Urinary metabolomics fingerprinting around parturition identifies metabolites that differentiate lame dairy cows from healthy ones

Lameness is a very important disorder of periparturient dairy cows with implications on milk production and composition as well as with consequences on reproductive performance. The aetiology of lameness is not clear although there have been various hypotheses suggested over the years. The objective of this study was to metabotype the urine of dairy cows prior to, during and after the onset of lameness by evaluating at weeks −8, −4 pre-calving, the week of lameness diagnosis, and +4 and +8 weeks post-calving. We used a metabolomics approach to analyse urine samples collected from dairy cows around calving (6 cows with lameness v. 20 healthy control cows). A total of 153 metabolites were identified and quantified using an in-house MS library and classified into 6 groups including: 11 amino acids (AAs), 39 acylcarnitines (ACs), 3 biogenic amines (BAs), 84 glycerophospholipids, 15 sphingolipids and hexose. A total of 23, 36, 40, 23 and 49 metabolites were observed to be significantly different between the lame and healthy cows at −8 and −4 weeks pre-calving, week of lameness diagnosis as well as at +4 and +8 weeks post-calving, respectively. It should be noted that most of the identified metabolites were elevated; however, a few of them were also lower in lame cows. Overall, ACs and glycerophospholipids, specifically phosphatidylcholines (PCs), were the metabolite groups displaying the strongest differences in the urine of pre-lame and lame cows. Lysophosphatidylcholines (LysoPCs), although to a lesser extent than PCs, were altered at all time points. Alterations in urinary AA concentrations were also observed during the current study for four time points. During the pre-calving period, there was an observed elevation of arginine (−8 week), tyrosine (−8 week) and aspartate (−4 week), as well as a depression of urinary glutamate (−4 weeks). In the current study, it was additionally observed that concentrations of several sphingomyelins and one BA were altered in pre-lame and lame cows. Symmetric dimethylarginine was elevated at both −8 weeks pre-calving and the week of lameness diagnosis. Data showed that urinary fingerprinting might be a reliable methodology to be used in the future to differentiate lame cows from healthy ones.     

Milk fatty acids as indicators of negative energy balance of dairy cows in early lactation

Most dairy cows experience negative energy balance (NEB) in early lactation because energy demand for milk synthesis is not met by energy intake. Excessive NEB may lead to metabolic disorders and impaired fertility. To optimize herd management, it is useful to detect cows in NEB in early lactation, but direct calculation of NEB is not feasible in commercial herds. Alternative methods rely on fat-to-protein ratio in milk or on concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) in blood. Here, we considered methods to assess energy balance (EB) of dairy cows based on the fatty acid (FA) composition in milk. Short- and medium-chain FAs (primarily, C14:0) are typically synthesized de novo in the mammary gland and their proportions in milk fat decrease during NEB. Long-chain FAs C18:0 and C18:1 cis-9 are typically released from body fat depots during NEB, and their proportions increase. In this study, these FAs were routinely determined by Fourier-transform infrared spectroscopy (FTIR) of individual milk samples. We performed an experiment on 85 dairy cows in early lactation, fed the same concentrate ration of up to 5 kg per day and forage ad libitum. Daily milk yield and feed intake were automatically recorded. During lactation weeks 2, 4, and 6 after calving, two milk samples were collected for FTIR spectroscopy, Tuesday evening and Wednesday morning, blood plasma samples were collected Thursday morning. Net energy content in feed and net energy required for maintenance and lactation were estimated to derive EB, which was used to compare alternative indicators of severe NEB. Linear univariate models for EB based on NEFA concentration (deviance explained = 0.13) and other metabolites in blood plasma were outperformed by models based on concentrations of metabolites in milk: fat (0.27), fat-to-protein ratio (0.18), BHB (0.20), and especially C18:0 (0.28) and C18:1 cis-9 (0.39). Analysis of generalized additive models (GAM) revealed that models based on milk variables performed better than those based on blood plasma (deviance explained 0.46 vs. 0.21). C18:0 and C18:1 cis-9 also performed better in severe NEB prediction for EB cut-off values ranging from −50 to 0 MJ NEL/d. Overall, concentrations of C18:0 and C18:1 cis-9 in milk, milk fat, and milk BHB were the best variables for early detection of cows in severe NEB. Thus, milk FA concentrations in whole milk can be useful to identify NEB in early-lactation cows.     

Metabolic hormones,grazing behaviour,offspring physical distance and productive response of beef cow grazing at two herbage allowances

Herbage allowance (HA) management during gestation–lactation cycle of cows grazing native grasslands improves pregnancy rates and calves’ weaning weight records. Those improvements were associated with greater herbage mass, and better body condition score (BCS) and metabolic status of the cows, which could affect grazing and maternal behaviour, particularly when temporary suckling restriction (TSR) and flushing (FL) is applied. The objective of this study was to evaluate the effect of HA during the gestation–lactation period on cows’ and calves’ liveweight (LW), BCS, milk yield, insulin, IGF-1 and leptin concentrations, as well as the proportion of diurnal grazing activities (grazing, ruminating, walking and idling), and maternal behaviour from −10 to 70 days relative to TSR (day 0 = initiation of TSR at 86 ± 10 days relative to calving). Thirty-three primiparous Hereford cows were allocated to HA treatments during gestation and lactation, which annually averaged 2.5 (low = LHA) and 4 kg DM/kg LW (high = HHA). The LW and BCS of cows did not differ during −10 to 50 days but were greater in HHA than LHA at the end of the study (P < 0.05). Concentrations of IGF-1 were greater in HHA compared to LHA, while insulin and leptin did not differ. Grazing was lower in HHA than LHA, and the opposite occurred with ruminating and idling (P < 0.05). Cow–calf physical distance was greater in LHA cows compared to HHA (P < 0.05) and increased greatly in the former group after FL, while this increase was lower and later in HHA cows. Milk yield was greater in HHA (P < 0.05), and calves’ weight did not differ between treatments from day −10 to 35, but was greater in HHA compared to LHA from 45 days until the end of the study. Thus, the HHA in a low herbage height and mass condition resulted in greater IGF-1 concentrations and milk yield, and induced changes in grazing and maternal behaviour that were associated with increased cows’ LW, BCS and calves’ weight at the end of the study.     

Effects of dietary conjugated linoleic acid on metabolic status,BW and expression of genes related to lipid metabolism in adipose tissue of dairy cows during peripartum

Conjugated linoleic acid (CLA) dietary supplementation reduces milk fat content and yield, but its effects on lipid metabolism and energy status remain controversial. The objective of this study was to investigate the effects of dietary CLA on adipose tissue (AT) mRNA abundance of genes related to lipid metabolism, plasma indicators of metabolic status, body condition score (BCS) and BW changes in dairy cows. Sixteen multiparous Holstein cows (3.2 ± 1.4 lactations, 615 ± 15 kg BW) were randomly assigned to treatments: 1) CLA; rumen-protected CLA (75 g/d) or 2) Control; equivalent amount of rumen inert fatty acid (FA) as the previous diet (78 g/d), from − 20.2 ± 3.2 (mean ± SEM) to 21 d relative to calving (d 0). Subcutaneous AT was biopsied from the tail-head region at d 21 to determine the mRNA abundance of genes related to lipid metabolism. Blood samples were collected at − 20.2 ± 3.2, 0, 7, 14 and 21 d relative to calving to determine plasma non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), insulin and glucose. Conjugated linoleic acid decreased milk fat yield and milk fat content by 15 and 16%, respectively. Cows fed CLA had lower plasma NEFA and BHBA and greater glucose and insulin concentrations (P < 0.05). Mean BCS at 21 d postpartum was greater (P < 0.01; 2.89 vs 2.25), and BCS loss from the day of enrollment to 21 d postpartum was reduced (P < 0.01; − 0.13 vs − 0.64) in the CLA group. The expression of acylcoenzyme A oxidase, carnitine palmitoyltransferase 1A, hormone-sensitive lipase, β2 adrenergic receptor and acetyl-CoA carboxylase was downregulated by CLA supplementation, whereas the expression of sterol regulatory element binding protein, lipoprotein lipase and peroxisome proliferator-activated receptor gamma was upregulated (P < 0.01). In summary, CLA-supplemented cows showed signs of better metabolic status and less severe fat mobilization. Moreover, CLA increased mRNA abundance of genes related to lipogenesis and decreased mRNA abundance of genes related to FA oxidation and lipolysis in the AT of dairy cows during early lactation.     

Genetic correlations between energy status indicator traits and female fertility in primiparous Nordic Red Dairy cattle

Inclusion of feed efficiency traits into the dairy cattle breeding programmes will require considering early lactation energy status to avoid deterioration in health and fertility of dairy cows. In this regard, energy status indicator (ESI) traits, for example, blood metabolites or milk fatty acids (FAs), are of interest. These indicators can be predicted from routine milk samples by mid-IR reflectance spectroscopy (MIR). In this study, we estimated genetic variation in ESI traits and their genetic correlation with female fertility in early lactation. The data consisted of 37 424 primiparous Nordic Red Dairy cows with milk test-day records between 8 and 91 days in milk (DIM). Routine test-day milk samples were analysed by MIR using previously developed calibration equations for blood plasma non-esterified FA (NEFA), milk FAs, milk beta-hydroxybutyrate (BHB) and milk acetone concentrations. Six ESI traits were considered and included: plasma NEFA concentration (mmol/l) either predicted by multiple linear regression including DIM, milk fat to protein ratio (FPR) and FAs C10:0, C14:0, C18:1 cis-9, C14:0 * C18:1 cis-9 (NEFA_FA) or directly from milk MIR spectra (NEFA_MIR), C18:1 cis-9 (g/100 ml milk), FPR, BHB (mmol/l milk) and acetone (mmol/l milk). The interval from calving to first insemination (ICF) was considered as the fertility trait. Data were analysed using linear mixed models. Heritability estimates varied during the first three lactation months from 0.13 to 0.19, 0.10 to 0.17, 0.09 to 0.14, 0.07 to 0.10, 0.13 to 0.17 and 0.13 to 0.18 for NEFA_MIR, NEFA_FA, C18:1 cis-9, FPR, milk BHB and acetone, respectively. Genetic correlations between all ESI traits and ICF were from 0.18 to 0.40 in the first lactation period (8 to 35 DIM), in general somewhat lower (0.03 to 0.43) in the second period (36 to 63 DIM) and decreased clearly (−0.02 to 0.19) in the third period (64 to 91 DIM). Our results indicate that genetic variation in energy status of cows in early lactation can be determined using MIR-predicted indicators. In addition, the markedly lower genetic correlation between ESI traits and fertility in the third lactation month indicated that energy status should be determined from the first test-day milk samples during the first 2 months of lactation.     

Effects of intravenous arginine infusion on inflammation and metabolic indices of dairy cows in early lactation

Enhancing the supply of arginine (Arg), a semi-essential amino acid, has positive effects on immune function in dairy cattle experiencing metabolic stress during early lactation. Our objective was to determine the effects of Arg supplementation on biomarkers of liver damage and inflammation in cows during early lactation. Six Chinese Holstein lactating cows with similar BW (508 ± 14 kg), body condition score (3.0), parity (4.0 ± 0), milk yield (30.6 ± 1.8 kg) and days in milk (20 ± days) were randomly assigned to three treatments in a replicated 3 × 3 Latin square design balanced for carryover effects. Each period was 21 days with 7 days for infusion and 14 days for washout. Treatments were (1) Control: saline; (2) Arg group: saline + 0.216 mol/day l-Arg; and (3) Alanine (Ala) group: saline + 0.868 mol/day l-Ala (iso-nitrogenous to the Arg group). Blood and milk samples from the experimental cows were collected on the last day of each infusion period and analyzed for indices of liver damage and inflammation, and the count and composition of somatic cells in milk. Compared with the Control, the infusion of Arg led to greater concentrations of total protein, immunoglobulin M and high density lipoprotein cholesterol coupled with lower concentrations of haptoglobin and tumor necrosis factor-α, and activity of aspartate aminotransferase in serum. Infusion of Ala had no effect on those biomarkers compared with the Control. Although milk somatic cell count was not affected, the concentration of granulocytes was lower in response to Arg infusion compared with the Control or Ala group. Overall, the biomarker analyses indicated that the supplementation of Arg via the jugular vein during early lactation alleviated inflammation and metabolic stress.     

Effects of nutrient restriction on the metabolic profile of Bos indicus-influenced and B. taurus suckled beef cows

Recent research from our group demonstrated that Bos indicus-influenced suckled beef cows had greater resilience to withstand nutrient restriction and establish pregnancy compared with B. taurus cows exposed to the same conditions. To further understand these findings, differences in metabolic profile between these same B. indicus-influenced and B. taurus females were explored. Suckled beef cows (n = 134) were enrolled in a completely randomized design with a 2 × 2 factorial arrangement of treatments. On day − 21, Angus (AN; Bos taurus) and Brangus (BN; B. indicus-influenced) cows were randomly assigned to 1) a diet that met daily energy maintenance requirements (MAINT), or 2) a diet that restricted intake to 70% of the daily energy maintenance requirements (RESTR). Cows were exposed to an estrus synchronization protocol and received an embryo 7 d after ovulation was pharmacologically induced on day 0. Blood samples were collected on days − 21 and 19 to determine circulating concentrations of non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), insulin, glucose, and IGF-1. Pregnancy status after embryo transfer was determined on day 28. As a consequence of the proposed diets, cows in the RESTR diet had less body condition score (BCS) on day 19 (P = 0.008) across breed types. Moreover, BCS change from day − 21 to 19 was included as independent covariate into subsequent analyses, allowing for the comparison of breed types under an equivalent level of body reserve mobilization. A breed × diet interaction was observed for plasma insulin (P = 0.03) and IGF-1 (P = 0.04) on day 19, where AN–RESTR cows had less plasma concentrations on day 19 compared with AN–MAINT cows. Diets did not impact (P > 0.10) plasma insulin and IGF-1 concentrations in BN cows. No diet or breed effects were observed in circulating concentrations of NEFA, BHB, and glucose (P > 0.10). Across breed types and nutritional treatment, there was positive linear effect (P ≤ 0.04) of plasma concentrations of insulin and IGF-1 on the probability of pregnancy to fixed-time embryo transfer. In summary, the negative impacts of nutrient restriction on the somatotropic axis, independently of body tissue mobilization, were heightened in Bos taurus females compared with B. indicus-influenced cohorts, which corroborate with the differences observed in fertility between these subspecies.     

The potential interaction between body condition score at calving and dietary starch content on productive and reproductive performance of early-lactating dairy cows

Improving reproductive performance is one of the most important factors affecting the profitability of dairy herds. This study investigated the effect of feeding a high starch (HS) diet and body condition score (BCS) at calving on blood metabolites, fertility and ovarian function and milk production in Holstein dairy cows. One hundred seventy-four multiparous cows were fed common close-up and early lactation diets during the first 15 days in milk (DIM). Cows were randomly assigned to 1 of 2 experimental diets from 16 until 50 DIM (n = 87 per group); normal starch (228 g/kg diet DM; NS) or HS (270 g/kg diet DM; HS) diets. Each treatment group was further subdivided based on BCS at calving as normal BCS (BCS ⩽ 3.5; normal BCS (NBCS); n = 45) or high BCS (HBCS) (BCS ⩾ 3.75; HBCS; n = 42). A significant difference was detected for increased milk production (47.24 v. 44.55 kg/day) and decreased milk fat (33.93 v. 36.33 g/kg) in cows fed HS or NS, respectively. Plasma glucose and insulin concentrations were significantly higher in cows fed the HS compared to the NS diet. Diets significantly affected DIM at first artificial insemination (AI, 79.51 ± 3.83 v. 90.40 ± 3.83 days for cows fed HS and NS diets, respectively). High BCS groups had greater milk fat content and elevated plasma nonesterified fatty acids (NEFA), β hydroxybutyrate (BHB) and bilirubin concentrations. In general, feeding higher starch diets to normal BCS cows during the first 50 DIM improved productive and reproductive performance of early-lactating dairy cows.     

Analyses of the genetic relationships between lactose,somatic cell score,and growth traits in Simmental cattle

Lactose and somatic cell score (SCS) are major economic traits of milk. However, for many countries, they are typically not directly considered in the national genetic evaluation of Simmental cattle. This study aimed to estimate the genetic relationships between lactose, SCS, and growth traits of Simmental cattle to provide information for the national genetic evaluation of the selection of traits of this cattle population. The data of 1781 animals with 6519 records obtained over a period of 41 years (1975–2016) were collected from Xinjiang Hutubi Farm, China. The analyzed traits included 305 days of milk yield (305MY), milk fat percentage (MFP), milk protein percentage (MPP), milk lactose percentage (MLP), total solids (TS), SCS, body height (BH), body length (BL), chest girth (CG), abdominal circumference (AC), rump width (RW), rump length (RL), leg circumference (LC), and cannon circumference (CC). The multiple-trait repeatability model was adopted to estimate (co)variance components using the average information-restricted maximum likelihood method implemented using the DMU statistical package. The heritability estimates for milk components and growth traits ranged from 0.09 (SCS) to 0.51 (BH). Genetic correlations for milk components ranged from 0.03 ± 0.14 (MFP and MLP) to 0.81 ± 0.08 (MFP and MPP). Genetic correlation between MLP and SCS was moderate and negative (− 0.50 ± 0.15) compared with that among other traits. Genetic correlations between the milk components and growth traits ranged from 0.00 ± 0.07 (305MY and RW) to − 0.64 ± 0.15 (MLP and BL). Genetic correlations of BL, LC, RW, and RL with MLP were moderate to high and negative ranging from − 0.39 to − 0.64. Somatic cell score showed the highest correlation with BL (0.41) followed by LC (0.21). An increase in MLP would result in an increase in 305MY or TS and a decrease in BL, LC, RW, and RL. Additionally, a decrease in SCS would occur with the selection of increased MLP and reduced BL. We conclude that selection based on easily and inexpensively measured growth traits could improve the milk quality from Simmental cattle.     

Use of milk electrical conductivity for the differentiation of mastitis causing pathogens in Holstein cows

Mastitis is one of the most prevalent and costly diseases in dairy cattle. Key components for adequate mastitis control are the detection of early stages of infection, as well as the selection of appropriate management interventions and therapies based on the causal pathogens associated with the infection. The objective was to characterize the pattern of electrical conductivity (EC) in milk during intramammary infection, considering specific mastitis-causing pathogen groups involvement. Cows (n = 200) identified by an in-line mastitis detection system with a positive deviation ≥15% in the manufacturer’s proprietary algorithm for EC (high electrical conductivity (HEC)) were considered cases and enrolled in the study at the subsequent milking. One control (CON) cow, within normal ranges for EC, was matched to each case. A composite milk sample was collected aseptically from each cow for bacteriological culture. Milk yield (MY) and EC were recorded for each milking during ±7 days relative to enrollment. Milk cultures were categorized into gram positive (GP), gram negative (GN), other (OTH) and no growth (NOG). Data were submitted for repeated-measures analysis with EC as the dependent variable and EC status at day −1, bacteriological culture category, parity number, stage of lactation and days relative to sampling as main independent variables. Average (± standard error (SE)) EC was greater in HEC than in CON cows (12.5 ± 0.5 v. 10.8 ± 0.5 mS/cm) on the day of identification (day −1). Milk yield on day −1 was greater in CON than in HEC (37.6 ± 5.1 v. 33.5 ± 5.2 kg). For practical management purposes, average EC on day −1 was similar for the different bacteriological culture categories: 11.4 ± 0.6, 11.7 ± 0.5, 12.3 ± 0.8 and 11.7 ± 0.5 mS/cm in GN, GP, OTH and NOG, respectively. Parity number was only associated with day −1 EC in HEC group, with the greatest EC values in parity 3 (12.3 ± 0.3 mS/cm), followed by parity 2 (11.9 ± 0.2 mS/cm), parity >3 (11.6 ± 0.5 mS/cm) and primiparous cows (11.2 ± 0.2 mS/cm). An effect on EC for the interaction of day relative to identification by pathogen gram category was observed. The same interaction effect was observed on daily MY. Overall, the level of variation for MY and EC between- and within-cows was substantial, and as indicated by the model diagnostic procedures, the magnitude of the variance in the cows in the CON group resulted in deviations from normality in the residuals. We concluded that characteristic temporal patterns in EC and MY in particular pathogen groups may provide indications for differentiation of groups of mastitis-causing pathogens. Further research to build detection models including EC, MY and cow-level factors is required for accurate differentiation.     

Diurnal variation of milk fatty acids in early-lactation Holstein cows with and without hyperketonemia

Estimates of milk constituents by Fourier-transform mid-infrared (FTIR) analysis have been shown to be a useful tool in monitoring energy deficit in early-lactation dairy cows. Our objectives were to describe the diurnal variation in milk fatty acids (FAs) and estimate the association of hyperketonemia with concentrations and diurnal patterns of FTIR estimates of milk FA. Blood samples were collected via jugular catheters bihourly for 5 d from multiparous Holstein cows (n = 28) enrolled between 3 and 9 days in milk. Milk samples were collected thrice daily at 0600, 1400, and 2200 h for d 2, 3, and 4 of the study period. Cows were retrospectively classified as hyperketonemic (HYK; n = 13) or non-HYK (n = 15) based on blood beta-hydroxybutyrate (bBHB) concentrations analyzed during the study period. Cows were classified as HYK if bBHB was ≥ 1.2 mmol/l for ≥ 50% (22/44) of bihourly timepoints; cows were classified as non-HYK if bBHB was ≥ 1.2 mmol/l for < 50% of bihourly timepoints. The HYK cows had bBHB ≥ 1.2 mmol/l for 31.4 ± 6.8 timepoints while the non-HYK cows had bBHB ≥ 1.2 mmol/l for 8.0 ± 3.9 timepoints. We used generalized linear mixed models to analyze concentrations of milk FA over time and differences between HYK groups. The relative percentage of de novo, mixed, and preformed FAs all followed diurnal patterns, however only the yield of preformed FA diurnally cycled, reaching a nadir at 0600 h and peaking at 1400 h. The yield per milking of preformed FA was also greater in the HYK cows than in the non-HYK cows. Oleic acid in milk followed a similar diurnal pattern to the yield of preformed FA, likely driving the cyclical nature of preformed FA. Finally, stearic acid was greater in HYK cows. Our results suggest that FTIR estimates of milk FA offer the potential to provide insight on the energy status of early-lactation cows, and when interested in understanding the absolute concentrations and yields of milk FA, diurnal variation should be considered.     

Exploration of robustness indicators using adaptive responses to short-term feed restriction in suckling primiparous beef cows

Animal robustness is a complex trait of importance for livestock production systems and genetic selection. Phenotyping is essential for evaluation of the adaptation of different genotypes to changing environments. This study tested an experimental framework to induce marked deviations in the adaptive responses of suckling beef cows and to identify relevant indicators of responses to characterise individual differences in the robustness of cows. The production and metabolic responses of primiparous suckling Charolais cows to two periods of feed restriction (FR, 50% of their net energy requirements) of different durations were monitored. After calving, 13 cows (aged 39 ± 2 months, BW of 680 ± 42 kg at calving) had ad libitum access to a diet composed of hay and supplemented with concentrate to meet their energy and protein requirements. Starting at 54 ± 6 days postcalving, the cows underwent two periods of FR: 4 days of FR (FR4), which was followed by 17 days of ad libitum intake to study the recovery from FR4, and 10 days of FR (FR10), which was followed by 18 days of ad libitum intake to study the recovery from FR10. The milk yield (MY), BW, body condition score and plasma non-esterified fatty acid (NEFA), β-hydroxybutyrate, glucose and urea concentrations were measured before, during and after each FR. Among all measured variables, the MY and NEFA concentrations showed the most significant changes in response to FR. A functional data analysis approach was applied to the MY and NEFA data to model the adaptive responses and extract quantifiable indicators of deviation and recovery. Linear correlations (P < 0.03–0.07) between FR4 and FR10 were found for some indicators describing MY and NEFA levels before and after FR. The overall repeatability of MY and NEFA responses between both FR accounted for 46% based on quartile analysis performed on average responses. Moreover, the variance in both the MY and NEFA variables did not differ significantly between FR4 and FR10, despite a trend for higher variances in FR10. Altogether, (1) the calculated variables derived from the functional data analysis of the time patterns of the MY and NEFA accounted for the differences in the cow responses to FR, and (2) the animal responses appeared to show concordance between FR4 and FR10. In conclusion, short-term FR is a relevant framework for studying productive and metabolic adaptive responses in suckling cows and allows the identification of potential robustness indicators.     

Effects of micronutrient supplementation on performance and epigenetic status in dairy cows

The postpartum period is crucial in dairy cows and is marked by major physiological and metabolic changes that affect milk production, immune response and fertility. Nutrition remains the most important lever for limiting the negative energy balance and its consequences on general health status in highly selected dairy cows. In order to analyze the effect of a commercial micronutrient on intrinsic parameters, performances and the epigenome of dairy cows, 2 groups of 12 Holstein cows were used: 1 fed a standard diet (mainly composed of corn silage, soybean meal and non-mineral supplement) and the other 1 fed the same diet supplemented with the commercial micronutrient (µ-nutrient supplementation) for 4 weeks before calving and 8 weeks thereafter. Milk production and composition, BW, body condition score (BCS), DM intake (DMI) and health (calving score, metritis and mastitis) were recorded over the study period. Milk samples were collected on D15 and D60 post-calving for analyses of casein, Na⁺ and K⁺ contents and metalloprotease activity. Milk leukocytes and milk mammary epithelial cells (mMECs) were purified and counted. The viability of mMECs was assessed, together with their activity, through an analysis of gene expression. At the same time points, peripheral blood mononuclear cells (PBMCs) were purified and counted. Using genomic DNA extracted from PBMCs, mMECs and milk leukocytes, we assessed global DNA methylation (Me-CCGG) to evaluate the epigenetic imprinting associated with the µ-nutrient-supplemented diet. The µ-nutrient supplementation increased BCS and BW without modifying DMI or milk yield and composition. It also improved calving condition, reducing the time interval between calving and first service. Each easily collectable cell type displayed a specific pattern of Me-CCGG with only subtle changes associated with lactation stages in PBMCs. In conclusion, the response to the µ-nutrient supplementation improved the body condition without alteration of global epigenetic status in dairy cows.     

Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows

Cows undergoing a negative energy balance (NEB) often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed to evaluate the impact of a folic acid and vitamin B₁₂ supplement and feed restriction on several immune parameters. Sixteen cows at 45 ± 3 days in milk were assigned to 8 blocks of 2 cows each according to each cow’s milk production in the previous week, and within each block, the cows randomly received weekly intramuscular injections of either saline or 320 mg of folic acid and 10 mg of vitamin B₁₂ for 5 weeks. During week 5, the cows were fed 75% of their ad libitum intake for 4 days. Blood samples were taken before the beginning of the experiment, just before feed restriction and after 3 days of feed restriction, in order to evaluate blood cell populations, the phagocytosis capacity and oxidative burst of polymorphonuclear leukocytes (PMNs), the proliferation of peripheral blood mononuclear cells (PBMCs) and concentrations of non-esterified fatty acids (NEFAs) and β-hydroxybutyrate. The vitamin supplement did not affect any of the tested variables except milk fat and lactose content. Feed restriction reduced milk production and increased the concentration of NEFAs. Feed restriction did not affect blood cell populations but did reduce the percentage of PMN positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate. The proliferation of PBMCs was reduced when the cell culture medium was supplemented with sera collected during the feed restriction. In conclusion, feed restriction affected the functions of PMN and PBMC and this effect was not prevented by the folic acid and vitamin B₁₂ supplement. These results support the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to impaired immune cell functions by high circulating concentration of NEFAs.     

Short-term physiological responses to moderate heat stress in grazing dairy cows in temperate climate

Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit short-term physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals’ vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na⁺, K⁺, and Cl^? concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 °C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows’ maximal daily vaginal temperature (VT_MAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCI_MEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VT_MAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na⁺ in milk was lower, and the concentration of K⁺ in milk tended to be higher in cows with increased VT_MAX. For beta-hydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na⁺ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.     

Relationship between body condition score loss and mRNA of genes related to fatty acid metabolism and the endocannabinoid system in adipose tissue of periparturient cows

The endocannabinoid system (ECS) controls feed intake and energy balance in nonruminants. Recent studies suggested that dietary management alters the expression of members of the ECS in the liver and endometrium of dairy cows. The aim of this study was to determine the relationship between body condition score (BCS) loss and the mRNA abundance of genes related to fatty acid metabolism and the ECS in the subcutaneous adipose tissue (AT) of dairy cows. The BCS was determined in multiparous (3.2 ± 0.5 lactations) Holstein cows at −21 and 42 days relative to calving (designated as d = 0). Cows were grouped into three categories according to BCS loss between both assessments as follows: (1) lost ≤0.25 unit (n = 8, low BCS loss (LBL)), (2) lost between 0.5 and 0.75 units (n = 8, moderate BCS loss (MBL)) and (3) lost ≥1 unit (n = 8, high BCS loss (HBL)). Concentrations of haptoglobin and non-esterified fatty acids (NEFAs) were determined in plasma. Real-time PCR was used to determine mRNA abundance of key genes related to fatty acid metabolism, inflammation and ECS in AT. Milk yield (kg/day) between week 2 and 6 post-calving was greater in the LBL group (49.4 ± 0.75) compared to MBL (47.9 ± 0.56) and HBL (47.4 ± 0.62) groups (P < 0.05). The overall mean plasma haptoglobin and NEFA concentrations were greater in MBL and HBL groups compared with the LBL group (P < 0.05). The mRNA abundance of TNF-α, Interleukin-6 (IL-6) and IL-1β was greatest at 21 and 42 days post-calving in HBL, intermediate in MBL and lowest in LBL groups, respectively. Cows in the HBL group had the greatest AT gene expression for carnitine palmitoyltransferase 1A, hormone sensitive lipase and adipose triglyceride lipase at 21 and 42 days post-calving (P < 0.05). Overall, mRNA abundance for very long chain acyl-CoA dehydrogenase and peroxisome proliferator-activated receptor gamma, which are related to NEFA oxidation, were greater in MBL and HBL groups compared to the LBL group at 42 days post-calving. However, mRNA abundance of fatty acid amide hydrolase was lower at 21 and 42 days post-calving in HBL cows than in LBL cows (P < 0.05). In summary, results showed a positive association between increased degree of BCS loss, inflammation and activation of the ECS network in AT of dairy cows. Findings suggest that the ECS might play an important role in fatty acid metabolism, development of inflammation and cow’s adaptation to onset of lactation.     

Effects of continuous milking during a field trial on productivity,milk protein yield and health in dairy cows

The objective of this field study with an automatic milking system was to evaluate the effects of omitting the dry period on health and productivity during the subsequent lactation in dairy cows. A total of 98 German Simmental cows of six Southern German farms were assigned randomly to two experimental groups: The first group was dried-off 56 days before calving (D for dried-off, n=49), and the second group was milked continuously during this period until calving (CM for continuous milking, n=49). From the latter a third group emerged, including cows that dried-off themselves spontaneously (DS for dried-off spontaneously, n=14). Blood serum values of glucose, β-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA) and IGF-1 showed most pronounced fluctuations in D cows. Over the entire study period, the concentrations of BHBA and NEFA were markedly lower in the CM and DS groups. Furthermore, IGF-1 concentration was lowest for D cows and also decrease in back fat thickness was more pronounced. Mean concentration of milk protein was markedly higher in CM and DS cows (3.70% and 3.71%) compared with D cows (3.38%). Owing to the lower 305-day milk yield (−15.6%) and the lower total milk yield (−3.1%), the total amount of produced protein in the subsequent lactation was 2.5% (6.8 kg) lower, although the additional protein amount in CM cows from week −8 to calving was 35.7 kg. The greatest benefit resulted from positive effects on fertility and the lower incidence of diseases: CM cows had their first oestrus 1 week earlier compared with D cows, they also conceived earlier and showed a significantly lower risk of developing hypocalcaemia, ketosis and puerperal disorders. The present study showed that the costs of medical treatment and milk losses were twice as high in D cows, compared with CM and DS cows, and thus the reduced costs because of the more stable health outweighed the financial losses of milk yield by +18.49 € per cow and lactation.     

Milk fat depression and plasma lipids in dairy cows and goats

This study examines the effects of diets supplemented with various lipids selected to induce divergent milk fat content responses (including a milk fat depression) between dairy cows and goats on plasma lipid composition. The objective was to better understand the mechanisms behind the regulation of milk fat secretion in these two ruminant species. Twelve Holstein cows and 12 Alpine goats were fed a basal diet not supplemented (CTL) or supplemented with corn oil plus wheat starch (COS, 5% DM intake (DMI)), marine algae powder of Schizochytrium sp. (MAP, 1.5% DMI), or hydrogenated palm oil (HPO, 3% DMI), in a replicated 4 × 4 Latin square design, during 28 days. On day 27, blood samples were collected for lipid analysis. Plasma lipid classes were quantified by high-performance thin-layer chromatography, with triacylglycerol (TAG) and free fatty acid (FFA) fractions analysed for FA composition by GLC. Plasma molecular species of TAG and ceramides were determined by HPLC–high-resolution MS and by liquid chromatography–triple quadrupole, respectively. Irrespective of diet, plasma total lipid content was higher in cows than goats (+61%), and TAG concentration was higher in goats than cows (+157%). In cows, conversely to goats, COS increased the trans-10 C18:1 proportion in the free FA (+248%) and the TAG (+195%) fractions. In cows and goats, MAP induced increases in cholesterol esters, cholesterol and phospholipids compared to CTL and changes in the plasma free FA and FA of TAG profiles. In both ruminant species, the concentrations of the lipid fractions were unchanged by HPO compared to CTL. Our results point to species specificities and different diet effects in plasma concentrations and compositions of lipid fractions in cows and goats. These new data highlight how diets, that induce large variations in milk fat secretions, affect the plasma lipid classes available for milk fat synthesis.     

Saturated fat supplemented in the form of triglycerides decreased digestibility and reduced performance of dairy cows as compared to calcium salt of fatty acids

The two most popular rumen-protected fatty acid supplements in dairy cow rations are calcium salts of palm oil fatty acid calcium salts of palm oil fatty acid (CSFA) and prilled saturated fatty acids (SFAs). The objectives of this study were to determine the effects of supplementing SFA in the form of triglycerides (TSFA), as compared to CSFA, on yields, efficiency and diet digestibility in high-yielding dairy cows. Twenty-eight (14 cows in each group) multiparous cows were fed a basal diet supplemented (on DM basis) with either 12 g/kg TSFA (~350 g/cow per day – contained 980 g/kg fat; 882.3 g/kg SFAs) or 14 g/kg CSFA (~440 g/cow per day – contained 800 g/kg fat; 566.4 g/kg SFAs). The supplement amounts in the diet were balanced according to fat content. Rumen samples were taken for measurements of ammonia and volatile fatty acids concentrations, and fecal samples were taken for digestibility measurements. The CSFA cows produced 3% higher milk yields (47.6 v. 46.2 kg/day; P < 0.0001) and 4.7% higher 4% fat-corrected milk (FCM; 44.7 v. 42.7 kg/day; P = 0.02) than the TSFA cows. No difference in milk-fat content was observed, but milk-protein content was higher in the TSFA than CSFA cows. Yields of fat and protein were similar, but lactose yields were higher in TSFA cows. There were no differences in dry matter intake or efficiency calculations between groups. The ruminal ammonia concentrations were similar between groups, whereas acetate concentrations and acetate : propionate ratio were greater for CSFA than TSFA cows. The apparent total-tract digestibility of dry (P < 0.0007) and organic matters (P < 0.0003), fat (P < 0.0001), NDF and ADF (P = 0.02) were lower in the TSFA v. CSFA cows. In conclusion, the CSFA-supplemented cows produced 3% higher milk and 4.7% higher 4% FCM than the TSFA cows. However, TSFA supplementation did not depress milk-protein content. The apparent total-tract digestibility was lower for all dietary components in the TSFA cows, which was probably due to the effects of both degree of saturation and triglyceride form of the TSFA supplement. Considering that diets were balanced according to the fat content of the supplements, the lower yields of milk and FCM observed in the TSFA than CSFA cows were likely due to the lower digestibility of the fat and other nutrients in the TSFA cows, which might have negatively influenced the dietary energy content.