Hypertonic NaCl infusions affect milk composition in goats

The capability of goats to maintain milk production during water deprivation is remarkable and not yet fully understood. The aim of the present study was to investigate whether intravenous infusions of hypertonic NaCl cause release of both vasopressin and oxytocin and whether the peptides, in combination with the hyperosmolality, affect milk flow and milk composition. Six Swedish domestic landrace goats in their first to third lactation were milked every 30 min during experiments. Hypertonic NaCl (HNaCl) or isotonic NaCl (IsoNaCl) were infused for 90 min. Goats were not allowed to drink during infusions. Plasma vasopressin concentration increased during HNaCl infusions, and did not change in response to IsoNaCl infusions. Plasma oxytocin concentration did not change during either infusion. Milk flow was maintained during the infusions. Milk fat concentration decreased in the three samples taken before onset of the infusions, but then increased gradually during HNaCl infusions, while it continued to fall during the IsoNaCl infusions. Milk osmolality followed the rise in plasma osmolality during the HNaCl infusions and did not change in IsoNaCl experiments. Milk lactose concentration increased throughout both series of experiments, the concentration being higher during HNaCl infusions. Milk protein concentration did not change during HNaCl infusions, but fell in the IsoNaCl experiments. It is concluded that the hyperosmolality in combination with elevated plasma vasopressin levels did not disturb the secretory activity of the mammary cells, but rather facilitated emptying of the alveolar milk. Such a mechanism may help to explain the sustained milk production in water deprived goats.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows

In addition to plasma metabolites and hormones participating as humoral signals in the control of feed intake, oxidative metabolic processes in peripheral organs also generate signals to terminate feeding. Although the degree of oxidation over longer periods is relatively constant, recent work suggests that the periprandial pattern of fuel oxidation is involved in regulating feeding behavior in the bovine. However, the association between periprandial oxidative metabolism and feed intake of dairy cows has not yet been studied. Therefore, the aim of this study was to elucidate possible associations existing between single feed intake events and whole-body net fat and net carbohydrate oxidation as well as their relation to plasma metabolite concentrations. To this end, 4 late-lactating cows equipped with jugular catheters were kept in respiratory chambers with continuous and simultaneous recording of gas exchange and feed intake. Animals were fed ad libitum (AL) for 24h and then feed restricted (RE) to 50% of the previous AL intake for a further 24h. Blood samples were collected hourly to analyze β-hydroxybutyrate (BHBA), glucose, nonesterified fatty acids (NEFA), insulin, and acylated ghrelin concentrations. Cross-correlation analysis revealed an offset ranging between 30 and 42 min between the maximum of a feed intake event and the lowest level of postprandial net fat oxidation (FOX(net)) and the maximum level of postprandial net carbohydrate oxidation (COX(net)), respectively. During the AL period, FOX(net) did not increase above -0.2g/min, whereas COX(net) did not decrease below 6g/min before the start of the next feed intake event. A strong inverse cross-correlation was obtained between COX(net) and plasma glucose concentration. Direct cross-correlations were observed between COXnet and insulin, between heat production and BHBA, between insulin and glucose, and between BHBA and ghrelin. We found no cross-correlation between FOX(net) and NEFA. During RE, FOX(net) increased with an exponential slope, exceeded the threshold of -0.2g/min as indicated by increasing plasma NEFA concentrations, and approached a maximum rate of 0.1g/min, whereas COX(net) decayed in an exponential manner, approaching a minimal COX(net) rate of about 2.5 g/min in all cows. Our novel findings suggest that, in late-lactating cows, postprandial increases in metabolic oxidative processes seem to signal suppression of feed intake, whereas preprandially an accelerated FOX(net) rate and a decelerated COX(net) rate initiate feed intake.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous β-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 μg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.

Trans-10,cis-12 conjugated linoleic acid (CLA) supplementation causes milk fat depression in dairy cows, but CLA effects on glucose metabolism are not clear. The objective of the study was to investigate glucose metabolism, especially endogenous glucose production (eGP) and glucose oxidation (GOx), as well as hepatic genes involved in endogenous glucose production in Holstein cows supplemented either with 50 g of rumen-protected CLA (9% trans-10,cis-12 and 10% cis-9,trans-11; CLA; n=10) or 50 g of control fat (24% C18:2; Ctrl; n=10) from wk 2 before parturition to wk 9 of lactation. Animal performance data were recorded and blood metabolites and hormones were taken weekly from 2 wk before to 12 wk after parturition. During wk 3 and 9 after parturition, glucose tolerance tests were performed and eGP and GOx were measured by [U-(13)C] glucose infusion. Liver biopsies were taken at the same time to measure total fat and glycogen concentrations and gene expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and carnitine palmitoyl-transferase 1. Conjugated linoleic acid feeding reduced milk fat, but increased milk lactose output; milk yield was higher starting 5 wk after parturition in CLA-fed cows than in Ctrl-fed cows. Energy balance was more negative during CLA supplementation, and plasma concentrations of glucose were higher immediately after calving in CLA-fed cows. Conjugated linoleic acid supplementation did not affect insulin release during glucose tolerance tests, but reduced eGP in wk 3, and eGP and GOx increased with time after parturition. Hepatic gene expression of cytosolic phosphoenolpyruvate carboxykinase tended to be lower in CLA-fed cows than in Ctrl-fed cows. In spite of lower eGP in CLA-fed cows, lactose output and plasma glucose concentrations were greater in CLA-fed cows than in Ctrl-fed cows. This suggests a CLA-related glucose sparing effect most likely due to lower glucose utilization for milk fat synthesis and probably because of a more efficient whole-body energy utilization in CLA-fed cows.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Long-term elevation of β-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma β-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-β-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.