Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2±0.24) and the DP (12.5±0.22) as compared with during early lactation (10.2±0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9±24.8 and 49.0±4.96% of cows, respectively), basal (56.2±28.1 and 54.6±7.78% of cows, respectively), or entire cytoplasm (56.8±13.4 and 61.6±14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.

Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk

A novel real-time quantitative PCR assay for detecting the pathogenic and contagious Staphylococcus aureus genotype B (GTB) in bulk tank milk was developed and evaluated. The detection of this pathogen in bulk tank milk would greatly facilitate its control, as it is responsible for great economic loss in Swiss dairy herds. The assay is based on the simultaneous detection of 3 GTB-typical target sequences, including 2 enterotoxin genes and a polymorphism within the leucotoxin E gene. A variety of mastitis-associated bacteria was used to validate the assays, resulting in an analytical specificity of 100% and high repeatability. The analytical sensitivity in milk was 40 cfu/mL. An exponential association between simulated cow prevalence and quantitative PCR result was observed. An initial field study revealed 1 GTB-positive herd among the 33 studied herds. This novel assay for bulk tank milk analysis is suitable for routine purposes and is expected to be an effective tool for minimizing Staph. aureus GTB in Swiss dairy herds.

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein x Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of alpha-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.

Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation

Homeorhetic and homeostatic controls in dairy cows are essential for adapting to alterations in physiological and environmental conditions. To study the different mechanisms during adaptation processes, effects of a deliberately induced negative energy balance (NEB) by feed restriction near 100 d in milk (DIM) on performance and metabolic measures were compared with lactation energy deficiency after parturition. Fifty multiparous cows were studied in 3 periods (1=early lactation up to 12 wk postpartum; 2=feed restriction for 3 wk beginning at 98+/-7 DIM with a feed-restricted and control group; and 3=a subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, despite NEB in early lactation [-42 MJ of net energy for lactation (NE(L))/d, wk 1 to 3] up to wk 9, milk yield increased from 27.5+/-0.7 kg to a maximum of 39.5+/-0.8 kg (wk 6). For period 2, the NEB was induced by individual limitation of feed quantity and reduction of dietary energy density. Feed-restricted cows experienced a greater NEB (-63 MJ of NEL/d) than did cows in early lactation. Feed-restricted cows in period 2 showed only a small decline in milk yield of -3.1+/-1.1 kg and milk protein content of -0.2+/-0.1% compared with control cows (30.5+/-1.1 kg and 3.8+/-0.1%, respectively). In feed-restricted cows (period 2), plasma glucose was lower (-0.2+/-0.0 mmol/L) and nonesterified fatty acids higher (+0.1+/-0.1 mmol/L) compared with control cows. Compared with the NEB in period 1, the decreases in body weight due to the deliberately induced NEB (period 2) were greater (56+/-4 vs. 23+/-3 kg), but decreases in body condition score (0.16+/-0.03 vs. 0.34+/-0.04) and muscle diameter (2.0+/-0.4 vs. 3.5+/-0.4 mm) were lesser. The changes in metabolic measures in period 2 were marginal compared with the adjustments directly after parturition in period 1. Despite the greater induced energy deficiency at 100 DIM than the early lactation NEB, the metabolic load experienced by the dairy cows was not as high as that observed in early lactation. The different effects of energy deficiency at the 2 stages in lactation show that metabolic problems in early lactating dairy cows are not due only to the NEB, but mainly to the specific metabolic regulation during this period.

Endocrine changes and liver mRNA abundance of somatotropic axis and insulin system constituents during negative energy balance at different stages of lactation in dairy cows

The liver has an important role in metabolic regulation and control of the somatotropic axis to adapt successfully to physiological and environmental changes in dairy cows. The aim of this study was to investigate the adaptation to negative energy balance (NEB) at parturition and to a deliberately induced NEB by feed restriction at 100 days in milk. The hepatic gene expression and the endocrine system of the somatotropic axis and related parameters were compared between the early and late NEB period. Fifty multiparous cows were subjected to 3 periods (1=early lactation up to 12 wk postpartum, 2=feed restriction for 3 wk beginning at around 100 days in milk with a feed-restricted and a control group, and 3=subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, plasma growth hormone reached a maximum in early lactation, whereas insulin-like growth factor-I (IGF-I), leptin, the thyroid hormones, insulin, and the revised quantitative insulin sensitivity check index increased gradually after a nadir in early lactation. Three days after parturition, hepatic mRNA abundance of growth hormone receptor 1A, IGF-I, IGF-I receptor and IGF-binding protein-3 (IGFBP-3) were decreased, whereas mRNA of IGFBP-1 and -2 and insulin receptor were upregulated as compared with wk 3 antepartum. During period 2, feed-restricted cows showed decreased plasma concentrations of IGF-I and leptin compared with those of control cows. The revised quantitative insulin sensitivity check index was lower for feed-restricted cows (period 2) than for control cows. Compared with the NEB in period 1, the changes due to the deliberately induced NEB (period 2) in hormones were less pronounced. At the end of the 3-wk feed restriction, the mRNA abundance of IGF-I, IGFBP-1, -2, -3, and insulin receptor was increased as compared with the control group. The different effects of energy deficiency at the 2 stages in lactation show that the endocrine regulation changes qualitatively and quantitatively during the course of lactation.

First report about the mode of action of combined butafosfan and cyanocobalamin on hepatic metabolism in nonketotic early lactating cows

The primary aim was to investigate the effect of combined butafosfan and cyanocobalamin on liver metabolism in early lactating cows through mRNA expression measurements of genes encoding 31 enzymes and transport proteins of major metabolic processes in the liver using 16 multiparous early lactating dairy cows. The treatments included i.v. injection of 10 mL/100 kg of body weight combined butafosfan and cyanocobalamin (TG, n = 8) on 3 d consecutively at 25 +/- 3 d in milk or injection with physiological saline solution similarly applied (CG, n = 8). Results include a higher daily milk production for TG cows (41.1 +/- 0.9 kg, mean +/- SEM) compared with CG cows (39.5 +/- 0.7 kg). In plasma, the concentration of inorganic phosphorus was lower in the TG cows (1.25 +/- 0.08 mmol/L) after the treatment than in the CG cows (1.33 +/- 0.07 mmol/L). The plasma beta-hydroxybutyrate concentration was 0.65 +/- 0.13 mmol/L for all cows before the treatment, and remained unaffected post treatment. The unique result was that in the liver, the mRNA abundance of acyl-coenzyme A synthetase long-chain family member 1, involved in fatty acid oxidation and biosynthesis, was lower across time points after the treatment for TG compared with CG cows (17.5 +/- 0.15 versus 18.1 +/- 0.24 cycle threshold, log(2), respectively). In conclusion, certain effects of combined butafosfan and cyanocobalamin were observed on mRNA abundance of a gene in the liver of nonketotic early lactating cows.

Induced hypoglycemia for 48 hours indicates differential glucose and insulin effects on liver metabolism in dairy cows

Hypoglycemia is a characteristic condition of early lactation dairy cows and is subsequently dependent on, and may affect, metabolism in the liver. The objective of the present study was to investigate the effects of induced hypoglycemia, maintained for 48 h, on metabolic parameters in plasma and liver of mid-lactation dairy cows. The experiment involved 3 treatments, including a hyperinsulinemic hypoglycemic clamp (HypoG, n=6) to obtain a glucose concentration of 2.5 mmol/L, a hyperinsulinemic euglycemic clamp (EuG, n=6) in which the effect of insulin was studied, and a control treatment with a 0.9% saline solution (NaCl, n=6). Blood samples for measurements of insulin, metabolites, and enzymes were taken at least once per hour. Milk yield was recorded and milk samples were collected before and after treatment. Liver biopsies were obtained before and after treatment to measure mRNA abundance by real-time, quantitative reverse transcription-PCR of 12 candidate genes involved in the main metabolic pathways. Milk yield decreased in HypoG and NaCl cows, whereas it remained unaffected in EuG cows. Energy-corrected milk yield (kg/d) was only decreased in HypoG cows. In plasma, concentration of beta-hydroxybutyrate decreased in response to treatment in EuG cows and was lower (0.41+/-0.04 mmol/L) on d 2 of the treatment compared with that in HypoG and NaCl cows (on average 0.61+/-0.03 mmol/L, respectively). Nonesterified fatty acids remained unaffected in all treatments. In the liver, differences between treatments for their effects were only observed in case of mitochondrial phosphoenolpyruvate carboxykinase (PEPCKm) and glucose-6-phosphatase (G6PC). In HypoG, mRNA abundance of PEPCKm was upregulated, whereas in EuG and NaCl cows, it was downregulated. The EuG treatment downregulated mRNA expression of G6PC, a marked effect compared with the unchanged transcript expression in NaCl. The mRNA abundance of the insulin receptor remained unaffected in all treatments, and no significant treatment differences were observed for genes related to lipid metabolism. In conclusion, low glucose concentrations in dairy cows affect liver metabolism at a molecular level through upregulation of PEPCKm mRNA abundance. Metabolic regulatory events in the liver are directed, apart from hormones, by the level of metabolites, either in excess (e.g., free fatty acids) or in shortage (e.g., glucose).

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1x) and the other twice a day (2x). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2x but not in the half milked 1x. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Maturation of endogenous glucose production in preterm and term calves

Glucose disposability is often impaired in neonatal calves and even more in preterm calves. The objective of this study was to investigate ontogenic maturation of endogenous glucose production (eGP) in calves and its effects on postnatal glucose homeostasis. Calves (n = 7 per group) were born preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4 d (TC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT; T) or on d 4 of life (TC) to determine metabolic and endocrine changes. After birth (PT and T) or on d 3 of life (TC), fasted calves were gavaged with deuterium-labeled water to determine gluconeogenesis (GNG) and intravenously infused with [U(13)C]-glucose to measure eGP and glucose oxidation (GOx) in blood plasma. After slaughter at 26h after birth (PT, T) or on d 4 of life (TC), glycogen concentrations in liver and hepatic mRNA concentrations and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase were measured. Preterm calves had the lowest plasma concentrations of cortisol and 3,5,3'-triiodothyronine at birth. Plasma glucose concentrations from d 1 to 2 decreased more, but plasma concentrations of lactate and urea and glucagon:insulin ratio were higher in PT than in T and TC calves. The eGP, GNG, GOx, as well as hepatic glycogen concentrations and PEPCK activities, were lowest in PT calves. Results indicate impaired glucose homeostasis due to decreased eGP in PT calves and maturation of eGP with ontogenic development.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Effects of housing, perches, genetics, and 25-hydroxycholecalciferol on keel bone deformities in laying hens

Several studies have shown a high prevalence of keel bone deformities in commercial laying hens. The aim of this project was to assess the effects of perch material, a vitamin D feed additive (25-hydroxyvitamin D(3); HyD, DSM Nutritional Products, Basel, Switzerland), and genetics on keel bone pathology. The study consisted of 2 experiments. In the first experiment, 4,000 Lohmann Selected Leghorn hens were raised in aviary systems until 18 wk of age. Two factors were investigated: perch material (plastic or rubber-coated metal) and feed (with and without HyD). Afterward, the hens were moved to a layer house with 8 pens with 2 aviary systems. Daily feed consumption, egg production, mortality, and feather condition were evaluated. Every 6 wk, the keel bones of 10 randomly selected birds per pen were palpated and scored. In the second experiment, 2,000 Lohmann Brown (LB) hens and 2,000 Lohmann Brown parent stock (LBPS) hens were raised in a manner identical to the first experiment. During the laying period, the hens were kept in 24 identical floor pens but equipped with different perch material (plastic or rubber-coated metal). The same variables were investigated as in the first experiment. No keel bone deformities were found during the rearing period in either experiment. During the laying period, deformities gradually appeared and reached a prevalence of 35% in the first experiment and 43.8% in the second experiment at the age of 65 and 62 wk, respectively. In the first experiment, neither HyD nor the aviary system had any significant effect on the prevalence of keel bone deformities. In the second experiment, LBPS had significantly fewer moderate and severe deformities than LB, and rubber-coated metal perches were associated with a higher prevalence of keel bone deformities compared with plastic perches. The LBPS laid more but smaller eggs than the LB. Again, HyD did not affect the prevalence of keel bone deformities. However, the significant effect of breed affiliation strongly indicates a sizeable genetic component that may provide a basis for targeted selection.

Gastrointestinal tract mucosal histomorphometry and epithelial cell proliferation and apoptosis in neonatal and adult dogs

In this study, the hypothesis was tested that the size of gastrointestinal tract (GIT) mucosal components and rates of epithelial cell proliferation and apoptosis change with increasing age. The aims were to quantitatively examine GIT histomorphology and to determine mucosal epithelial cell proliferation and apoptosis rates in neonatal (<48 h old) and adult (8 to 11.5 yr old) dogs. Morphometrical analyses were performed by light microscopy with a video-based, computer-linked system. Cell proliferation and apoptosis of the GIT epithelium were evaluated by counting the number of Ki-67 and caspase-3-positive cells, respectively, using immunohistochemical methods. Thickness of mucosal, glandular, subglandular, submucosal and muscular layers, crypt depths, villus heights, and villus widths were consistently greater (P < 0.05 to P < 0.001), whereas villus height/crypt depth ratios were smaller (P < 0.001) in adult than in neonatal dogs. The number of Ki-67-positive cells in stomach, small intestine, and colon crypts, but not in villi, was consistently greater (P < 0.01) in neonatal than in adult dogs. In contrast, the number of caspase-3-positive cells in crypts of the stomach, small intestine, and colon and in villi was not significantly influenced by age. In conclusion, canine GIT mucosal morphology and epithelial cell proliferation rates, but not apoptosis rates, change markedly from birth until adulthood is reached.

Milk flow-dependent vacuum loss in high-line milking systems: effects on milking characteristics and teat tissue condition

To study the effects of a milking system that partially compensates for milk flow-dependent vacuum loss compared with a standard (high-line) milking unit in a tie-stall barn, milk flow and vacuum patterns were recorded in 10 cows during machine milking with 2 milking systems in a crossover design for 7 d each. Before and after each treatment period postmilking teat condition was recorded by ultrasound cross-sectioning. Additionally, 2 methods to measure teat tissue condition were compared: longitudinal teat ultrasound cross-sectioning and teat tissue density measurements with the spring-loaded caliper (cutimeter method). The partial compensation of milk flow-dependent vacuum loss caused an elevation of the peak flow rate (4.74+/-0.08 vs. 4.29+/-0.07 kg/min) and a shorter duration of plateau (1.57+/-0.06 vs. 1.96+/-0.07 min) compared with the standard milking system. Total milk yield, duration of incline and decline of milk flow, average milk flow, time until peak flow rate, main milking time, and total milking time did not differ between treatments (overall means: 13.75+/-0.17 kg; 0.65+/-0.01 min; 2.88+/-0.09 min; 2.82+/-0.05 kg/min; 1.65+/-0.03 min; 5.23+/-0.09 min, and 5.30+/-0.10 min, respectively). The vacuum drop in the short milk tube during periods of high milk flow was less in the compensating vacuum than in the standard milking system (11+/-1.1 vs. 15+/-0.7 kPa). Teat measures as determined by ultrasound remained unchanged over the entire experimental period with both milking systems. Postmilking teat tissue measures including their recovery within 20 min after the end of milking show a correlation (0.85 and 0.71, respectively) between the methods used (ultrasound and cutimeter method). In conclusion, a more constant vacuum at the teat tip (within the short milk tube) during periods of high milk flow affected milk flow patterns, mainly increasing peak flow rate. However, the reduced vacuum loss did not increase the overall speed of milking. In addition, effects of higher vacuum stability on teat condition and udder health were not obvious.