Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 mumol/l [n = 8, high NEFA (HNEFA)] and <140 mumol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFalpha), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, beta-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFalpha concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement.

Variation in hepatic regulation of metabolism during the dry period and in early lactation in dairy cows

The purpose of this study was to investigate variations in hepatic regulation of metabolism during the dry period, after parturition, and in early lactation in dairy cows. For this evaluation, cows were divided into 2 groups based on the plasma concentration of beta-hydroxybutyric acid (BHBA) in wk 4 postpartum (PP; group HB, BHBA >0.75 mmol/L; group LB, BHBA <0.75 mmol/L, respectively). Liver biopsies were obtained from 28 cows at drying off (mean 59 +/- 8 d antepartum), on d 1, and in wk 4 and 14 PP. Blood samples were collected every 2 wk during this entire period. Liver samples were analyzed for mRNA abundance of genes related to carbohydrate metabolism (pyruvate carboxylase, PC; phosphoenolpyruvate carboxykinase, PEPCK; citrate synthase, CS), fatty acid biosynthesis (ATP citrate lyase, ACLY) and oxidation (acyl-CoA synthetase long-chain, ACSL; carnitine palmitoyltransferase 1A, CPT 1A; carnitine palmitoyltransferase 2, CPT 2; acyl-coenzyme A dehydrogenase very long chain, ACADVL), cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, HMGCS1), ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, HMGCS2), and of genes encoding the transcription factors peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and sterol regulatory element binding factor 1 (SREBF1). Blood plasma was assayed for concentrations of glucose, BHBA, nonesterified fatty acids, cholesterol, triglycerides, insulin, insulin-like growth factor-I, and thyroid hormones. In both groups, plasma parameters followed a pattern usually observed in dairy cows. However, changes were moderate and the energy balance in cows turned positive in wk 7 PP for both groups. Additionally, the energy balance and milk yield were similar for both groups after parturition onwards. Significant group effects were found at drying off, when plasma concentrations of triglycerides were higher in LB than in HB, and in wk 4 PP, when plasma concentrations of glucose and IGF-I were lower in HB than in LB. Similarly, moderate changes in mRNA expression of hepatic genes between the different time points were observed, although HB cows showed more adaptive performance than LB cows based on changes in mRNA expression of PEPCKc, PEPCKm, CS, CPT 1A, CPT 2, and PPARalpha. Part of the variation measured in this study was explained by parity. Significant Spearman rank correlation coefficients between the variables were not similar at each time point and were not similar between the groups at each time point, suggesting that metabolic regulation differs between cows. In conclusion, metabolic regulation in dairy cows is a dynamic system, and differs obviously between cows at different metabolic stages related to parturition.

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.

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.

Liver fat content and lipid metabolism in dairy cows during early lactation and during a mid-lactation feed restriction.

During the transition period, the lipid metabolism of dairy cows is markedly affected by energy status. Fatty liver is one of the main health disorders after parturition. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages in lactation [NEB at the onset of lactation postpartum (p.p.) and a deliberately induced NEB by feed restriction near 100 d in milk] on liver triglyceride content and parameters of lipid metabolism in plasma and liver based on mRNA abundance of associated genes. Fifty multiparous dairy cows were studied from wk 3 antepartum to approximately wk 17 p.p. in 2 periods. According to their energy balance in period 1 (parturition to wk 12 p.p.), cows were allocated to a control (CON; n=25) or a restriction group (RES; 70% of energy requirements; n=25) for 3 wk in mid lactation starting at around 100 d in milk (period 2). Liver triglyceride (TG) content, plasma nonesterified fatty acids (NEFA), and β-hydroxybutyrate were highest in wk 1 p.p. and decreased thereafter. During period 2, feed restriction did not affect liver TG and β-hydroxybutyrate concentration, whereas NEFA concentration was increased in RES cows as compared with CON cows. Hepatic mRNA abundances of tumor necrosis factor α, ATP citrate lyase, mitochondrial glycerol-3-phosphate acyltransferase, and glycerol-3-phosphate dehydrogenase 2 were not altered by lactational and energy status during both experimental periods. The expression of fatty acid synthase was higher in period 2 compared with period 1, but did not differ between RES and CON groups. The mRNA abundance of acetyl-coenzyme A-carboxylase showed a tendency toward higher expression during period 2 compared with period 1. The solute carrier family 27 (fatty acid transporter), member 1 (SLC27A1) was upregulated in wk 1 p.p. and also during feed restriction in RES cows. In conclusion, the present study shows that a NEB has different effects on hepatic lipid metabolism and TG concentration in the liver of dairy cows at early and later lactation. Therefore, the homeorhetic adaptations during the periparturient period trigger excessive responses in metabolism, whereas during the homeostatic control of endocrine and metabolic systems after established lactation, as during the period of feed restriction in the present study, organs are well adapted to metabolic and environmental changes.

Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

Different adaptation of IGF-I and its IGFBPs in dairy cows during a negative energy balance in early lactation and a negative energy balance induced by feed restriction in mid-lactation

Control of metabolic pathways is a major task of the somatotropic axis and its constituents. Insulinlike growth-factor binding proteins (IGFBPs) bind IGF-I and -II and act as carriers and regulators of their activities in blood, body fluids and tissues. Over two periods of physiological adaptation, this study investigated the binding pattern of IGF-I to IGFBPs in the plasma of 50 multiparous Holstein dairy cows and identified relationships with the hepatic mRNA abundance of IGFBPs and plasma IGF-I during the lactational negative energy balance (NEB) and during a deliberately induced NEB by feed restriction. Period 1 lasted from week 3 antepartum (a.p.) to week 12 postpartum (p.p.) and period 2, the period of feed restriction, started at around 100 DIM and lasted for three weeks with a control (C) and a restricted group (R). Blood samples and liver biopsies were collected in week 3 a.p., and in weeks 1 and 4 p.p. of period 1 and in weeks 0 and 3 of period 2. For column chromatography of IGFBPs, plasma samples of all animals were pooled by group and time points of sampling. Plasma IGF-I dropped from week 3 a.p. to week 1 p.p. and thereafter increased until week 0 (period 2) and did not change up to week 3 of period 2. The binding of IGF-I to plasma IGFBP-1 and -2 increased in period 1 from week 3 a.p. to week 4 p.p., while at the same time it decreased for IGFBP-3. During period 2, the binding of IGF-I to plasma IGFBP-1 and -2 decreased for both groups, but less for R cows. In C cows, the IGF-I binding to IGFBP-3 in plasma increased from week 0 to week 3 of period 2, whereas R cows showed a slight decrease. In period 1, hepatic mRNA abundance of IGFBP-3 followed the plasma IGFBP-3 binding in contrast to the mRNA abundances of IGFBP-1 and -2. The latter increased from week 3 a.p. to week 1 p.p. and decreased afterwards whereas IGF-I binding to IGFBP-1 and -2 increased. In week 3 of period 2, the binding of IGF-I to IGFBP-1 and -2 and their hepatic mRNA abundance were higher in R cows compared to C cows. Hepatic mRNA abundance of IGF-I was consistently positively correlated with plasma IGF-I, especially pronounced during the NEBs in week 1 p.p. (period 1) and in week 3 (period 2) in R cows. While no distinct relation between mRNA abundance of IGFBP-1 and plasma IGF-I was evident, the mRNA abundance of IGFBP-2 was inversely related to plasma IGF-I over all experimental time points independent of treatment. The mRNA abundance of IGFBP-3 was particularly correlated with plasma IGF-I during the 2 experimental stages of a NEB. Obviously IGFBP-3, but not IGFBP-1 and -2, binding in plasma closely followed the respective pattern of hepatic mRNA abundance during the entire experimental period. The fact that changes in the different plasma IGFBPs during altering metabolic stages in different stages of lactation do not always strictly follow their mRNA abundance in liver suggests tissues other than the liver flexibly contributing to the IGFBP pool in plasma as well as a partially post-transcriptional regulation of IGFBP synthesis.

Comparison of hepatic adaptation in extreme metabolic phenotypes observed in early lactation dairy cows on-farm

The aim was to study the variation in metabolic responses in early-lactating dairy cows (n = 232) on-farm that were pre-selected for a high milk fat content (>45 g/l) and a high fat/protein ratio in milk (>1.5) in their previous lactation. Blood was assayed for concentrations of metabolites and hormones. Liver was measured for mRNA abundance of 25 candidate genes encoding enzymes and receptors involved in gluconeogenesis (6), fatty acid β-oxidation (6), fatty acid and triglyceride synthesis (5), cholesterol synthesis (4), ketogenesis (2) and the urea cycle (2). Two groups of cows were formed based on the plasma concentrations of glucose, non-esterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA) (GRP+, high metabolic load; glucose <3.0 mm, NEFA >300 μm and BHBA >1.0 mm, n = 30; GRP-, low metabolic load; glucose >3.0 mm, NEFA <300 μm and BHBA <1.0 mm, n = 30). No differences were found between GRP+ and GRP- for the milk yield at 3 weeks post-partum, but milk fat content was higher (p < 0.01) for GRP+ than for GRP-. In week 8 post-partum, milk yield was higher in GRP+ in relation to GRP- (37.5 vs. 32.5 kg/d; p < 0.01). GRP+ in relation to GRP- had higher (p < 0.001) NEFA and BHBA and lower glucose, insulin, IGF-I, T3 , T4 concentrations (p < 0.01). The mRNA abundance of genes related to gluconeogenesis, fatty acid β-oxidation, fatty acid and triglyceride synthesis, cholesterol synthesis and the urea cycle was different in GRP+ compared to GRP- (p < 0.05), although gene transcripts related to ketogenesis were similar between GRP+ and GRP-. In conclusion, high metabolic load post-partum in dairy cows on-farm corresponds to differences in the liver in relation to dairy cows with low metabolic load, even though all cows were pre-selected for a high milk fat content and fat/protein ratio in milk in their previous lactation.

Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

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.

Short communication: Circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows

Adiponectin, one of the most abundant adipokines in circulation, is known for its role in regulation of body metabolism. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages of lactation (lactational NEB at the onset of lactation and an induced NEB by feed restriction near 100 d of lactation) on circulating adiponectin concentrations. We also investigated the effect of feed restriction on adiponectin concentrations in milk and the relationships of blood and milk adiponectin with selected plasma or milk variables and with measures of body condition. Plasma adiponectin was measured in 50 multiparous Holstein dairy cows throughout 3 experimental periods [i.e., period 1=3 wk antepartum up to 12 wk postpartum, period 2=3 wk of feed restriction starting at around 100 d in milk with a control (n=25) and feed-restricted group (50% of energy requirements; n=25), and period 3=subsequent realimentation period for 8 wk]. Milk adiponectin was investigated among 21 multiparous cows at wk 2 and wk 12 of period 1 and wk 2 of period 2. Adiponectin concentrations in plasma and skim milk were measured using an in-house ELISA specific for bovine adiponectin. Major changes in circulating adiponectin concentrations were observed during the periparturient period, whereas energy deficiency during established lactation at around 100 d in milk and subsequent refeeding did not affect plasma adiponectin. Together with lower adiponectin concentrations in milk (µg/mL), the reduction in milk yield led to decreased adiponectin secretion via milk (mg/d) at the second week of feed restriction. Irrespective of time and treatment, milk adiponectin represented about 0.002% of total milk protein. Mean adiponectin concentrations in milk (0.61 ± 0.03 µg/mL) were about 92% lower than the mean plasma adiponectin concentrations (32.1 ± 1.0 µg/mL). The proportion of the steady-state plasma adiponectin pool secreted daily via milk was 2.7%. In view of the similar extent of NEB in both periods of energy deficiency, decreasing adiponectin concentrations seems important for accomplishing the adaptation to the rapidly increasing metabolic rates in early lactation, whereas the lipolytic reaction toward feed restriction-induced NEB during established lactation seems to occur largely independent of changes in circulating adiponectin.

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.

Milk fatty acid profile related to energy balance in dairy cows

Milk fatty acid (FA) profile is a dynamic pattern influenced by lactational stage, energy balance and dietary composition. In the first part of this study, effects of the energy balance during the proceeding lactation [weeks 1-21 post partum (pp)] on milk FA profile of 30 dairy cows were evaluated under a constant feeding regimen. In the second part, effects of a negative energy balance (NEB) induced by feed restriction on milk FA profile were studied in 40 multiparous dairy cows (20 feed-restricted and 20 control). Feed restriction (energy balance of -63 MJ NEL/d, restriction of 49 % of energy requirements) lasted 3 weeks starting at around 100 days in milk. Milk FA profile changed markedly from week 1 pp up to week 12 pp and remained unchanged thereafter. The proportion of saturated FA (predominantly 10:0, 12:0, 14:0 and 16:0) increased from week 1 pp up to week 12 pp, whereas monounsaturated FA, predominantly the proportion of 18:1,9c decreased as NEB in early lactation became less severe. During the induced NEB, milk FA profile showed a similarly directed pattern as during the NEB in early lactation, although changes were less marked for most FA. Milk FA composition changed rapidly within one week after initiation of feed restriction and tended to adjust to the initial composition despite maintenance of a high NEB. C18:1,9c was increased significantly during the induced NEB indicating mobilization of a considerable amount of adipose tissue. Besides 18:1,9c, changes in saturated FA, monounsaturated FA, de-novo synthesized and preformed FA (sum of FA >C16) reflected energy status in dairy cows and indicated the NEB in early lactation as well as the induced NEB by feed restriction.

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.