Farming systems with a reduced or absent concentrate supplementation, as postulated by organic agriculture associations, require adapted dairy cows. The aim of this experiment was to examine the impact of concentrate supplementation on milk production, grazing and rumination behaviour, feed intake, physical activity and blood traits with two Holstein-Friesian cow strains and to conclude the consequences for sustainable and organic farming. The experiment was a cross-over study and took place on an organic farm in Switzerland. In all, 12 Swiss Holstein-Friesian (HCH) cows and 12 New Zealand Holstein-Friesian (HNZ) cows, which were paired according to lactation number, days in milk and age for primiparous cows, were used. All cows grazed full time and were supplemented either with 6 kg/day of a commercial, organic cereal-grain mix or received no supplement. After an adaptation period of 21 days, a measurement period of 7 days followed, where milk yield and composition, pasture dry matter intake estimated with the n-alkane double-indicator technique, physical activity based on pedometer measurements, grazing behaviour recorded by automatic jaw movement recorder and blood samples were investigated. Non-supplemented cows had a lower milk yield and supplemented HCH cows produced more milk than supplemented HNZ cows. Grazing time and physical activity were greater for non-supplemented cows. Supplementation had no effect on rumination behaviour, but HNZ cows spent longer ruminating compared with HCH cows. Pasture dry matter intake decreased with the concentrate supplementation. Results of blood analysis did not indicate a strong negative energy balance for either non-supplemented or supplemented cows. Minor differences between cow strains in this short-term study indicated that both cow strains are equally suited for an organic pasture-based production system with no concentrate supplementation. Many factors such as milk yield potential, animal welfare and health, efficiency, grazing behaviour and social aspects influence the decision to supplement grazing dairy cows with concentrates.As ruminants are able to digest fibre efficiently and assuming that competition for feed  In the future, competition between feed and food will increase. Ruminants like dairy cows are able to digest forage fibre efficiently. As concentrate supplementation is limited in organic dairy farming, restrictions may cause health problems as energy requirements for high-yielding dairy cows may not be met from forage-only rations. The aim of this study was to verify how supplementation changes the behaviour and production of grazing dairy cows under organic conditions. The two Holstein cow strains investigated in this short-term study are equally suited for an organic, pasture-based farming system with no concentrate supplementation.et al., et al., et al., In Switzerland, the milk yield per cow has increased steadily over the last years  by the Animal Care Committee of the Canton of Fribourg, Switzerland. Before selecting the cows for the experiment, a medical examination was performed. The experiment consisted of two measurement periods, each consisting of a 21-day adaption period and a 7-day measurement period. For the flow of work and equipment reasons, the cows were equally divided into two consecutive data collection periods of 7 days/measurement period. The experiment took place on the organic farm ‘Ferme École de Sorens’, located 824 m above sea level in Sorens, Switzerland.A total of 24 HF cows, including 12 Swiss Holstein-Friesian (HCH) cows and 12 HF cows of New Zealand origin (HNZ), were used for the experiment. In all, 14 of them were multiparous and 10 were primiparous. Matched pairs of HCH and HNZ cows were formed according to the number of lactation, days in milk (DIM) and age for primiparous cows. The average economic breeding value  was 985 (SD 74) for HCH cows. The average economic breeding value for HNZ cows was 806 (SD 70), but this excluded four animals as no breeding value was available.At the start of the first data collection period, HCH cows had an average number of lactations of 2.3 (SD 1.6), had been 109 (SD 17.9) DIM, had an average BW of 609 (SD 90.1) kg, a BCS of 2.4 (SD 0.28) and were producing 27.6 (SD 3.77) kg milk/day. The HNZ cows had an average number of lactations of 2.7 (SD 2.0), had been 114 (SD 16.7) DIM, had an average BW of 560 (SD 72.3) kg, a BCS of 2.9 (SD 0.24) and were producing 24.1 (SD 5.27) kg milk/day.n=5) mm, corresponding to 1024 (SD 209.3) kg DM/ha above 50 mm in the first measurement period and 122  mm, corresponding to 941 (SD 143.8) kg DM/ha for the second measurement period. The average post-grazing sward surface height was 57  mm in the first measurement period and 71  mm in the second measurement period. Herbage mass above 50 mm (kg DM/ha) was calculated according to: −624.5+12.8×sward height (mm). This regression was calibrated for the pastures of the organic farm ‘Ferme École de Sorens’ . The pastures were long established and composed predominantly of grasses , but also of clover (mainly Trifolium repens) and herbs . The pastures were fertilized once per year with 25 m3/ha of farm-produced manure . The chemical composition of the pasture during the measurement periods is presented in The experiment was carried out in a rotational grazing system from 7 May to 8 July 2012. All 24 experimental cows were managed as a single group separated from the rest of the lactating herd and grazed on pasture from 0800 to 1400 h and from 1800 to 0430 h the following morning. Meanwhile, cows were milked and housed in a free-stall barn. Paddocks were rotationally grazed for 2 to 5 days based on decision rules considering sward height with a reference of 130-mm pre-grazing equivalent to an herbage mass of ~1000 kg dry matter (DM)/ha above 50 mm until a post-grazing sward surface height of 50 mm from ground level. The sward surface height was measured with a pasture meter  before cows entered a new paddock and after leaving the paddock. The average pre-grazing sward height was 129 , 0 or 6 kg (as-fed basis), took place. The pelleted concentrate was offered to six HCH–HNZ cow pairs in two equal meals (3 kg at 0600 h and 3 kg at 1700 h) after milking in the free-stall barn using separate buckets for each cow. During the measurement period, all 6 kg of concentrate was ingested by all cows with no refusals. The other six HCH–HNZ cow pairs received no concentrate in addition to pasture. Fresh water was always available and a mineral block was available in the barn.The ambient outdoor temperature was recorded daily by the meteorological station in Grangeneuve , located about 15 km north of the experimental pastures. During the first measurement period, the average temperature was 16°C  and 19°C  in the second measurement period.et al.  was recorded daily and milk composition was analysed from a pooled sample of the morning and evening milk on days 1, 4 and 7 of each data collection period. Milk samples were preserved in tubes containing Broad Spectrum Microtabs II  at 8°C. The BW was recorded twice daily after milking and BCS was assessed before each adaptation period and before and after each data collection period according to the five-point system of Edmonson et al. .n-alkane double-indicator technique was used  on a carrier of dried fruit pomace, were administered manually with an applicator twice per day starting 6 days before the data collection periods. During the data collection period, a daily spot sample of faeces was taken from each cow with or without stimulus between 0700 and 0800 h. Samples were pooled by cow and collection period and stored at −20°C. Collection of pasture samples started and ended 1 day before the faeces sampling. Pasture sample collection was carried out as described by Graf et al. . The pedometer was attached to the right hind leg of the cow at the metatarsus level and recorded acceleration in three dimensions at 0.1 s intervals for 72 h. Using the software program IceTag-Analyser , the data were downloaded and compiled over 60 s intervals. Walking was defined as >3 steps/min, as suggested by Thanner et al. . Plasma for the analysis of hormones was retrieved using Vacuette® EDTA tubes (Greiner Bio-One GmbH). After sampling, these tubes were cooled in ice water until they were centrifuged at room temperature (20°C) for 15 min at 3000×g. For analyses of blood metabolites and enzymes, Vacuette® serum tubes (Greiner Bio-One GmbH) were stored upside down for 1 h at room temperature (20°C) before centrifugation at 3000×g for 15 min and then at 4000×g for an additional 5 min  for contents of fat, protein and lactose  were determined as described by Peiretti et al. . Concentrate, pasture and faeces samples were milled through a 1.0-mm screen . Subsamples were dried for 3 h at 105°C to determine DM and subsequently incinerated at 550°C until they reached a stable mass to assess the ash contents. Mineral residues in the ash were dissolved with nitric acid and analysed for Ca, Na, P, Mg and K with inductively coupled plasma optical emission spectrometry  based on European Standard: EN 155510:2008. The contents of i et al. . The N ci et al.  on a C/Ni et al. . Acid-dei et al.  was detei et al.  and NDF i et al.  was asseγ-glutamyltransferase , glutamate dehydrogenase , total protein , urea , triglyceride , non-esterified fatty acids  and glucose .Metabolite concentrations and enzyme activities were determined using the following commercial test kits: albumin , alkaline phosphatase , alanine aminotransferase , aspartate aminotransferase , β-hydroxybutyrate , cholesterol , creatinine , creatine kinase , et al. .Plasma insulin and IGF-1 concentrations were quantified using radioimmunoassay as described by Vicari et al. . 3,5,3'-n-alkanes HC32 and HC33 on the basis of the equation proposed by Mayes et al. (Net energy for lactation (NEL) and the absorbable protein in the small intestine when rumen fermentable energy is limiting microbial protein synthesis in the rumen were calculated according to Agroscope . The enes et al. .conc0) and with concentrate supplementation (equation (2), DMIconc6) was additionally calculated according to Baker (0.75 is the metabolic body size (kg BW to the power 0.75), ECM (kg/d) the energy-corrected milk yield, NEL Conc the NEL content of the concentrate (MJ/kg DM) and NEL pasture the NEL content of the pasture (MJ/kg DM).To double check the intake estimation, the pasture intake without (equation (1), DMIto Baker  based onto Baker . ChangesYijklm is the response, μ the least-squares mean, τithe fixed effect of cow strain i , φjthe fixed effect of the treatment j , Pk the fixed effect of the period , (τp)ik the effect of the interaction between cow strain i and period k, (τφ)ij the effect of the interaction between cow strain i and treatment j, Plthe random effect of cow pair l , Km the random effect of the cow m  and εijklmthe random error. Models of this type were recommended by Tempelman (2004) with variance components as variance–covariance structure for the repeated measurements. As there were only two periods no alternative variance–covariance structure was envisaged in this study. Not normally distributed data were either logarithmically transformed to fit with normal distribution (NEFA and creatinine kinase) or analysed using R . Data for milk yield and composition, rumination and grazing behaviour, physical activity, feed intake and blood traits were collected over several days and averaged per cow and measurement period. They were analysed using following linear mixed model:et al., et al., Due to the high proportion of insulin ‘non-detects’ , descriptive statistics was performed as described by Helsel  using R P⩽0.05. A value of 0.05<P<0.10 was considered a trend.The effects were considered significant at P<0.001) milk and less (P<0.001) ECM compared with supplemented cows and HNZ cows had a lower (P=0.04) milk yield compared with HCH cows, but no difference between cow strains was observed for ECM (P=0.02) between cow strain and supplementation was reported for milk yield. The HNZ cows produced less (P=0.04) milk per kg concentrate than HCH cows.Non-supplemented cows produced less ( for ECM . An inteP<0.001) for non-supplemented cows, but milk protein content was not influenced by supplementation. The HNZ cows had a greater (P<0.01) protein content and in tendency a greater milk fat content than HCH cows. No differences were recorded for the lactose content relative to concentrate supplementation or cow strain. The milk contents of acetone and urea of non-supplemented cows were greater (P<0.001) compared with supplemented cows, but no differences were observed between cow strains.Milk fat content was greater  than pasture DMI for supplemented cows, but total DMI estimated with alkanes was lower (P<0.001) for non-supplemented cows than for supplemented cows. New Zealand Holstein cows tended to have lower pasture DMI and total DMI compared with HCH cows. Calculated pasture DMI based on the requirements was greater (P<0.001) for non-supplemented cows, but supplementation had no effect on calculated total DMI and no differences between cow strains were observed for calculated pasture and total DMI.For non-supplemented cows, pasture dry matter intake (DMI) estimated with n) and mastication rate (n/min) were greater (P<0.001) for non-supplemented cows compared with supplemented cows and no differences occurred between cow strains (P<0.001) duration of eating bouts compared with supplemented cows, but no difference between cow strains was reported.Grazing time, mastication ( strains . The numP<0.005) time ruminating, had a greater (P<0.005) number of mastication and a greater (P<0.005) number of boli compared with HCH cows. No difference between cow strains was observed for mastication rate, mastication per boli and rumination bouts. New Zealand Holstein cows tended to have longer duration of rumination bouts compared with HCH cows.Concentrate supplementation had no effect on traits describing rumination behaviour, but HNZ cows spent more (P<0.001) time lying down, but stood, moved and walked more (P<0.001). There was a trend recorded for HNZ to stand and move less and lie down more, but no difference was observed for walking. No significant interaction of concentrate supplementation and cow strain was observed.Non-supplemented cows spent less (P<0.01) serum glucose concentration but greater (P<0.001) concentration of serum BHBA, NEFA and urea compared with supplemented cows (P=0.04) and activity of AP was lower (P<0.001) for non-supplemented cows.Non-supplemented cows had lower  concentrations for non-supplemented cows. Non-supplemented cows had a lower (P=0.03) plasma insulin and lower (P<0.001) IGF-1 concentration compared with supplemented cows.Concentrate supplementation had an impact on the plasma concentration of the hormones T3 and T4 with lower (P<0.01) concentration for HCH cows. Furthermore, HCH cows had a lower (P<0.01) activity of ALAT and a lower (P<0.05) concentration of IGF-1. For other blood traits no differences between cow strains were recorded. No interactions of concentrate supplementation and cow strain for blood traits were observed.Differences between cow strains were recorded for serum glucose concentration with lower  pasture DMI were lowered by concentrate supplementation. In contrast, total DMI of supplemented cows was greater or at least the same as the total DMI of non-supplemented cows. However, with reduced pasture DMI for supplemented cows, cheap pasture is substituted by expensive concentrate which is an economic aspect for the farmer. Furthermore, importing ingredients of a commercial organic concentrate mix disturbs the holistic approach of organic guidelines. In the current study, the energy:protein ratio may be balanced as concentrations of urea in blood and milk were in the normal range, with elevated values for non-supplemented cows. In organic farming, the risk of excessive protein intake from pasture might be reduced as the CP content of organic pasture is lower compared with conventional pasture . The results are in accordance with missing differences for ECM.The substitution rate of pasture DMI is linked to milk response with a lower substitution rate for cows with high genetic potential for milk yield (Bargo et al., et al. (et al. (et al., Because no differences were observed between cow strains in blood concentration of insulin, NEFA and BHBA, which are indicators of the energy balance of dairy cows (Reist , et al. , HNZ cow (et al.  reported et al., .et al. (The increase in ALAT activity is in accordance with the results of Thanner et al. , which mMinor differences between cow strains in the current study indicate that both cow strains are equally suited for an organic, pasture-based feeding system. However, the current study was a short-term experiment performed with a small number of animals after the peak of lactation without considering BCS losses, fertility and health traits during a whole lactation, which are important aspects for continuous and successful organic farming. Just choosing a different cow breed or cow strain may not ensure a well-working, low-input organic system. Careful selection of cows that have a high grazing drive ensures the efficient use of pasture for milk production, whereas cows that can deal with lower energy intake at the beginning of lactation and show properties of good health and fertility may be well-suited organic cows.


SPP1), were evaluated. Mean OPN levels were higher in patients compared to healthy controls. Carriers of rs11728697*T allele  were more frequent in the poorly GC responsive group compared to the GC responsive group indicating an association of rs11728697*T allele with GC non-responsiveness. One risk haplotype (AGTACT) was identified associated with GC non-responsiveness compared with GC responsive MG group. Genetic variations of SPP1 were found associated with the response to GC among MG patients.Biomarkers that assess treatment response for patients with the autoimmune disorder, myasthenia gravis (MG), have not been evaluated to a significant extent. We hypothesized the pro-inflammatory cytokine, osteopontin (OPN), may be associated with variability of response to glucocorticoids (GCs) in patients with MG. A cohort of 250 MG patients treated with standardized protocol of GCs was recruited, and plasma OPN and polymorphisms of its gene, secreted phosphoprotein 1 ( Myasthenia gravis (MG) is a chronic autoimmune disorder caused by antibodies directed against postsynaptic proteins, primarily the skeletal muscle nicotinic acetylcholine receptor (AChR). Oral glucocorticoids (GCs) are the primary therapy , 2, but SPP1 gene polymorphisms have been identified as being associated with GC responsiveness in boys with Duchenne muscular dystrophy (SPP1) gene, which encodes OPN, are associated with differences in GC responsiveness of patients with MG.Osteopontin (OPN) is a pro-inflammatory cytokine, and increased circulating levels have been associated with inflammatory muscle diseases and muscular dystrophy –13 as weystrophy . We hypoSPP1 gene and response to a standardized 3-month GC treatment protocol with prospectively collected outcome data. As our primary outcome assessment, we used the quantitative MG score (QMGS), which is a validated scale and was recommended by the MG Foundation of America as the primary clinical outcome measure for clinical trials . Antibodies against AChR (AChRAb) was detected by using ELISA kit  . The tesSPP1 gene region by linkage disequilibrium (LD). Among the 12 SNPs, 1 tag SNP (rs2853749) was selected using the HapMap database with the software as previously described, and 11 SNPs  were previously reported (Twelve SNPs (variations in single base pairs in a DNA sequence) were selected based on previous reports and information from NCBI dbSNP and HapMap database , in an attempt to cover the majority of the reported –33. Ten TM Kit . For quality assurance, 3.87% (28/724) of the total samples were randomly repeated. Concordance for duplicate samples was 100% for all assays.Single nucleotide polymorphism genotyping was performed using a custom-designed SNPscanp < 0.05 was considered statistically significant. The database, constructed using SQL server, contains data pertaining to the SNPs, clinical features and treatment, and clinical follow-up of patients.Statistical analysis was performed with SPSS Statistics 13 , SHEsis software , Haploview 4.2 software, and GraphPad Prism 5 . A two-side comparison with t test or the Mann–Whitney U-test for continuous variables and by chi-square test or Fisher exact test for categorical variables. The association between the GC efficacy and MG phenotype and SPP1 genotypes was examined by multivariate regression analysis. In this pilot study, we did not make a correction for multiple comparisons. The Haploview 4.2 software was used to calculate pairwise LD of SNPs and construct haplotype blocks. Haplotypes are defined as genetic variations that are inherited together. Haplotype frequencies were estimated with Partition–Ligation–Combination–Subdivision Expectation Maximization algorithm implemented in SHEsis software.The normality of the data was tested using the method of Kolmogorov and Smirnov. Continuous variables were presented as mean ± SD or median, 25th and 75th percentiles; categorical variables were presented as a percentage. Differences between groups were analyzed with independent sampled Functional annotations of SNPs were investigated using RegulomeDB, a database which provides assessment of whether SNPs are located in known or predicted regulatory elements, including regions of DNase I hypersensitivity, binding sites for transcription factors (TFs), and promoter regions that regulate transcription .With the aim of exploring the molecular basis of the observed associations, eQTLs analysis was performed by using published cell-specific eQTL dataset .The study cohort consisted of 250 patients of which 154 (61.6%) were women (Table p < 0.0001). The change in QMGS ranged from −2 to 18 .Quantitative MG score ranged from 1 to 35  at study onset, and after 3 months of treatment, a significant reduction in QMGS was observed ranging from 0 to 29 points  (Table Two hundred thirty-one patients (92.4%) were considered GC responsive, and 19 (7.6%) were considered GC non-responsive. Clinical characteristics of the patients are summarized in Table p = 0.013; Figure p = 0.006, Figure p = 0.667, 0.227, 0.641, 1.00, 1.00, 0.373, 0.606, respectively, Figures Prior to initiation of immunotherapy, mean OPN plasma levels were greater among MG patients (68.33 ± 43.03 ng/ml) compared to healthy controls , indicating association with a poor response to GCs. No statistically significant differences were observed for the remaining SNPs (Table p = 0.99).The rs11728697 T carriers (C/T + T/T genotypes) were more frequent in the GC non-responsive group compared to the GC responsive group .The association between SPP1 gene is shown in Figure SPP1 gene consists of two blocks. The first block ranges from rs2728127 to rs11728697 , the second from rs4754 to rs9138 . A total of four common haplotypes were identified across the first block, ranging in frequency from 35.5 to 7.3% in all patients . One risk haplotype (AGTACT) in the first block was identified  in the GC non-responsive MG group compared with GC responsive MG group (15.8 vs 6.5%) (Table p = 0.127)  with evidence for eQTL, TF binding, a matched TF binding motif, and location within DNase-sensitive site. rs11728697 is within the region of the binding site of TF regulatory factor X 3 that was detected by Chip-Seq analysis in K562 cell line (D′ = 0.958 and R2 = 0.724), which was identified as score 1f with evidence for mapping to a predicted TF binding site and/or within a DNase I sensitivity peak and correlating with gene expression. rs12502049 is known to regulate the expression of SPP1 gene that was detected by eQTL analysis in a lymphoblastoid cell line , which was associated a poor response to GC treatment among patients with MG. One risk haplotype (AGTACT) containing a mutation at this SNP was identified among the GC non-responsive patients compared with the GC responsive patients. The carrier of the variant does not differ between MG patients and the controls, indicating the association of rs11728697 T with GC response is not determined by its involvement in pathogenesis of MG but associates with GC treatment response. Presently, there is no evidence of a direct interaction between OPN and GC response pathways; however, OPN influences T and B cell function, which provides indirect pathways that could be associated with GC treatment responsiveness . LK receives grant support from the Muscular Dystrophy Association. All other authors report no disclosures.