to start their second gestation. Their udders still contained significant quantities of fluid (40-60% of the gross volume), but parenchyma volume was also greater (by 4.7-fold) than in goats beginning their first gestation. By Week 15 of gestation there was no longer a parity difference in parenchyma; the udders of first-gestation goats had grown significantly, but those of second-gestation goats had not. Conversely, between gestation Week 15 and lactation Week 2 mammary growth was significantly more rapid in the second cycle, such that the udder was larger at the start of the second lactation.     

Lactogenic effects of hyperinsulinemic-euglycaemic clamp in goats

In the experiment performed on lactating goats, insulin was infused into the jugular vein over during 2 days every day at the rate 2 mg/kg/hour during 6 hour synchronously with glucose at variable rate to maintain euglycaemia; the transport activity (T, in clearance units) was estimated using the equation: T = Q x E/ (1-E), where Q is plasma flow and E is extraction efficiency. At the end of infusion of the 1st and 2nd days, insulin level in the blood was increased by 63 and 82%, mammary plasma flow by 38 and 78%, milk secretion rate by 23.7 and 31.3 %, milk protein yield by 21.4 and 40%, transport activity of glucose by 63 %, and amino acids by 18% (all p < 0.05) compared to control, respectively. The data obtained suggest that productive effect resulted from elevated metabolic activity of secretory cells and increased mammary blood flow.     

Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows

Lysine is usually taken up in excess by the mammary gland (MG) relative to milk protein output, allowing for mammary synthesis of non-essential (NE) amino acids (AA) from Lys-N. It is unclear whether this NEAA synthesis from Lys is obligate or whether more efficient use of Lys can be made under limiting conditions. Six multi-catheterized dairy cows received a basal diet low in protein plus an abomasal infusion of AA (560 g/day) with or without Lys (50.3 g/day), in a crossover design with 7-day periods. On day 7, all cows received a 7.5-h jugular infusion of [2-15N]lysine. Six blood samples were collected from arterial, portal, hepatic and mammary vessels at 45 min intervals. In addition, cows were milked at 6 and 7 h with the milk casein plus arterial and mammary plasma collected at 7 h analyzed for AA enrichment. Milk protein concentration and casein yield tended (P < 0.10) to decrease with Lys deletion, while Lys secretion in milk protein was lowered (P < 0.05). The addition of Lys in the AA mixture increased the net portal absorption of Lys by the amount infused, suggesting limited oxidation of this extra supply by the gut. Net liver flux of Lys was unaltered by treatment and, therefore, net splanchnic release of Lys reflected closely the amounts absorbed. For both treatments, however, post-liver supply was greater than mammary uptake, which exceeded milk output. Nonetheless, while Lys deletion decreased mammary uptake by 10.1 mmol/h, Lys in milk protein secretion was reduced by only 3.9 mmol/h. On a net basis, there was no evidence of the additional uptake of any other measured AA during the Lys deletion. The mammary uptake to output ratio of Lys decreased from 1.37 to 1.12, but still showed an excess with Lys deletion. The total amount of 15N in milk protein did not change with treatment but the distribution into AA was altered. In conditions that simulated normal feeding (Lys infused), 83% of the 15N was present as Lys, with Glx, Asx, Ser and Ala harvesting, respectively, 6.8%, 2.4%, 2.1% and 1.0%. With Lys depletion, N-transfers from Lys to other AA within the MG were still present, but rates were considerably lower. This would suggest that part, at least, of Lys catabolism in the MG is either needed or cannot be prevented completely, even at low supply of Lys. Such catabolism will provide N to support the synthesis of NEAA.