Little is known about the biological role of the phospholipase A2 receptor (PLA2R1) transmembrane protein. In recent years, PLA2R1 has been shown to have an important role in regulating tumor-suppressive responses via JAK2 activation, but the underlying mechanisms are largely undeciphered. In this study, we observed that PLA2R1 increases the mitochondrial content, judged by increased levels of numerous mitochondrial proteins, of the mitochondrial structural component cardiolipin, of the mitochondrial DNA content, and of the mitochondrial DNA replication and transcription factor TFAM. This effect of PLA2R1 relies on a transcriptional program controlled by the estrogen-related receptor alpha1 (ERRα) mitochondrial master regulator. Expression of ERRα and of its nucleus-encoded mitochondrial targets is upregulated upon PLA2R1 ectopic expression, and this effect is mediated by JAK2. Conversely, downregulation of PLA2R1 decreases the level of ERRα and of its nucleus-encoded mitochondrial targets. Finally, blocking the ERRα-controlled mitochondrial program largely inhibits the PLA2R1-induced tumor-suppressive response. Together, our data document ERRα and its mitochondrial program as downstream effectors of the PLA2R1-JAK2 pathway leading to oncosuppression.

Two experiments were conducted to determine the effect of an energy restriction (80% (ER) vs. 100% (CON) of total energy requirements; 140d: P1) followed by a re-alimentation (ad libitum intake; 70d; P2) on feed intake, body weight (BW) change, and feed efficiency in double-muscled Belgian Blue cows. Regression analysis based on feed intake and BW change during P1 was used to deduce energy requirements for maintenance by setting BW change to zero. The diet consisted of maize silage and 0.5kg mineral–vitamin premix in experiment 1, where dietary crude protein (CP) concentration was constant (105g/kg dry matter (DM)) during the whole experiment. A similar diet was fed during the restriction period in experiment 2 with 97gCP/kgDM. Extra soybean meal and urea were fed to all cows during the re-alimentation period of experiment 2, resulting in 198gCP/kgDM. Both experiments showed that BW loss of ER cows during P1 was not compensated during P2, so that BW gain of ER cows during the entire experiment remained lower compared to CON cows (P<0.05). Similar results were obtained for feed intake. Feed conversion ratio was not different between treatments during P2, but it tended to be worse for ER cows vs. CON cows for the entire experiment (P<0.10). Extra dietary crude protein during P2 in experiment 2 did not modify the effect of treatment on animal performance compared to experiment 1. Therefore, feeding double-muscled Belgian Blue cows below their energy requirements for maintenance, and growth in case of first and second-calf cows should be strongly discouraged. Metabolisable and net energy requirements for maintenance were estimated at 0.586MJ and 0.338MJ/kgBW0.75, respectively.

The aim of the present study was to determine to what extent an energy restriction, followed by a re-alimentation, affects body weight (BW), feed efficiency and concentrations of blood metabolites and IGF-1 (insulin-like growth factor 1). Two experiments were conducted with 20 (initial BW 621±18(SE)kg) and 13 (initial BW 613±29kg) non-pregnant dry Belgian Blue double-muscled cows, respectively. A similar diet was fed in both experiments, consisting of maize silage, 0.5kg per day of a vitamin–mineral premix, and urea to prevent a negative rumen degradable nitrogen balance. In experiment 1, cows were divided into 2 groups and fed an energy level (EL) corresponding to their requirements (EL100; 224 days), or 0.7 of their requirements (EL70–130) during the first 112 days (Phase 1), and 1.3 times the requirements during the next 112 days (Phase 2). Cows were weighed, scored for body condition, and blood samples were collected at 8-week intervals. Glucose, non-esterified fatty acids (NEFA), creatinine, IGF-1, IGFBP (IGF Binding Protein)-2 and IGFBP-3 were analyzed. EL70 resulted in a larger BW change compared to EL100 (−10.2% of the initial BW; P<0.001) during phase 1, but BW loss was completely compensated during phase 2. Efficiency of nutrient use (gain/intake) for the restriction and the re-alimentation period as a whole was similar for both treatments (P>0.10). Blood concentrations of metabolites and IGF-1, IGFBP-2 and IGFBP-3 were not affected by energy restriction (P>0.10). Concentrations of glucose, NEFA, creatinine and IGFBP-3 were affected by sampling day, while a treatment×sampling day interaction was found for NEFA. In experiment 2, cows were fed at 0.8 or 1.0 of their energy requirements during 140 days, followed by a 70-day re-alimentation period, where all cows were fed ad libitum. EL80 resulted in a BW loss of 7.9% of the initial BW during phase 1. Average growth rate for the restriction period and the re-alimentation period together remained lower (P=0.014) in comparison with cows fed according to their requirements. Furthermore, efficiency of nutrient use for the whole period was adversely affected P<0.05). Therefore, feeding Belgian Blue double-muscled cows below their energy requirements should be discouraged.

The use of culled potatoes was investigated in Belgian Blue double-muscled finishing cows, confined in tie stalls. The control diet (Treatment 1) consisted of concentrate and maize silage (50/50 on a dry matter (DM) basis). Potatoes either replaced 60% maize silage (Treatment 2) or 60% concentrate (Treatment 3). Diets were formulated to be isocaloric and isonitrogenous. They were fed ad libitum. Approximately 18 kg potatoes were fed daily in Treatments 2 and 3. Daily gain was not significantly altered; it decreased from 1.09 kg (Treatment 1) to 1.04 kg (Treatment 2) or increased to 1.20 kg (Treatment 3), although potatoes stimulated DM intake by 5% to 8% (P < 0.05). Feed conversion was unaffected in comparison with the control diet, when expressed in terms of DM, but energy efficiency (MJ/kg live weight gain) was substantially lower for Treatment 2 compared with Treatment 1 (89.9 v. 79.0; P = 0.046). Carcass weight, grading and composition were not affected by treatments, but potatoes increased dressing percentage (P = 0.009). Treatment had no significant effect on meat quality parameters. However, potatoes (Treatments 2 and 3) tended to decrease moisture content (P = 0.090) and tended to increase drip loss (P = 0.059) compared with Treatment 1. Because of a better animal performance and a lower feed cost, it is most appropriate to use potatoes as a replacement for concentrate. Feeding large amounts of potatoes besides concentrate may have an adverse effect on the fibrousness of the diet, resulting in a tendency (-5%) for a reduced daily gain and a lower energy efficiency (P < 0.05).

Six experiments were conducted to investigate the effect of a feed supplement on the performance of grazing Belgian Blue double-muscled (BBDM) heifers with an initial weight and age of 195 ± 43 kg and 190 ± 52 days. Treatments included were: Exp. 1: supplementation with beet pulp (BP): 2 kg/day per head v. ad libitum intake; Exp. 2: supplementation ad libitum with BP v. a mixture of BP and soybean meal (SBM; BP/SBM ratio of 80/20; FW (fresh weight) basis); Exp. 3: supplementation with 4 kg/day per head of a mixture of BP/SBM (80/20; FW basis) v. BP/formaldehyde-treated SBM (BP/FSBM); Exp. 4: supplementation with 4 kg/day per head of a mixture with a similar protein content (125 g DVE per kg dry matter (DM)), consisting of 80/20 BP/SBM v. 92/8 BP/FSBM; Exp. 5: supplementation with 3 kg/day per head of a mixture of BP/SBM (80/20; FW basis) v. BP/DDGS (dried distillers grains and solubles; 70/30, FW basis); and Exp. 6: supplementation with 3 kg/day per head of 80/20 BP/SBM v. maize silage (MS) and SBM, on the basis of a similar protein concentration in the DM as the 80/20 BP/SBM supplement, and fed at a similar amount of DM as in the BP/SBM group. Supplementing BP ad libitum did not affect daily gain (0.54 v. 0.48 kg) and partial feed conversion (3.62 kg on average) compared with 2 kg/day. Supplying SBM besides BP increased growth rate compared with BP (0.87 v. 0.62 kg/day; P < 0.001), but partial feed conversion was similar. Supplying FSBM did not affect growth rate and partial feed conversion (P > 0.10), but blood urea levels were reduced by FSBM (P < 0.05). DDGS tended to increase growth rate (0.77 v. 0.59 kg/day; P < 0.10) compared with BP/SBM, without effect on partial feed conversion. Replacing BP by MS did not affect daily gain, but partial feed conversion tended to be higher (3.21 v. 3.60 kg/kg body weight (BW) gain; P = 0.062). Increasing the supplement (80/20 BP/SBM) level from 3 to 4 kg daily, corresponding to 1.02% and 1.18% of the mean BW, respectively, resulted in a tendency (P = 0.121) for an increased growth rate. Grazing BBDM heifers of <1 year of age necessitate extra protein besides an energy supplement to improve their performance. DDGS can replace SBM and BP can be replaced by MS.

A palatable concentrate may stimulate intake and ease weaning stress. This study investigated the effect of the incorporation of spelt in the concentrate on calf performance (Experiment 1) and the palatability (Experiment 2).Sixty-six newborn Belgian Blue double-muscled female calves were involved in Experiment 1 to investigate the effect of incorporating 30% spelt grain in the concentrate (S+) as a replacement for 15% barley and 15% wheat (S-) during a 143-day rearing period. Calves received reconstituted milk at 10% of their birth weight. They were weaned when concentrate intake achieved 0.75 kg/day. Grass hay was fed ad lib. After the rearing period all calves were adapted during 2 weeks to the same diet, fed to appetite, and consisting of maize silage and concentrate without spelt. Performances were investigated during 4 weeks. Spelt reduced the in vitro organic matter digestibility and the net energy content of the concentrate from 91.5% and 7.4 MJ/kg to 88.1% and 7.0 MJ/kg, respectively. It stimulated concentrate intake initially, so that calves were weaned after 58 days compared to 67 days for calves fed S- concentrate (P=0.012). A higher amount of dry matter (P=0.056) was required per kg live-weight gain for S+ concentrate, but energy efficiency was similar for both treatments (P=0.415). Daily live-weight gain for the entire rearing period was not affected (P=0.970) and averaged 0.77 kg. Daily live-weight gain and feed intake were lower for the S+ treatment during the transition period (P < 0.001), but neither age at 1st calving nor post-partum live-weight was affected.Five Holstein-Friesian females and five Belgian Blue double-muscled females, aging 203 +/- 8 (mean standard error) days initially, and unaccustomed to eating S- and S+ concentrates, were involved in a preference test (Experiment 2). Both concentrates were administered free choice. Grass hay was fed ad lib. Concentrate intake was registered during 4 consecutive days at 0.5, 1, 3, 5, 8 and 24 h after the morning feeding. Concentrate containing spelt was preferred by Holstein-Friesian calves but not by Belgian Blue calves, resulting in a concentrate x breed interaction. This interaction emphasised the complex role of sensory and metabolic signals in the control of feed intake.It was concluded that spelt can stimulate concentrate intake, so that rearing calves can be weaned earlier. However, the preference of concentrate containing spelt exerted no major effect on animal performance during the entire rearing period. (C) 2012 Elsevier B.V. All rights reserved.

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90–Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1–STRAD and LKB1–Hsp90–Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1–Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90–Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90–Cdc37 and Hsp/Hsc70–CHIP, finely control the cellular level of LKB1 protein.

In this review, we summarize available data regarding bone phenotypes in estrogen receptors α and β, androgen receptor, and aromatase enzyme-deficient mice. We examine sex differences in the trabecular and cortical bone compartments and we discuss these findings in relation to known estrogen effects in humans. We also report how estrogen influences the responsiveness of the skeleton to exercise. Although uncertainties remain, it is clear that both estrogen and androgen are important for both male and female skeleton. Estrogen receptor α mainly through its classical signaling pathway is particularly important for the male mice skeleton while both estrogen receptors α and β are required for female mice skeleton. These deletions also induce major hormonal alterations themselves impacting on bone metabolism. More investigations are needed to fully understand the respective role of all these receptors in periosteal expansion in both sexes and the way they affect the mechanical sensitivity of the periosteum.

Four groups of five non-lactating and non-pregnant Belgian Blue double-muscled (BBDM) cows were used to investigate the effect of energy level (E) on digestion, and blood and urine metabolites. The energy levels of the groups, applied indoors during a 140-day restriction period, were 100%, 90%, 80% or 70% of their energy requirements (E100, E90, E80, E70) respectively. Afterwards, animals grazed on the same swards for 203 days (re-alimentation period). Balance trials were conducted at the end of the restriction period (BT1) and at the end of the re-alimentation period (BT2). Blood was sampled at the end of these trials. Diets consisted of maize silage and straw (80/20 on a dry matter basis) and a mineral-vitamin premix, fed at the appropriate E during BT1, or maize silage and a mineral-vitamin premix, fed at 125% of the maintenance requirements, during BT2. Significant increases of the digestibility coefficients were found during BT1 when E decreased, resulting in a better net energy capture of 7% for E70 compared with E100 (p < 0.05). Slightly, but non-significantly higher digestibility coefficients were observed for decreasing E during BT2. Plasma concentrations of glucose and creatinine did not differ between treatments during BT1, while differences were found for triacylglycerols and alpha-amino nitrogen. A tendency for a linear increase was observed for non-esterified fatty acids with decreasing E. Differences in blood metabolite concentrations disappeared in BT2. Urinary creatinine excretion was not affected by E, while body nitrogen loss increased linearly with energy restriction in BT1. No differences were found during BT2, suggesting that non-lactating and non-pregnant BBDM cows are able to adapt to a cyclic change of body weight and body reserves. These data show that restricted cows mobilized body fat as well as body protein. It is concluded that the qualitative aspects of metabolism during energy restriction are comparable in double-muscled cows with those in non-double-muscled animals, although the magnitude of the effects may be different.

Milk urea content (MUC) is used to manage protein nutrition and predict nitrogen excretion of dairy cows. However, MUC might depend on the roughage type offered and hence, for comparable MUC values, different N-excretions might be found. To evaluate this, three diets were compared in a feeding trial with 18 lactating Holstein cows in a Latin square design with as roughages 100% maize silage (treatment 100 MS), 50%/50% maize silage/prewilted grass silage (treatment 50 MS) and 100% prewilted grass silage (treatment 100 PGS). For all treatments, cows were fed to supply 105% of their net energy and digestible protein requirements and to have a daily rumen degraded protein balance (RDPB) intake of 100g. This was only possible by feeding soybean meal as a protein corrector to 100 MS and 50 MS and by feeding citruspulp as an energy corrector in 100 PGS. The same balanced concentrate was fed to all groups. In a separate trial, N-balance was determined for both 100% rations.In the feeding trial, the MUC of 100MS (230mg/1) and 50MS treatment (214mg/1) were significantly (P < 0.001) different from that of 100PGS (171 mg/l). Cows on treatments 50 MS and 100 PGS ingested the same amount of RDPB (71 and 73g/day), but when fed lOOMS cows ingested -16g/day. After correction for differences in energy and protein supply, MUC of the 100 MS was 71 mg/l higher than that of 100 PGS.N-balances indicated that total N-excretion (faecal, urinary and milk) was almost identical for both treatments: 392 for 100MS versus 389g/day for 100PGS, as was environmental N-excretion (faecal and urinary): 259 for 100 MS versus 272g/day for 100 PGS. However, the MUC content for 100MS was significantly higher: 248mg/l versus 180mg/l for 100PGS. From a correction for differences in energy and protein supply, this difference increased up to 84mg/l between 100MS and 100PGS.These results suggest that MUC is roughage dependent and that a system to predict N-excretion should account for these differences. Therefore the exact mechanism behind the determined roughage influence should be investigated further. (c) 2006 Elsevier B.V. All rights reserved.

We have investigated the effects of receptor-interacting protein 140 (RIP140) on transcriptional regulation by estrogen receptor-related receptors (ERRs). We first show that RIP140 inhibits transactivation by ERRalpha, beta, and gamma on natural or artificial reporter genes containing different types of response elements. This repression correlates with a strong in vitro binding between several regions of RIP140 and the three ERR isoforms. Surprisingly, although RIP140 inhibits transactivation of the thyroid hormone receptor-alpha gene by ERRbeta, it significantly increases its regulation by ERRalpha and ERRgamma. Mutagenesis and transient transfections in SL2 cells indicate that thyroid hormone receptor-alpha promoter expression involved Sp1 sites. In support of this observation, we demonstrate that RIP140 also positively regulates ERRs transactivation of other known Sp1 targets such as the p21 gene. This effect requires the two proximal Sp1 binding sites of the promoter and is partially dependent on the activation function 2 domain of ERRs. Finally, we provide evidences for a role of histone deacetylases in the regulation of p21 promoter by RIP140. Altogether, these data indicate that RIP140 differentially regulates ERR activity depending on the target sequence on the promoters.

Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen‐receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM‐homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRα) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.

The potential of a gas production technique (GPT) to predict fractions of rumen fermentable organic matter (OMf), crude protein (CP) and starch escaping rumen degradation (EP and ESTA), as well as in vitro organic matter digestibility (OMd) were studied with 30 samples of maize silage. Rumen degradation parameters were determined with four cannulated lactating cows by incubating finely cut fresh silage in nylon bags for 0, 12, 24, 48, 72 and 288 h. In vitro OMd was measured according to Tilley and Terry [Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc. 18, 104–111], using rumen fluid from two sheep. Gas production (GP) was continuously measured by incubating dried and ground samples in buffered rumen fluid from two sheep for 72 h, and GP curves were analysed with a three-phase model. Parameters studied were GP in the first 3 h of fermentation (GP3 h), between 3 and 20 h (GP3–20 h), the time to reach half of these values (b1 and b2), the maximum fermentation rate during the second phase (R2 max) and total GP, and OM fermented after 72 h of incubation (GP72 h and OMf72 h). Multiple regression was used to predict OMf, EP, ESTA and OMd based on GP parameters and chemical composition (i.e., DM, NDF, starch, CP). Fractions of OMf, EP, ESTA and OMd were 0.580 ± 0.040, 0.350 ± 0.062, 0.260 ± 0.106 and 0.732 ± 0.028, respectively. The GP3 h, GP3–20 h and GP72 h were 52 ± 5, 177 ± 13 and 323 ± 15 ml/g OM, respectively, b1 and b2 were 0.9 ± 0.2 and 8.7 ± 0.6 h, respectively, and R2 max was 15.6 ± 1.6 ml/h. All rumen degradability parameters could be predicted by a two-term regression with b2 as most important parameter. For OMf, the combination of b2 and GP3 h resulted in a determination coefficient (R2) of 50.4% and a residual standard deviation (R.S.D.) of 0.028. The EP could be predicted by b2 and CP (R2 = 68.9%; R.S.D. = 0.035) and ESTA by b2 and DM (R2 = 74.3%; R.S.D. = 0.054). In the case of OMd, OMf72 h was the single explaining variable (R2 = 77.9%; R.S.D. = 0.013).

Gastrulation is a process involving cellular commitment and movements whereby the three fundamental germ layers are established in vertebrates embryos. Estrogen Receptor-Related (ERR) alpha is a nuclear receptor displaying high sequence identity to the Estrogen Receptors (ERs). However, ERRalpha is unable to bind and to be regulated by estrogens or any natural ligand to date. Whereas recent studies have suggested roles for ERRalpha in bone and adipose tissue metabolism in the mouse, little is known about its roles during embryonic development. In zebrafish embryos, ERRalpha is expressed from the beginning of gastrulation at the margin of the blastoderm that represents the presumptive mesendoderm. Using loss of function (morpholinos or a dominant-negative version of the protein) and gain of function (mRNA injection) strategies, we show here that ERRalpha is involved in epiboly and convergent-extension (CE) processes in the zebrafish. Altogether, these results propose ERRalpha as a new regulator of morphogenetic movement during gastrulation, independently of cell fate determination.

Twenty-four nonlactating and nonpregnant Belgian Blue double-muscled cows, with diverging parities (one to seven), body conditions and body weights (436 to 903 kg), were used to investigate empty body (EB) composition. Direct measurements of EB composition, such as water, fat, protein, ash and energy, were carried out after slaughter. EB weight (EBW) averaged 624.7 kg and consisted of 393.3 kg water, 122.3 kg protein, 84.5 kg fat and 24.6 kg ash and was characterized by an energy content of 6158 MJ. Relationships between body weight (BW), body condition score (BCS), chest girth, dressing percentage, carcass grading score, EBW, rib-cut components and EB composition were determined. Significant regression equations (P<0.001) with a coefficient of determination (R2) of more than 0.9 were obtained between BW or BW and BCS and EB water, EB fat and EB energy. The prediction of EB ash was less accurate (R2<0.75). The relationship could further be improved by inclusion of carcass characteristics and rib-cut components (R2>0.95). Energy contents of EB lipids and protein amounted to 39.3 and 23.2 MJ/kg. EB protein (197 g/kg) was higher in the present double-muscled cows than reported for non-double-muscled animals, while EB fat (126 g/kg) and EB energy (9.5 MJ/kg) were lower. One BCS unit corresponded with 26.7 kg EB fat (P<0.001; R2=0.659). It can be concluded that simple live animal measurements as BW and BCS can be considered as potentially useful predictors of EB composition in double-muscled cows. Theoretical calculations based on the present observed data indicated that body reserves were lower in Belgian Blue double-muscled cows than in most other breeds. Body reserve tissue may be limited in young primiparous suckling cows so that energy restriction may be detrimental for reproductive performance.

For 10 batches of fresh grass, 11 of grass silage and 7 of grass hay, rumen degradation kinetics (soluble, potentially degradable and undegradable fraction and the degradation rate) of the organic matter (OM), crude protein (CP) and neutral detergent fibre (NDF), as well as the digestibility of rumen escape protein in the intestines (EPdi) were measured with 3 or 4 cows with cannulae in the rumen and duodenum. Data were used to calculate the fermentable OM content (FOM), the fraction of protein escaping the rumen (EP), the true protein digestible in the small intestine (DVE) and the degraded protein balance in the rumen (OEB) according to the Dutch protein evaluation system. The nutritive quality of fresh grass and silage was generally better than that of hay. On average, the FOM content of fresh grass, silage and hay amounted to 618, 571 and 552g/kg DM, respectively. The EP of hay was almost double that of fresh grass and silage (0.535, 0.287 and 0.244 respectively). The EPdi did not differ greatly among grassland products, with 0.849 for fresh grass, 0.794 for silage and 0.816 for hay. The DVE content of fresh grass was similar to that of hay (86 and 85g/kg DM) and higher than that of silage (68g/kg DM). The OEB content was similar and positive for both fresh grass and silage (24 and 26g/kg DM), whereas that of hay was negative (−30g/kg DM). In order to predict in situ values, regression equations with growth days, chemical parameters, OM digestibility (OMd) and the washable CP fraction (WCP) were developed for the three grassland products separately, as well as pooled. Most significant variables were OMd, WCP, CP and ash and separate equations for the three grassland products were more accurate than a general equation. Use of the best product-specific equations resulted in a residual standard deviation (RSD) for the 28 studied samples of 8g/kg DM for FOM, 0.016 for EP, 6g/kg DM for DVE and 6g/kg DM for OEB. For EPdi, a general equation for the pooled grassland products gave an RSD of 0.042. With near-infrared reflection spectroscopy (NIRS), the number of samples was too small to develop product-specific calibrations. A global calibration for the three grassland products gave cross-validation errors of 20g/kg DM, 0.057, 0.049, 9g/kg DM and 14g/kg DM, for respectively FOM, EP, EPdi, DVE and OEB, clearly less accurate than with laboratory measurements.

The effect of feeding a total mixed ration (TMR) and the effect of protein balance in the rumen (OEB) on milk urea concentration (MUC) was studied. Eighteen Holstein cows were offered three diets in a 3×3 Latin square design. Separate feeding (rations 1 and 3) of maize silage in the morning and prewilted grass silage in the evening was compared with a TMR (ration 2). Rations 1 and 2 provided a normal OEB level (±150 g/day), whereas ration 3 a high OEB level (±400 g/day). All diets were formulated to supply the same level of true protein digested in the small intestine (DVE) and net energy lactation (VEM). The MUC of the morning milk was higher than that of the evening milk with separate feeding, whereas no difference was observed with the TMR. However, feeding strategy had no significant effect on the mean daily MUC, which was 247 and 240 mg/l for ration 1 and 2, respectively. The high daily OEB supply resulted in a MUC of 331 mg/l, corresponding with an increase in MUC of 0.33 mg/l per g higher OEB. The ammonia (NH3) concentration in rumen fluid of three fistulated lactating cows was determined for each ration in a Latin-square design (3×3). The NH3 concentration reached a maximum about 2 h postfeeding. For the diets with equal OEB, peak NH3 levels were similar. The NH3 concentration took longer to decrease after prewilted grass silage was fed than after feeding maize silage. The highest NH3 concentrations were measured for the diet with the high OEB supply. Average daily rumen pH values were equal for all diets.

Chemical composition, digestibility, nutritive value and intake of hay from an agri-environmental management (EH) were compared with those from hay (Lolium perenne) from an intensive management (IH). IH was of low to moderate quality because of unfavourable weather conditions. EH was harvested mid-June of 2000 (EH1) and 2001 (EH2) on the same sward that had not received mineral fertilizer for 10 years. The EH was characterized by a species-rich botanical composition. On average, it had lower contents of protein (32%), NDF (9%) and ash (35%), and a higher concentration of water-soluble carbohydrates (117%) than IH. Digestibility of dry and organic matter, determined with sheep, was not different between IH and EH and averaged 59 and 63%, respectively. Crude fibre and NDF digestibility were lower in EH (58 and 57%, respectively) than in IH (70 and 69%, respectively). Net energy value for lactation did not differ between IH and EH and amounted to 4.78 MJ per kg DM. True protein digested in the small intestine and rumen degraded protein balance were lower in EH (63 and − 60 g per kg DM) than in IH (71 and − 33 g per kg DM). Intake of hay was investigated in Holstein-Friesian heifers and Belgian Blue double-muscled heifers (mean BW 280 ± 22 kg and 269 ± 21 kg, respectively), and in Belgian Blue non-lactating and non-pregnant double-muscled cows (initial BW 642 ± 82 kg), using a cross-over design. Hay was freely available. It was supplemented with 1 kg concentrate daily. Dry matter intake from hay was higher for EH than for IH in heifers (4% and 13%, respectively in Holstein-Friesian and Belgian Blue heifers) and in cows (22%). Hay from an agri-environmental management may be used for low-performing animals, as energy intake only exceeded maintenance requirements by 20 to 35%. Several characteristics of EH were different between years, such as dry matter digestibility, net energy value for lactation and fermentable organic matter content.

Circadian gene expression has been demonstrated in many tissues and involves both positive and negative regulatory loops. The potential interferences of circadian rhythmicity with other well-known biologic rhythms, such as the ovarian cycle, at least in part controlled by estrogens, has not been questioned. The estrogen receptor-related receptor (ERR)alpha is an orphan nuclear receptor that is widely expressed in estrogen-responsive tissues such as liver, uterus and bone. In addition, expression of the ERRalpha gene has been proposed to be transcriptionally controlled by estrogens in the uterus. Here we show that the expression of ERRalpha displays a circadian rhythmicity in liver, bone and uterus. This is in contrast to other uterine estrogen-regulated genes. Analysis of clock/clock mutant mice shows that ERRalpha is an output gene of the circadian clock oscillator. The expression of clock-control genes, such as Bmal1 and Rev-erbalpha, also displays diurnal oscillations in the uterus, but not in bone. In this tissue, however, Per2 displayed a rhythmic expression, altogether suggesting unconventional loops in the regulation of circadian rhythm in bone.