Effects of transport age (14 d vs. 28 d of age) on blood total cholesterol, insulin and IGF-1 concentrations of veal calves.

The main aim of the current study was to find biomarkers of health in calves transported at different ages. The selected blood parameters were total cholesterol, insulin and IGF-1 and the longitudinal study investigated whether or not these concentrations were different between calves that were transported from the dairy farm to the veal farm at 14 d or 28 d of age. Relationships between these blood variables and health characteristics of veal calves were investigated. In a 34-wk study period, a total of 683 calves originating from 13 Dutch dairy farms were transported at an age of 14 or 28 d to 8 Dutch veal farms. Calves were blood sampled the first wk after birth (mean and SD: 4.4 ± 2.1 d), a day before transport (mean and SD: 25.8 ± 7.3 d) and in wk 2 post-transport (mean and SD: 36.7 ± 12.2 d). In these samples, insulin, IGF-1 and total cholesterol were determined and analyzed with a linear mixed model (LMM). Individual medical treatments were recorded from birth until the day of transport at the dairy farm, and from the moment of arrival at the veal farm until slaughter, and analyzed as a binary response variable (calf treated or not) with a generalized linear mixed model. Fecal (calf with or without loose or liquid manure) and navel (calves with or without swollen and inflamed navel) scores measured during a single visit in wk 2 post-transport were also analyzed as binary response variables, whereas carcass weights at slaughter age were analyzed with a LMM. Cholesterol, insulin and IGF-1 were included as covariates in the previous models to test their relationships with the likelihood of calves being medically treated, fecal and navel scores, and carcass weights. One day before transport 28 d-old calves had higher blood cholesterol (Δ = 0.40 mmol/l) and IGF-1 (Δ = 53.6 ng/ml) concentrations, and evidence of higher insulin (Δ = 12.2 µU/ml) compared with 14 d-old calves. In wk 2 post-transport, 28-d old calves had higher blood IGF-1 (Δ = 21.1 ng/ml), with evidence of higher insulin (Δ = 12.2 µU/ml) concentrations compared with 14-d old calves. Cholesterol concentration measured one day before transport and in wk 2 post-transport had a positive relationship with carcass weight at slaughter (ß = 4.8 and 7.7 kg/mmol/l, respectively). Blood cholesterol concentration in wk 2 post-transport was negatively associated with the fecal score measured at the same sampling moment (ß = -0.55/mmol/l), with the likelihood of a calf of being treated with antibiotics (ß = -0.36/mmol/l) and other medicines (ß = -0.45/mmol/l) at the veal farm. Blood IGF-1 concentration in wk 2 post-transport was negatively associated with the likelihood of a calf of being treated with antibiotics and other medicines (both ß = -0.01/ng/ml) at the veal farm, and with fecal score recorded in wk 2 post-transport (ß = -0.004/ng/ml). When looking at the blood indicators, it appeared that calves transported at 28 d of age were more developed compared with 14 d old calves, thus transport at an older age might be more beneficial for the animals. It can be concluded that both blood cholesterol and IGF-1 concentrations seemed to be valuable biomarkers of health and energy availability in veal calves.

Dry period length affects rumen adaptation in dairy cattle precalving and during the first weeks after calving.

Omitting or shortening the dry period may result in a fairly constant ration throughout the transition period of dairy cows, reducing the need for adaptation of cow metabolism and rumen function to a new lactation. The objective of this study was to determine the effect of dry period length on rumen adaptation and cow metabolic state during the transition period. Twelve pregnant, rumen-cannulated Holstein Friesian dairy cows at the end of their first lactation were assigned to one of 3 treatments: a conventional (60 d), short (30 d) or no dry period (0 d). At dry-off, cows received a dry cow ration until calving. Lactating cows received a lactation ration. Cows were monitored from 8 wk before calving until 8 wk after calving for milk yield and dry matter intake (DMI). Rumen biopsies were taken from 3 locations in the rumen at 60, 40 and 10 d before calving and 3, 7, 14, 28 and 56 d after calving to assess papillae dimensions. Blood was sampled weekly from 3 wk before until 8 wk after calving, and liver biopsies were taken at wk -2, wk 2 and wk 4 relative to calving. Prepartum, DMI and milk yield were greater for cows with a short or no dry period, compared with cows with a conventional dry period. Postpartum, DMI was greater for cows with a short dry period compared with cows with a conventional dry period. Plasma glucose concentration was greater for cows without a dry period, compared with the other dry period lengths postpartum. Plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate, and liver triglyceride content, did not differ among dry period. Rumen papillae differed in size based on biopsy location, but there was no interaction between biopsy location and the effect of dry period length. Rumen papillae surface area for cows managed for a 30 d or 60 d dry period decreased toward calving. At 40 d prepartum, papillae surface area was greater for short and no dry period treatment compared with a conventional dry period. At 10 d prepartum, papillae surface area was greater for the no dry period treatment compared with both other treatments, and this difference was still present 3 d postpartum. Cows managed for a short dry period showed faster increase in papillae dimensions after calving compared with cows managed for a conventional dry period. From d 28 onwards, no differences in papillae surface area were observed. The faster rumen adaptation postpartum may be related to the increased DMI during the first weeks postpartum for cows managed for a short dry period. However, this did not result in improved metabolic status or milk yield. The results from the present study demonstrate that the dietary changes related to a conventional dry period length affected rumen papillae development, not only prepartum but also early postpartum. Further optimization of dry period length as well as dietary composition throughout the transition period may support cows in their adaptation to a new lactation.

Behavioural responses related to increasing core body temperature of grazing dairy cows experiencing moderate heat stress.

Exposure to direct solar radiation, high ambient temperature, lack of wind movement, coupled with own metabolic heat production, makes grazing dairy cows vulnerable to heat stress. In pastures, it would be beneficial to monitor heat stress by observable changes in behaviour. We hypothesised that grazing dairy cows exhibit behavioural changes due to increasing heat load in temperate climate. Over two consecutive summers, 38 full-time grazing Holstein dairy cows were investigated in 12 experimental periods of up to 3 consecutive days where the cows were repeatedly exposed to various levels of moderate heat load determined by the comprehensive climate index (CCI). The CCI defines the ambient climate conditions, combining air temperature, relative humidity, solar radiation and wind speed. Vaginal temperature (VT) was automatically measured as an indicator of heat stress. In addition, as a less invasive method, we investigated if reticular temperature (RET) can be indicative of heat stress on pastures. Walking activity, lying-, feeding, and ruminating durations were recorded continuously with sensors. Respiration rate (RR), proximity to and competition at the water trough, social licking, self-licking, inter-individual distance, and fly intensity were directly observed. Data were analysed in the morning (0900-1100 h) and during the hottest time of day when cows were on pasture (1230-1430 h). The VT and RET showed similar patterns in relation to the CCI, suggesting that RET can be suitable for continuous monitoring of heat stress on pastures. In the morning, the cow's VT and RET did not relevantly react to the CCI. During the period 1230-1430 h, the cow's mean VT (mean vaginal temperature (VTMEAN); range: 37.7-40.3 °C) and mean RET (mean reticular temperature; range: 37.0-41.1 °C) were positively related to the mean CCI (mean comprehensive climate index) in this period (mean ± SD: 25.9 ± 5.71 °C). For cows with greater VTMEAN, an increased mean RR and decreased durations of walking, lying, feeding, and ruminating were found. These cows were also more likely to be in proximity to the water trough and to have small inter-individual distances. Changes in these traits seem to reflect behavioural adaptations to heat stress in a temperate climate and could be used to detect the heat stress in individual dairy cows on pastures.

Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets.

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin ß 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

Relationship between periparturient diseases, metabolic markers and the dynamics of hair cortisol concentrations in dairy cows.

Hair cortisol concentration (HCC) might represent a promising marker for retrospective welfare assessment of dairy cows. The objective of the study was to explore the dynamics of HCC in diseased and healthy cows from eight-week ante partum (AP) to eight-week post partum (PP). Twenty-four pregnant cows were followed from drying off to week eight PP. Tail hair was used to measure cortisol at five different time points. The occurrence of peripartum diseases, lameness and the body condition score (BCS) were monitored on a weekly basis. Blood ß-hydroxybutyric acid, non-esterified fatty acids, calcium and insulin-like growth factor-1 (IGF-1) concentrations were measured. The temperature-humidity index (THI) was continuously recorded. The median values of HCC in all cows were 0.4, 0.3, 0.6, 0.8 and 0.5 pg/mg at weeks eight, four AP, calving, weeks four, eight PP, respectively. There was no association between HCC and the occurrence of peripartum diseases (P ≥ 0.05). A positive correlation between HCC and BCS loss (P < 0.01) and THI (P < 0.05) was observed. The occurrence of peripartum diseases was associated with low IGF-1 during the study period but no relationship was found between cortisol and IGF-1 levels (P ≥ 0.05). Brown Swiss cows showed higher HCC (P < 0.01) at weeks eight, four AP, and week four PP and lower average milk yield (P < 0.05) than Holstein-Friesian cows. In conclusion, HCC was not a suitable marker for peripartum diseases but it could reflect a stress response, which is linked to BCS loss, heat stress and breed.

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 (VTMAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VTMAX 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 VTMAX. 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.

Effect of different dietary regimens at dry-off on performance, metabolism, and immune system in dairy cows.

Concentrate withdrawal and feed restriction are commonly used to reduce milk production and to facilitate dry-off, but may impair immune function in dairy cows. We investigated the effect of feed rations providing different amounts of nutrients in combination with feed restriction on performance, endocrine, and metabolic responses, as well as on leukocyte function before and after abrupt dry-off. Forty-three cows were studied from d 12 before until d 6 after dry-off (56 d before scheduled calving). Cows were fed experimental concentrates rich in crude protein (nitrogenic, n = 14), glucogenic precursors (glucogenic, n = 14), or lipids (lipogenic, n = 15). On d 3 before dry-off, total feed allowance was restricted to 50% in half of the animals of each dietary group, whereas feed allowance remained unchanged in the other animals. Performance parameters (milk yield, milk composition, and dry matter intake) were recorded, and daily blood and milk samples were taken and analyzed for various metabolic and endocrine parameters. Additionally, activity and mRNA abundance of several genes in leukocytes were measured at selected time points before and after feed restriction and dry-off, respectively. Feed restriction immediately resulted in a negative energy balance and decreased milk production. Concomitantly, concentrations of nonesterified fatty acids increased, whereas insulin, insulin-like growth factor-1, and glucagon decreased. After dry-off, energy balance turned positive and plasma nonesterified fatty acids decreased. Plasma glucose, insulin, and insulin-like growth factor-1 concentrations increased in all groups after dry-off. Glucose, insulin, and glucagon concentrations in plasma were higher in nonrestricted compared with restricted animals after dry-off. The experimental concentrate types marginally affected the investigated metabolic and endocrine factors, with the exception of elevated milk and plasma urea concentrations in cows fed the nitrogenic concentrate. Chemotactic and phagocytic activity of leukocytes were not affected by diets, feed restriction, or dry-off. Likewise, blood leukocyte mRNA abundance encoding for tumor necrosis factor α (TNF), heat shock protein family A (HSP70), and the glucose transporters (GLUT) 1 and 3 remained unchanged throughout the study period. Overall, the short-term negative energy balance induced by feed restriction was temporarily accompanied by metabolic adaptations, but did not alter the studied factors related to the immune system. Metabolic and endocrine adaptations supporting milk synthesis were continued during the first days after dry-off despite cessation of milking. Thus, the abrupt dry-off resulted in a short-term increase of glucose and triglyceride concentrations, with a delayed endocrine response to re-establish nutrient homeostasis in blood.

Suitability of milk lactate dehydrogenase and serum albumin for pathogen-specific mastitis detection in automatic milking systems.

In response to intramammary infection (IMI), blood-derived leukocytes are transferred into milk, which can be measured as an increase of somatic cell count (SCC). Additionally, pathogen-dependent IgG increases in milk following infection. The IgG transfer into milk is associated with the opening of the blood-milk barrier, which is much more pronounced during gram-negative than gram-positive IMI. Thus, milk IgG concentration may help to predict the pathogen type causing IMI. Likewise, lactate dehydrogenase (LDH) and serum albumin (SA) cross the blood-milk barrier with IgG if its integrity is reduced. Because exact IgG analysis is complicated and difficult to automate, LDH activity and SA concentration aid as markers to predict the IgG transfer into milk in automatic milking systems (AMS). This study was conducted to test the hypothesis that LDH and SA in milk correlate with the IgG transfer, and in combination with SCC these factors allow the differentiation between gram-positive and gram-negative IMI or even more precisely the infection-causing pathogen. Further, the expression of these parameters in foremilk before (BME) and after (AME) milk ejection was tested. In the AMS, quarter milk samples (n = 686) from 48 Holstein-Friesian cows were collected manually BME and AME, followed by an aseptic sample for bacteriological culture. Mixed models were used to (1) predict the concentration of IgG transmitted from blood into milk based on LDH and SA; (2) use principal component analysis to evaluate joint patterns of SCC (cells/mL), IgG (mg/mL), LDH (U/L), and SA (mg/mL) and use the principal component scores to compare gram-positive, gram-negative, and control IMI types and BME versus AME samples; and (3) predict gram-positive and gram-negative IMI by inclusion of combined SCC-LDH and SCC-SA as predictors in the model. Overall, the SA and LDH had similar ability to predict IgG transmission from blood into milk. Comparing the areas under the curve (AUC) of the receiver operator characteristic curves, the SCC-LDH versus SCC-SA had lower gram-positive (AUC = 0.984 vs. 0.986) but similar gram-negative (AUC = 0.995 vs. 0.998) IMI prediction ability. The SCC, IgG, LDH, and SA were greater in gram-negative than in gram-positive IMI (BME and AME) in early lactation. All measured factors had higher values in milk samples taken BME than AME. In conclusion, LDH and SA could be used as replacement markers to indicate the presence of IgG transfer from blood into milk; in combination with SCC, both SA and LDH are suitable for differentiating IMI type, and BME is better for mastitis detection in AMS.

Assessment of anti-Müllerian hormone in mares' transitional period and in relation to fertility in elderly mares.

Anti-Müllerian Hormone (AMH) has been linked to reproductive tract abnormalities in mares and stallions. This study aimed at evaluating AMH as a biomarker for two reproductive conditions in mares. In the first part of this study, plasma AMH was evaluated as an early indicator of the onset of cyclicity in mares in the transitional period from the anovulatory phase during winter anoestrus to the cyclic phase during the breeding season. Ten mares between 8 and 17 years old were included in the experiment which lasted from mid-February until the end of April. Ovarian activity was monitored with ultrasonography three times per week, the detection of a corpus haemorrhagicum/luteum was documented and antral follicle counts (AFC) were recorded. Blood samples were collected weekly by jugular venipuncture during the whole study period to compare AMH concentrations before and after the first ovulation of the year. The second objective was to evaluate if plasma AMH concentrations in middle-aged mares are linked to fertility and could serve as a prognostic marker in that age group. A total of 41 privately-owned clinically sound mares aged between 12 and 21 years of various breeds were inseminated with fresh or frozen semen. Mares were scanned between day 14 and 20 and the "early pregnancy rate" included only positive pregnancy examinations after the first observed cycle in the season of each mare. Potential associations between the early pregnancy rate in the first cycle and the explanatory factors AMH concentrations, age, status of the mare, stud, development of post-breeding endometritis, number of inseminations and semen type were analysed using logistic regression models. In the first part of the study, correlation between AMH and AFC for the whole study period (P = 0.0002, ρ = 0.55) as well as prior to (P = 0.008, ρ = 0.58) and after the first ovulation (P = 0.0007, ρ = 0.69) were observed. However, AMH concentrations before and after the first ovulation of the year were not statistically different. The second part of the study revealed no association between early pregnancy rate and AMH concentrations or any of the other mentioned factors. In conclusion, this study showed no evidence of a difference between AMH concentrations before and after the first ovulation of the year thus not supporting the use of AMH as a biomarker to predict the onset of cyclicity in mares. We could furthermore not show a relationship between plasma AMH concentrations and early pregnancy rates in this cohort of animals.

Metabolic and endocrine responses to short-term nutrient imbalances in the feed ration of mid-lactation dairy cows.

Short-term imbalances of dietary nutrients occur during natural fluctuations in roughage quality (e.g. on pasture) or temporal shortages of supplementary feed components. In contrast to a deficiency, macronutrients (i.e. carbohydrates, proteins, lipids) beyond the adequate supply with other nutrients may, for instance, alter milk composition, increase BW or result in a greater excretion of nitrogen. Especially dairy cows with a moderate performance, in mid- or late lactation, or in extensive farming systems may be exposed to imbalanced rations. A better understanding of metabolic and endocrine responses depending on macronutrient supply may help to precisely feed dairy cows. The present study investigated short-term metabolic and endocrine responses to different levels of concentrates formulated to particularly provide one major macronutrient source (carbohydrates, proteins or lipids). Based on parity number, lactational stage, milk yield and BW, nine mid-lactating cows (211 ± 19 days in milk) were grouped into three blocks of three animals each. Concentrates (aminogenic: rich in CP and nitrogen sources; glucogenic: high content of carbohydrates and glucogenic precursors; lipogenic: high lipid content) were fed in addition to hay in a factorial arrangement at increasing levels from 2.5 to 7.5 kg/d during 9 d. Milk yield, BW and feed intake were recorded daily. Blood and milk were sampled every 3 d at the end of each concentrate level. Milk fat, protein, lactose and urea contents were determined. In blood, concentrations of various metabolites, endocrine factors and enzyme activities (e.g. glucose, non-esterified fatty acids (NEFAs), ß-hydroxybutyrate, urea, cholesterol, triglycerides, insulin, glucagon, aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT) and glutamate dehydrogenase activity (GLDH)) were measured. Milk yield, milk composition and BW were not affected by type and level of concentrates. Feed intake increased in cows with greater amounts of the aminogenic and lipogenic concentrate compared with the glucogenic concentrate. Milk and plasma urea concentrations were elevated in the aminogenic and to a lesser extent in the lipogenic treatment compared with the glucogenic treatment. Glucose concentrations in plasma were not affected by treatments, whereas insulin and glucagon increased, and NEFA concentrations decreased only in cows fed 7.5 kg/d aminogenic concentrate compared with the glucogenic and lipogenic treatment. Activities of ASAT, GGT and GLDH as well as the total antioxidant capacity were not affected by diets. In conclusion, immediate metabolic and endocrine responses were observed due to the short-term dietary changes. Particularly, a surplus of nitrogen supply via the aminogenic diet affected metabolic responses and stimulated insulin and glucagon secretion.

Pathogen dependent effects of high amounts of oxytocin on the bloodmilk barrier integrity during mastitis in dairy cows.

INTRODUCTION: The reduction of antibiotic use in food producing animals becomes increasingly important. Therefore, suitable alternatives for mastitis treatment in dairy cows have to be considered. Oxytocin (OT) induces milk ejection and hence supports milk removal from infected mammary quarters. Beyond udder emptying, the injection of very high dosages of OT causes increased somatic cell counts (SCC) in milk and enables the transfer of immunoglobulins (Ig) from blood into milk through a reduced blood-milk barrier integrity. The aim of the present study was to investigate pathogen-specific changes of SCC, the blood derived milk components lactate dehydrogenase (LDH), serum albumin (SA), and IgG in milk of cows suffering from mastitis caused by different pathogens treated with two intravenous injections of high dosages of OT (100 IU). Milk samples from 184 dairy cows from different farms were collected on day 1 (day of clinical examination and mastitis diagnosis) and on days 2, 3, 14, and 28. Bacteriological examination (day 1) identified involved pathogens. Cows were randomly assigned to treatment (OT injections on days 1 and 2) or control group (no OT). Independently of the assigned experimental group, cows received the common therapy protocol of the veterinary practice after sample collection if the general condition was affected. Milk SCC, LDH, SA, and IgG changed specifically depending on involved pathogens. Highest values of all three parameters were measured in mastitis caused by Streptococcus uberis. Changes were less pronounced with other Streptococci spp., Staphylococci spp. or Corynebacterium bovis. Oxytocin treatment did not affect any of the studied parameters independent of the involved pathogen. Only in quarters infected with Staphylococci other than Staphylococcus aureus a decreased SCC and increased IgG concentrations in quarters, where no pathogens were detected, were observed. Thus, high dosage OT administration is obviously not suitable as a stand-alone mastitis treatment in dairy cows.

INTRODUCTION: La réduction de l'utilisation d'antibiotiques chez les animaux destinés à l'alimentation devient de plus en plus importante. Par conséquent, des alternatives appropriées au traitement des mammites chez les vaches laitières doivent être envisagées. L'ocytocine (OT) induit l'éjection du lait et favorise donc l'élimination du lait des quartiers infectés. Au-delà de la vidange de la mamelle, l'injection de doses très élevées d'OT entraîne une augmentation du nombre de cellules somatiques (CSC) dans le lait et permet le transfert d'immunoglobulines (Ig) du sang vers le lait grâce à une réduction de l'intégrité de la barrière sang-lait. Le but de la présente étude était d'étudier les changements spécifiques aux agents pathogènes du CSC, les composants du lait dérivés du sang que sont la lactate déshydrogénase (LDH) et l'albumine sérique (SA) ainsi que les IgG dans le lait de vaches souffrant de mammites causées par différents agents pathogènes traités par deux injections intraveineuses de doses élevées d'OT (100 UI). Des échantillons de lait de 184 vaches laitières de différentes exploitations ont été prélevés au jour 1 (jour de l'examen clinique et diagnostic de mammite) et aux jours 2, 3, 14 et 28. L'examen bactériologique (jour 1) a identifié les agents pathogènes impliqués. Les vaches ont été assignées au hasard au traitement (injections d'OT les jours 1 et 2) ou au groupe témoin (pas d'OT). Indépendamment du groupe auquel elles étaient attribuées, les vaches ont reçu le protocole thérapeutique usuel du cabinet vétérinaire après le prélèvement de l'échantillon si leur état général était affecté. Le CSC, la LDH, la SA et les IgG du lait ont varié spécifiquement en fonction des agents pathogènes impliqués. Les valeurs les plus élevées des trois paramètres ont été mesurées dans les mammites causées par Streptococcus uberis. Les changements étaient moins prononcés avec d'autres Streptococci spp., Staphylococci spp. ou Corynebacterium bovis. Le traitement à l'ocytocine n'a affecté aucun des paramètres étudiés indépendamment de l'agent pathogène impliqué. On a uniquement observé, dans les mammites causées par des staphylocoques autres que Staphylococcus aureus, une diminution du CSC et, dans les mammites où aucun agent pathogène n'a été détecté, une augmentation des concentrations d'IgG dans les quartiers. Ainsi, l'administration d'OT à forte dose n'est pas appropriée comme traitement unique des mammites chez les vaches laitières.

Invited review: The role of the blood-milk barrier and its manipulation for the efficacy of the mammary immune response and milk production.

The intact blood-milk barrier (BMB) prevents an uncontrolled exchange of soluble and cellular components between blood and milk in the mammary gland. It enables the sustainability of the optimal milk composition for the nourishment of the offspring. Endothelial cells, connective tissue, the basal membrane, and mainly the epithelial cells provide the semipermeability of this barrier, allowing only a selective transfer of components necessary for milk production. The epithelial cells are closely connected to each other by different formations, in which the tight junctions are the most critical for separating the milk-containing compartments from the surrounding extracellular fluid and vasculature. During mastitis, the integrity of the BMB is reduced. This facilitates the transfer of immune cells and immune factors such as antibodies from blood into milk. Simultaneously, the transfer of soluble blood constituents without an obvious immune function into milk is promoted. Furthermore, a reduced BMB integrity causes a loss of milk constituents into the blood circulation. Different mechanisms are responsible for the barrier impairment including tight junction opening, but also cell degradation. To promote the cure of mastitis, the targeted manipulation of the BMB permeability may be a tool to optimize the immune function of the mammary gland. An intensified opening of the BMB supports the antibody transfer from blood into milk, which is supposed to increase the contribution of the specific immune system in the immune defense. On the contrary, a fast closure of the BMB during the recovery from mastitis can accelerate the normalization of milk composition and milk yield. Various agents have been experimentally shown to either open (e.g., pathogens and pathogen-associated molecular patterns, several nonsteroidal anti-inflammatory drugs, oxytocin, calcium chelators) or close (e.g., glucocorticoids, nonsteroidal anti-inflammatory drugs, natural anti-inflammatory drugs) the BMB.

Effect of maternal supplementation with essential fatty acids and conjugated linoleic acid on metabolic and endocrine development in neonatal calves.

We tested the hypothesis that the maternal supply of essential fatty acids (EFA), especially α-linolenic acid, and conjugated linoleic acid (CLA), affects glucose metabolism, the endocrine regulation of energy metabolism and growth, and the intestinal development of neonatal calves. We studied calves from dams that received an abomasal infusion of 76 g/d coconut oil (CTRL; n = 9), 78 g/d linseed oil and 4 g/d safflower oil (EFA; n = 9), 38 g/d Lutalin (BASF SE) containing 27% cis-9,trans-11 and trans-10,cis-12 CLA (CLA; n = 9), or a combination of EFA and CLA (EFA+CLA; n = 11) during the last 63 d of gestation and early lactation. Calves received colostrum and transition milk from their own dam for the first 5 d of life. Insulin-like growth factor (IGF)-I, leptin, and adiponectin concentrations were measured in milk. Blood samples were taken before first colostrum intake, 24 h after birth, and from d 3 to 5 of life before morning feeding to measure metabolic and endocrine traits in plasma. On d 3 of life, energy expenditure was evaluated by a bolus injection of NaH13CO3 and determination of CO2 appearance rate. On d 4, additional blood samples were taken to evaluate glucose first-pass uptake and 13CO2 enrichment after [13C6]-glucose feeding and intravenous [6,6-2H2]-glucose bolus injection, as well as postprandial changes in glucose, nonesterified fatty acids (NEFA), insulin, and glucagon. On d 5, calves were killed 2 h after feeding and samples of small intestinal mucosa were taken for histomorphometric measurements. The concentrations of IGF-I, adiponectin, and leptin in milk decreased during early lactation in all groups, and the concentrations of leptin in first colostrum was higher in EFA than in CTRL cows. Plasma glucose concentration before first colostrum intake was higher in EFA calves than in non-EFA calves and was lower in CLA calves than in non-CLA calves. Plasma IGF-I concentration was higher on d 1 before colostrum intake in EFA calves than in EFA+CLA calves and indicated an overall CLA effect, with lower plasma IGF-I in CLA than in non-CLA calves. Postprandial NEFA concentration was lowest in EFA and CLA calves. The postprandial rise in plasma insulin was higher in EFA than in non-EFA calves. Plasma adiponectin concentration increased from d 1 to d 2 in all groups and was higher on d 3 in CLA than in non-CLA calves. Plasma leptin concentration was higher on d 4 and 5 in EFA than in non-EFA calves. Maternal fatty acid treatment did not affect energy expenditure and first-pass glucose uptake, but glucose uptake on d 4 was faster in EFA than in non-EFA calves. Crypt depth was lower, and the ratio of villus height to crypt depth was higher in the ilea of CLA than non-CLA calves. Elevated plasma glucose and IGF-I in EFA calves immediately after birth may indicate an improved energetic status in calves when dams are supplemented with EFA. Maternal EFA and CLA supplementation influenced postprandial metabolic changes and affected factors related to the neonatal insulin response.

Glucose metabolism and the somatotropic axis in dairy cows after abomasal infusion of essential fatty acids together with conjugated linoleic acid during late gestation and early lactation.

Sufficient glucose availability is crucial for exploiting the genetic potential of milk production during early lactation, and endocrine changes are mainly related to repartitioning of nutrient supplies toward the mammary gland. Long-chain fatty acids, such as essential fatty acids (EFA) and conjugated linoleic acid (CLA), have the potential to improve negative energy balance and modify endocrine changes. In the present study, the hypothesis that combined CLA and EFA treatment supports glucose metabolism around the time of calving and stimulates insulin action and the somatotropic axis in cows in an additive manner was tested. Rumen-cannulated German Holstein cows (n = 40) were investigated from wk 9 antepartum (AP) until wk 9 postpartum (PP). The cows were abomasally supplemented with coconut oil (CTRL, 76 g/d); 78 g/d of linseed and 4 g/d of safflower oil (EFA); Lutalin (CLA, isomers cis-9,trans-11 and trans-10,cis-12 CLA, each 10 g/d); or the combination of EFA+CLA. Blood samples were collected several times AP and PP to determine the concentrations of plasma metabolites and hormones related to glucose metabolism and the somatotropic axis. Liver tissue samples were collected several days AP and PP to measure glycogen concentration and the mRNA abundance of genes related to gluconeogenesis and the somatotropic axis. On d 28 AP and 21 PP, endogenous glucose production (eGP) and glucose oxidation (GOx) were measured via tracer technique. The concentration of plasma glucose was higher in CLA than in non-CLA-treated cows, and the plasma ß-hydroxybutyrate concentration was higher in EFA than in non-EFA cows on d 21 PP. The eGP increased from AP to PP with elevated eGP in EFA and decreased eGP in CLA-treated cows; GOx was lower in CLA than in CTRL on d 21 PP. The plasma insulin concentration decreased after calving in all groups and was higher in CLA than in non-CLA cows at several time points. Plasma glucagon and cortisol concentrations on d 21 PP were lower in CLA than non-CLA groups. The glucagon/insulin and glucose/insulin ratios were higher in CTRL than in CLA group during the transition period. Plasma IGF-I concentration was lower in EFA than non-EFA cows on d 42 AP and was higher during the dry period and early lactation in CLA than in non-CLA cows. The IGF binding protein (IGFBP)-3/-2 ratio in blood plasma was higher in CLA than in non-CLA cows. Hepatic glycogen concentration on d 28 PP was higher, but the mRNA abundance of PC and IGFBP2 was lower in CLA than non-CLA cows on d 1 PP. The EFA treatment decreased the mRNA abundance of IGFBP3 AP and PCK1, PCK2, G6PC, PCCA, HMGCS2, IGFBP2, and INSR at several time points PP. Results indicated elevated concentrations of plasma glucose and insulin along with the stimulation of the somatotropic axis in cows treated with CLA, whereas EFA treatment stimulated eGP but not mRNA abundance related to eGP PP. The systemic effects of the combined EFA+CLA treatment were very similar to those of CLA treatment, but the effects on hepatic gene expression partially corresponded to those of EFA treatment.

Intrauterine infusion of killed semen adversely affects uterine blood flow and endometrial gene expression of inflammatory cytokines in mares susceptible to persistent breeding-induced endometritis.

Persistent breeding-induced endometritis (PBIE) is a leading cause of infertility in mares. The objective of the study was to assess genital perfusion and endometrial gene expression of inflammatory cytokines in mares classified as susceptible (n = 5) or resistant (n = 5) to PBIE. Ten mares were examined daily during estrus until 6 d after hCG-induced ovulation for two estrous cycles. Twenty-four hours after application of 1500 IU hCG, 4 mL of killed (by repeated freezing in liquid nitrogen and thawing at 50 °C) deep-frozen semen or sterile saline was instilled into the uterine body and examinations were carried out immediately before and 3, 6, and 12 h after intrauterine infusion. Examinations included blood sampling to determine plasma progesterone (P4) concentrations, and transrectal ultrasonography in B- and color Doppler mode to determine follicular and luteal size and blood flow, the extent of intrauterine fluid, as well as time-averaged maximum velocity (TAMV), blood flow volume (BFV), and blood flow resistance (expressed as pulsatility index, PI) of the uterine arteries. Additionally, endometrial biopsies were obtained at 24 h before, and 2 and 7 d after infusion, and mRNA expressions of IL1B, IL6, IL8, IL10, TNF, CASP3, and COX2 were determined by qRT-PCR. Statistical analyses were performed with mixed models. Intrauterine fluid retention (diameter >20 mm for at least 3 d) was found after infusion of killed semen in five susceptible mares. There was no treatment effect (semen vs saline; P > 0.05) on genital blood flow, plasma P4 concentration, and endometrial gene expression. In comparison to resistant mares, susceptible mares had an increased (P = 0.04) BFV of the uterine arteries at 24 h before intrauterine infusion of killed semen, and an increased (P = 0.03) PI at 2 d after infusion. The TAMV, plasma P4 concentrations, and follicular and luteal size and blood flow did not differ (P > 0.05) between resistant and susceptible mares. Endometrial mRNA expression of IL1B increased (P = 0.05) at 2 d after the infusion of killed semen in the susceptible mares, and the expression of IL10 increased (P = 0.003) at 7 d after the infusion within the resistant mares. Interleukin 6 mRNA was increased (P = 0.05) in susceptible compared to resistant mares at 2 d after infusion. In summary, an intrauterine infusion of killed semen increases uterine blood flow resistance and alters endometrial gene expression of inflammatory cytokines for at least 7 d but does not affect ovarian blood supply and luteal function in mares susceptible to PBIE.

Free and bound cortisol, corticosterone, and metabolic adaptations during the early inflammatory response to an intramammary lipopolysaccharide challenge in dairy cows.

Glucocorticoids, particularly cortisol and corticosterone, are key homeostatic regulators during metabolic and endocrine adaptations including inflammatory responses. Besides the established response of total cortisol (TC) concentrations during inflammatory processes in dairy cows, we investigated changes of corticosterone, free cortisol (FC), and serum albumin as the main protein of unspecific cortisol binding, in response to an intramammary lipopolysaccharide (LPS) challenge. Furthermore, we evaluated relationships of glucocorticoid responses with concomitant alterations of metabolites and their endocrine regulators, insulin and glucagon. Blood samples of 10 multiparous Holstein dairy cows (26.8 ± 3.4 d in milk, previous lactation yield: 7,601 ± 938 kg; mean ± SD) were obtained every 30 min up to 5 h after the LPS instillation, and rectal temperature and heart rate were measured in parallel. Corticosterone was measured by enzyme immunoassay, TC by radioimmunoassay, and the proportion of FC by ultrafiltration. A mixed model was used to evaluate differences within the investigated parameters among selected time points (0, 3.5, and 5 h relative to the intramammary LPS administration). Rectal temperature increased up to 41.6 ± 0.1°C at 5 h after the LPS application. Concentrations of TC and corticosterone increased until 3.5 h, and the proportion of FC relative to TC more than doubled until 3.5 h after LPS administration. Serum albumin concentration was reduced at 5 h compared with initial values, whereas concentrations of insulin, glucagon, and glucose were increased after 5 h compared with 0 h. In conclusion, the stimulation of the immune system by the intramammary LPS administration is accompanied by distinct metabolic and endocrine changes. Corticosterone and TC concentrations react similarly in response to the LPS challenge and earlier compared with metabolic adaptations. The increased need of active cortisol is covered by both increased secretion and a higher percentage of FC.

Short communication: Comparative estimation of colostrum quality by Brix refractometry in bovine, caprine, and ovine colostrum.

Newborn ungulates depend on the timely supply of colostrum containing sufficient immunoglobulins to obtain passive immunity against disease. Brix refractometry enables a rapid on-farm estimation of colostrum quality and has been intensively studied in bovines. However, the suitability of Brix refractometers for assessing colostrum quality in goats and ewes has been scarcely evaluated. The present study compared bovine, caprine, and ovine colostrum quality estimation using an optical Brix refractometer. In addition, between-species variations in the relationships between Brix values and colostrum constituents (IgG, fat, protein, and lactose) and the accuracy of Brix refractometry at different cutoff values were evaluated by a receiver operating characteristic curve analysis. We measured the Brix value and contents of IgG, fat, protein, and lactose in 324 colostrum samples (108 cows, 116 does, and 100 ewes). Thresholds for classification of good colostrum quality (as determined by ELISA) were set at 50 mg IgG/mL in cows and 20 mg/mL in does and ewes. Bovine colostrum showed the greatest IgG concentrations compared with caprine and ovine colostrum. Fat and protein content was higher in sheep colostrum compared with the other species, whereas the highest lactose concentrations were detected in goat colostrum. Brix values ranged from 11.4 to 34.6% (22.1 ± 4.2%; mean ± standard deviation), 15.4 to 40.0% (28.5 ± 6.8%), and 8.8 to 39.8% (21.6 ± 5.3%) in bovine, ovine, and caprine colostrum, respectively. In all 3 species, Brix was highly correlated with IgG and protein concentrations (cows, r = 0.83 and 0.98; goats, r = 0.83 and 0.89; sheep, r = 0.75 and 0.87). Optimal cutoff points for greatest accuracy of Brix measurements were 19.3% Brix in cows [with 87.1% sensitivity (Se) and 100% specificity (Sp)], 20.7% Brix in does (with 53.5% Se and 100% Sp), and 26.5% Brix in ewes (with 75% Se and 91.3% Sp). In conclusion, Brix refractometry is an acceptable tool for on-farm estimations of colostrum quality in does and ewes despite distinct between-species variations in colostrum composition.

Effects of local or systemic administration of meloxicam on mammary gland inflammatory responses to lipopolysaccharide-induced mastitis in dairy cows.

Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used in combination with antimicrobial mastitis treatments to reduce pain. Little is known about whether meloxicam, an NSAID designed for the preferential inhibition of cyclooxygenase-2 over cyclooxygenase-1, affects the mammary immune response. The objective of this study was to analyze the mammary immune response to intramammary (local) or intravenous (systemic) administration of meloxicam with or without immune activation by lipopolysaccharide (LPS). We challenged 108 quarters of 30 cows with or without a low or high dose of LPS from Escherichia coli (0.1 or 0.2 µg/quarter), with or without meloxicam via intramammary administration (50 mg/quarter) or intravenous injection (0.5 mg/kg of body weight; ~300 mg/cow). Intramammary administration of meloxicam alone did not trigger an acute inflammatory response, verified by unchanged somatic cell count (SCC) and lactate dehydrogenase (LDH), BSA, and IgG concentrations in milk, which are normally augmented during mastitis due to an opening of the blood-milk barrier. Similarly, intramammary meloxicam did not change the mRNA abundance of inflammatory factors in mammary gland tissue. As expected, quarters challenged with either dose of LPS showed increased leukocyte infiltration (SCC); increased LDH, BSA, IgG, Na, and Cl concentrations; and diminished K concentrations in milk. In contrast to our hypothesis, the addition of intramammary or intravenous meloxicam did not reduce these markers of mastitis in milk. Instead, intramammary meloxicam appeared to accelerate the SCC response to LPS, but only at the lower LPS dose. Moreover, the mRNA expression of inflammatory factors in mammary tissue was not modified by the intramammary application of meloxicam compared with the contralateral quarters that were challenged with LPS only. We demonstrated for the first time that intramammary meloxicam at a dose of 50 mg/quarter did not trigger an immune response in the mammary glands of dairy cows. At the doses we used, meloxicam (intramammary or systemic) did not lower inflammatory responses. The intramammary administration of meloxicam seemed to stimulate leukocyte recruitment into the milk in quarters challenged with a low dose of LPS. The integrity of the blood-milk barrier was not protected by meloxicam in LPS-stimulated quarters. This study provides the first indications that meloxicam does not limit the inflammatory response in the mammary gland, although it does not impair the mammary immune system.

Nonsteroidal anti-inflammatory drugs affect the mammary epithelial barrier during inflammation.

During inflammation of the mammary gland, the blood-milk barrier, which is predominantly composed of mammary epithelial cells, loses its integrity and gradients between blood and milk cannot be maintained. Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used systemically in combination with local administration of antimicrobials in mastitis treatments of dairy cows to improve the well-being of the cow during the disease. However, the knowledge about their effects on the blood-milk barrier is low. This study aimed to investigate effects of different NSAID, with different selectivity of cyclooxygenase-inhibition, on the transepithelial electrical resistance (TEER) and capacitance, cell viability, and expression of tumor necrosis factor α of bovine mammary epithelial barriers in vitro. Primary mammary epithelial cells of 3 different cows were challenged with lipopolysaccharide (LPS) from Escherichia coli with or without addition of ketoprofen (1.25 mg/mL or 4 mM), flunixin meglumine (1.0 mg/mL or 4 mM), meloxicam (0.25 mg/mL, 0.75 mg/mL, or 4 mM), diclofenac (0.75 mg/mL or 4 mM) or celecoxib (0.05 mg/mL) for 6 h. Concentrations were adapted to comparable relations of the recommended dosage for systemic application. Additionally, a similar molar concentration of all NSAID was used. Lipopolysaccharide with or without NSAID induced a decrease in TEER within 5 h, which returned to control level within 14 h. Viability of cells challenged with LPS only was not affected. However, the cell viability was decreased with increasing concentrations of NSAID and this effect was amplified with simultaneous LPS challenge. Ketoprofen at both dosages, flunixin meglumine at 1.0 mg/mL, and meloxicam at 0.75 mg/mL accelerated the recovery of TEER in comparison to LPS only (return to control level within 9 h). The comparison of NSAID effects at the same molecular quantity of 4 mM showed different effect on the barrier in which ketoprofen accelerated the recovery after LPS-induced barrier opening, whereas meloxicam and diclofenac slowed down the recovery (return to control level after 24 h). In conclusion, NSAID do not prevent the mammary epithelial barrier opening by LPS; however, ketoprofen, flunixin meglumine, and meloxicam obviously support the re-establishment of the barrier integrity. Used in mastitis therapy at an optimized dosage the tested NSAID would likely support the recovery of milk composition. However, an overdose of NSAID would likely cause tissue irritation and in turn, a delayed recovery of the barrier permeability.

Metabolic status is associated with the recovery of milk somatic cell count and milk secretion after lipopolysaccharide-induced mastitis in dairy cows.

Infections of the mammary gland in dairy cows are commonly accompanied by reduced milk production and feed intake and poor milk quality. The metabolic status of early-lactating cows is known to affect immune response to pathogens and imposed immune challenges. We investigated the extent to which metabolic status before an intramammary lipopolysaccharide (LPS) challenge (LPS-CH) is associated with immune response, milk production, and feed intake and the recovery thereof. In 15 Holstein cows, weekly blood sampling and daily recording of dry matter intake, milk yield, milk composition, and body weight (to calculate energy balance) was started immediately after parturition. In wk 4 after parturition, cows underwent an intramammary LPS-CH (50 µg of LPS into 1 quarter). Blood and milk samples were taken in parallel at 30- and 60-min intervals, respectively, until 10 h after the LPS application. Plasma concentrations of glucose, nonesterified fatty acids, ß-hydroxybutyrate (BHB), cortisol, and insulin were analyzed. In milk, serum albumin, IgG concentration, somatic cell count (SCC), and lactate dehydrogenase (LDH) activity were determined. Dry matter intake and milk yield were recorded for an additional 6 d. Milk of the LPS-treated quarter was sampled at every milking for 8 d after the challenge. Based on plasma glucose concentrations in wk 1 to 4 after parturition before the LPS-CH, cows were retrospectively grouped into a high-glucose group (HG; 3.34-3.93 mmol/L, n = 7) and a low-glucose group (LG; 2.87-3.31 mmol/L, n = 8). Data were evaluated using mixed models with time, group, and time × group interaction as fixed effects and cow as repeated subject. Glucose was lower and BHB was higher in LG compared with HG before LPS-CH, whereas dry matter intake, energy balance, and SCC did not differ. During LPS-CH, SCC and LDH increased similarly in HG and LG, body temperature increased less in HG, and BHB and nonesterified fatty acids were higher in LG compared with HG. Dry matter intake declined in both groups during the day of the LPS-CH but recovered to prechallenge values faster in HG. Milk yield recovered within 2 d after the LPS-CH with no differences in morning milkings, whereas evening milk yield increased faster in HG. During 8 d after LPS-CH, SCC, LDH, IgG, and serum albumin in milk were lower in HG compared with LG. In conclusion, the level of circulating glucose and BHB concentrations in cows was associated with metabolic responses during an LPS-CH as well as the recovery of udder health and performance thereafter.