Abomasal infusion of essential fatty acids and conjugated linoleic acid during late pregnancy and early lactation affects immunohematological and oxidative stress markers in dairy cows.

Oxidative stress and inflammation, as natural parts of metabolic adaptations during the transition from late gestation to early lactation, are critical indicators of dairy cows' metabolic health. This study was designed to investigate the effects of abomasal infusion of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) on plasma, erythrocyte, and liver markers of oxidative stress in dairy cows during the transition period. Rumen-cannulated German Holstein cows (n = 38) in their second lactation (11,101 ± 1,118 kg milk/305 d, mean ± standard deviation) were abomasally infused with one of the following treatments from d -63 antepartum until d 63 postpartum (PP): CTRL (n = 9; 76 g/d coconut oil); EFA (n = 9; 78 g/d linseed plus 4 g/d safflower oil); CLA (n = 10; isomers cis-9,trans-11 and trans-10,cis-12 CLA; 38 g/d); and EFA+CLA (n = 10; 120 g/d). Hematological parameters as well as markers of oxidative status were measured in plasma, erythrocytes, and liver before and after calving. Immunohematological parameters, including erythrocyte number, hematocrit, hemoglobin, mean corpuscular hemoglobin, leukocytes, and basophils, were affected by time, and their peak levels were observed on the day after calving. The oxidative stress markers glutathione peroxidase 1 and reactive oxygen metabolites in plasma and erythrocytes were both affected by time, exhibiting the highest levels on d 1 PP, whereas ß-carotene, retinol, and tocopherol were at their lowest levels at the same time. Immunohematological parameters were only marginally affected by fatty acid treatment in a time-dependent manner. As such, lymphocyte and atypical lymphocyte counts were both significantly highest in the groups that received EFA at d 1 PP. Moreover, EFA supplementation increased the mean corpuscular volume and showed a trend for induction of mean corpuscular hemoglobin compared with the CLA group during the transition period. The PP mean thrombocyte volume was higher in the EFA than in the CLA group (except for d 28) and both EFA and CLA reduced number of thrombocytes and thrombocrit at distinct time points. Hepatic mRNA abundance of markers related to oxidative status, including glutathione peroxidase (GPX-1) and catalase (CAT), was lower (P < 0.05) in EFA-treated than non-EFA-treated cows at d 28 PP. Dairy cows at the onset of lactation were characterized by induced markers of both oxidative stress and inflammation. Supplementing EFA and CLA had minor and time-dependent effects on markers of oxidative stress in plasma, erythrocytes, and liver. A comparison of EFA supplementation with CLA or CTRL showed higher immunohematological response at d 1 PP and lower hepatic antioxidant levels by d 28 PP. Supplementation with EFA+CLA had only a minor effect on oxidative markers, which were more similar to those with the EFA treatment. Altogether, despite the time-dependent differences, the current findings show only minor effects of EFA and CLA supplementation in the prevention of early lactation-induced oxidative stress.

The age of hair matters - the incorporation of cortisol by external contamination is enhanced in distal hair segments of pigs and cattle.

Hair cortisol concentration (HCC) is used as an indicator of long-term stress or pathologies in humans and increasingly in animals. Although the main mechanism for the incorporation of cortisol into the hair shaft is by diffusion from blood, cortisol may also be incorporated from external sources by contamination of the hair surface. In farm animals under conventional husbandry conditions and trapped animals, contamination of hair with cortisol-containing body fluids, especially with urine, was shown to be a considerable confounding factor when studying HCCs. We recently found that cattle and pigs exhibit elevated HCCs in distal hair segments and assume that the incorporation of external cortisol is facilitated in these older hair segments. Therefore, the aim of this study was to investigate the effects of urine contamination on HCC in different hair segments of pigs and cattle, and to determine whether different cleaning protocols can prevent contamination effects. In an in vivo experiment in pigs (n = 18) and an in vitro experiment in cattle (n = 12), hairs were repeatedly contaminated with urine of the respective species and then shaved or cut in segments. Cortisol concentrations in hair segments were analysed by enzyme immunoassay after washing with isopropanol and extraction with methanol. Results were compared with HCCs in untreated hairs or hairs treated with water. Moreover, additional bovine hair samples contaminated with urine were subjected to two further cleaning procedures. Contamination with urine generally increased HCCs, and it was demonstrated for the first time that this effect is more pronounced in distal compared to proximal hair segments in both species. The immersion of bovine hair in vitro in water caused a washout of cortisol, which was also more pronounced in distal hair segments. In general, the different cleaning protocols for cattle hair did not prevent contamination effects, so we assume that external cortisol not only adheres but is incorporated into the hair shaft. Structural damage of older, distal hair segments may facilitate permeability of the hair matrix and diffusion of cortisol from and into aqueous solutions. Thus, the validity of HCC as a marker of stress is compromised in animals where soiling of hair with body fluids is a risk factor. Therefore, hair samples should be collected from clean body regions and, if possible, using proximal hair segments.

The dark side of white hair? Artificial light irradiation reduces cortisol concentrations in white but not black hairs of cattle and pigs.

Analysing hair cortisol concentrations (HCCs) is a minimally invasive way to retrospectively assess long-term stress, and its application in studies of animal welfare and stress has attracted considerable interest. However, not only stress-related effects but also hair-specific characteristics and external influences can affect HCCs and interfere with the interpretation of results. Thus, it was the aim of this study to investigate the impact of daylight and UV irradiation on cortisol concentrations in the hairs of pigs and cattle. We also examined whether a potential irradiation effect on HCCs depended on the colour of the hair. For this purpose, black and white hair samples from 18 Saddleback pigs and 18 Holstein Friesian cattle were exposed to artificial light (both visible and UV) and compared with control hair samples from the same animals kept in the dark. Exposure to artificial light significantly decreased HCCs in both pigs (P < 0.05) and cattle (P < 0.001), and hair colour had an influence on HCCs, with black hair showing higher cortisol levels than white hair (cattle: P < 0.001, pigs: P = 0.07). The interaction between light exposure and hair colour was significant in both pigs (P < 0.01) and cattle (P < 0.001), so light exposure reduced HCCs in porcine white hair but not black hair. In cattle, light-exposed white hair exhibited lower hair cortisol levels than control white hair or black hair. These results demonstrate that artificial light irradiation degrades hair cortisol or favours its elimination by structural changes of the hair matrix. However, this effect was only detectable in white hair, indicating that the melanin pigments in black hair absorbed radiation, thereby reducing the effects of photodegradation. Compared with other known influencing factors on HCCs, such as age and body region, the influence of light irradiation was relatively low in this in vitro experiment. However, further studies should investigate this influence under real-life animal conditions, such as outdoor and indoor housing.

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.

Modulation of colostrum composition and fatty acid status in neonatal calves by maternal supplementation with essential fatty acids and conjugated linoleic acid starting in late lactation.

Sufficient maternal supply of essential fatty acids (EFA) to neonatal calves is critical for calf development. In the modern dairy cow, EFA supply has shifted from α-linolenic acid (ALA) to linoleic acid (LA) due to the replacement of pasture feeding by corn silage-based diets. As a consequence of reduced pasture feeding, conjugated linoleic acid (CLA) provision by rumen biohydrogenation was also reduced. The present study investigated the fatty acid (FA) status and performance of neonatal calves descended from dams receiving corn silage-based diets and random supplementation of either 76 g/d coconut oil (CTRL; n = 9), 78 g/d linseed oil and 4 g/d safflower oil (EFA; n-6/n-3 FA ratio = 1:3; n = 9), 38 g/d Lutalin (BASF SE, Ludwigshafen, Germany) providing 27% cis-9,trans-11 and trans-10,cis-12 CLA, respectively (CLA; n = 9), or a combination of EFA and CLA (EFA+CLA; n = 11) in the last 9 wk before parturition and following lactation. The experimental period comprised the first 5 d of life, during which calves received colostrum and transition milk from their own dam. The nutrient compositions of colostrum and transition milk were analyzed. Plasma samples were taken after birth and before first colostrum intake and on d 5 of life for FA analyses of the total plasma fat and lipid fractions. Maternal EFA and CLA supplementation partly affected colostrum and transition milk composition but did not change the body weights of calves. Most EFA in calves were found in the phospholipid (PL) and cholesterol ester (CE) fractions of the plasma fat. Maternal EFA supplementation increased the percentage of ALA in all lipid fractions of EFA and EFA+CLA compared with CTRL and CLA calves on d 1 and 5, and the increase was much greater on d 5 than on d 1. The LA concentration increased from d 1 to 5 in the plasma fat and lipid fractions of all groups. The concentrations of docosapentaenoic acid, docosahexaenoic acid, and arachidonic acid in plasma fat were higher on d 1 than on d 5, and the percentage of n-3 metabolites was mainly increased in PL if dams received EFA. The percentage of cis-9,trans-11 CLA was higher in the plasma fat of EFA+CLA than CTRL calves after birth. By d 5, the percentages of both CLA isomers increased, leading to higher proportions in plasma fat of CLA and EFA+CLA than in CTRL and EFA calves. Elevated cis-9,trans-11 CLA enrichment was observed on d 5 in PL, CE, and triglycerides of CLA-treated calves, whereas trans-10,cis-12 CLA could not be detected in individual plasma fractions. These results suggest that an altered maternal EFA and CLA supply can reach the calf via the placenta and particularly via the intake of colostrum and transition milk, whereas the n-3 and n-6 FA metabolites partly indicated a greater transfer via the placenta. Furthermore, the nutrient supply via colostrum and transition milk might be partly modulated by an altered maternal EFA and CLA supply but without consequences on calf performance during the first 5 d of life.

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.

Effects of a combined essential fatty acid and conjugated linoleic acid abomasal infusion on metabolic and endocrine traits, including the somatotropic axis, in dairy cows.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid (ALA), and conjugated linoleic acid (CLA) supplementation on metabolic and endocrine traits related to energy metabolism, including the somatotropic axis, in mid-lactation dairy cows. Four cows (126 ± 4 d in milk) were used in a dose-escalation study design and were abomasally infused with coconut oil (CTRL; 38.3 g/d; providing saturated fatty acids), linseed and safflower oils (EFA; 39.1 and 1.6 g/d; n-6:n-3 FA ratio = 1:3), Lutalin (CLA; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d of each), or EFA and CLA (EFA+CLA) for 6 wk. The initial dosage was doubled twice after 2 wk, resulting in 3 dosages (dosages 1, 2, and 3). Each cow received each fat treatment at different times. Cows were fed with a corn silage-based total mixed ration providing a low-fat content and a high n-6:n-3 fatty acid ratio. Plasma concentrations of metabolites and hormones (insulin-like growth factor-binding proteins only on wk 0 and 6) were analyzed at wk 0, 2, 4, and 6 of each treatment period. Liver biopsies were taken before starting the trial and at wk 6 of each treatment period to measure hepatic mRNA abundance of genes linked to glucose, cholesterol and lipid metabolism, and the somatotropic axis. The changes in the milk and blood fatty acid patterns and lactation performance of these cows have already been published in a companion paper. The plasma concentration of total cholesterol increased with dosage in all groups, except CLA, reaching the highest levels in EFA+CLA and CTRL compared with CLA. The high-density lipoprotein cholesterol plasma concentration increased in CTRL and was higher than that in EFA and CLA, whereas the concentration of low-density lipoprotein cholesterol increased in a dose-dependent manner in EFA and EFA+CLA, and was higher than that in CLA. Hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA synthase 1 was upregulated in all groups but was highest in EFA+CLA. Expression of sterol regulatory element-binding factor 1 tended to be lowest due to EFA treatment, whereas expression of long chain acyl-CoA-synthetase was lower in EFA than in CTRL. Hepatic mRNA expression of GHR1A tended to be higher in EFA+CLA than in CTRL. The plasma concentration of insulin-like growth factor I increased in CLA, and the plasma IGFBP-2 concentration was lower in EFA+CLA than in CTRL at wk 6. The plasma concentration of adiponectin decreased in EFA+CLA up to dosage 2. Plasma concentrations of albumin and urea were lower in CLA than in CTRL throughout the experimental period. Supplementation with EFA and CLA affected cholesterol and lipid metabolism and their regulation differently, indicating distinct stimulation after the combined EFA and CLA treatment. The decreased IGFBP-2 plasma concentration and upregulated hepatic mRNA abundance of GHR1A in EFA+CLA-supplemented cows indicated the beneficial effect of the combined EFA and CLA treatment on the somatotropic axis in mid-lactation dairy cows. Moreover, supplementation with CLA might affect protein metabolism in dairy cows.

Changes in fatty acids in plasma and association with the inflammatory response in dairy cows abomasally infused with essential fatty acids and conjugated linoleic acid during late and early lactation.

Dairy cows are exposed to increased inflammatory processes in the transition period from late pregnancy to early lactation. Essential fatty acids (EFA) and conjugated linoleic acid (CLA) are thought to modulate the inflammatory response in dairy cows. The present study investigated the effects of a combined EFA and CLA infusion on the fatty acid (FA) status in plasma lipids, and whether changes in the FA pattern were associated with the acute phase and inflammatory response during late pregnancy and early lactation. Rumen-cannulated Holstein cows (n = 40) were assigned from wk 9 antepartum to wk 9 postpartum to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 g/d of linseed oil and 4 g/d of safflower oil; ratio of oils = 19.5:1; n-6:n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; isomers cis-9,trans-11 and trans-10,cis-12; each 10 g/d), or both (EFA+CLA). Blood samples were taken to measure changes in FA in blood plasma on d -63, -42, 1, 28, and 56, and in plasma lipid fractions (cholesterol esters, free fatty acids, phospholipids, and triglycerides) on d -42, 1, and 56 relative to calving, and in erythrocyte membrane (EM) on d 56 after calving. Traits related to the acute phase response and inflammation were measured in blood throughout the study. Liver samples were obtained for biopsy on d -63, -21, 1, 28, and 63 relative to calving to measure the mRNA abundance of genes related to the inflammatory response. The concentrations of α-linolenic acid and n-3 FA metabolites increased in lipid fractions (especially phospholipids) and EM due to EFA supplementation with higher α-linolenic acid but lower n-3 metabolite concentrations in EFA+CLA than in EFA treatment only. Concentration of linoleic acid decreased in plasma fat toward calving and increased during early lactation in all groups. Concentration of plasma arachidonic acid was lower in EFA- than in non-EFA-treated groups in lipid fractions and EM. The cis-9,trans-11 CLA increased in all lipid fractions and EM after both CLA treatments. Plasma haptoglobin was lowered by EFA treatment before calving. Plasma bilirubin was lower in EFA and CLA than in CTRL at calving. Plasma concentration of IL-1ß was higher in EFA than in CTRL and EFA+CLA at certain time points before and after calving. Plasma fibrinogen dropped faster in CLA than in EFA and EFA+CLA on d 14 postpartum. Plasma paraoxonase tended to be elevated by EFA treatment, and was higher in EFA+CLA than in CTRL on d 49. Hepatic mRNA abundance revealed time changes but no treatment effects with respect to the inflammatory response. Our data confirmed the enrichment of n-3 FA in EM by EFA treatment and the inhibition of n-3 FA desaturation by CLA treatment. The elevated n-3 FA status and reduced n-6:n-3 ratio by EFA treatment indicated a more distinct effect on the inflammatory response during the transition period than the single CLA treatment, and the combined EFA+CLA treatment caused minor additional changes on the anti-inflammatory response.

Changes in fluid and acid-base status of diarrheic calves on different oral rehydration regimens.

The administration of oral rehydration solutions (ORS) is an effective method to treat dehydration and acidosis in calves suffering from diarrhea. The ORS can be prepared in water or milk. The aim of the present study was to elucidate how fluid and acid-base balance change after feeding milk compared with ORS prepared in water or milk to diarrheic calves. Calves (n = 30) with naturally acquired diarrhea were sequentially assigned in a 2:1 ratio to the following pretreatments: milk and water-ORS (pretreatment 1; n = 20 calves) or milk-ORS (pretreatment 2; n = 10 calves), respectively. The assignment was done on the day of diarrhea diagnosis. On d 3 ± 1 following assignment to pretreatment group, and after a fasting period of 9 h, diarrheic calves were subjected to the following treatments: 2 L of milk (pretreatment 1; n = 10 calves), water-ORS (pretreatment 1; n = 10 calves), or milk-ORS (pretreatment 2; n = 10 calves). Blood samples were taken before and at several time points until 6 h after feeding. Plasma protein, osmolality, and electrolytes were determined and a blood gas analysis was performed. Change in plasma volume was calculated according to plasma protein, and water intake during the experimental period was recorded. Plasma volume was increased 30 min after feeding water-ORS or milk but the increase was less pronounced after feeding milk compared with water-ORS. After feeding milk-ORS, no significant increase in plasma volume could be detected. Because of the pretreatment, plasma osmolality was higher in calves fed milk-ORS, but no change in plasma osmolality after feeding was detected. No difference in water consumption between the treatment groups was noted within the observed 6-h period. The pH was increased after feeding milk-ORS, whereas water-ORS and milk-feeding did not increase pH in blood. Pretreatment with milk-ORS resulted in higher baseline d-lactate concentration, but feeding milk-ORS reduced d-lactate values after feeding. In calves with diarrhea, plasma volume increased more quickly and to a greater extent after feeding water-ORS; thus, we recommend treating diarrheic calves with water-ORS before supplying milk. Nevertheless, diarrheic calves need milk to fulfill their energy needs. The administration of ORS in milk combined with free water access is more advisable than feeding milk exclusively because milk has no alkalinizing ability and contains less sodium. However, the effects of milk-ORS feeding on d-lactate levels in diarrheic calves need further elucidation.

Effects of abomasal infusion of essential fatty acids together with conjugated linoleic acid in late and early lactation on performance, milk and body composition, and plasma metabolites in dairy cows.

Rations including high amounts of corn silage are currently very common in dairy production. Diets with corn silage as forage source result in a low supply of essential fatty acids, such as α-linolenic acid, and may lead to low conjugated linoleic acid (CLA) production. The present study investigated the effects of abomasal infusion of essential fatty acids, especially α-linolenic acid, and CLA in dairy cows fed a corn silage-based diet on performance, milk composition, including fatty acid (FA) pattern, and lipid metabolism from late to early lactation. Rumen-cannulated Holstein cows (n = 40) were studied from wk 9 antepartum to wk 9 postpartum and dried off 6 wk before calving. The cows were assigned to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 and 4 g/d; linseed/safflower oil ratio = 19.5:1; n-6/n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; BASF SE, Ludwigshafen, Germany; isomers cis-9,trans-11 and trans-10,cis-12 each 10 g/d) or EFA+CLA. Milk composition was analyzed weekly, and blood samples were taken several times before and after parturition to determine plasma concentrations of metabolites related to lipid metabolism. Liver samples were obtained by biopsy on d 63 and 21 antepartum and on d 1, 28, and 63 postpartum to measure triglyceride concentration. Body composition was determined after slaughter. Supplementation of CLA reduced milk fat concentration, increased body fat mass, and improved energy balance (EB) in late and early lactation, but EB was lowest during late lactation in the EFA group. Cows with CLA treatment alone showed an elevated milk citrate concentration in early lactation, whereas EFA+CLA did not reveal higher milk citrate but did have increased acetone. Milk protein was increased in late lactation but was decreased in wk 1 postpartum in CLA and EFA+CLA. Milk urea was reduced by CLA treatment during the whole period. After calving, the increase of nonesterified fatty acids in plasma was less in CLA groups; liver triglycerides were raised lowest at d 28 in CLA groups. Our data confirm an improved metabolic status with CLA but not with exclusive EFA supplementation during early lactation. Increased milk citrate concentration in CLA cows points to reduced de novo FA synthesis in the mammary gland, but milk citrate was less affected in EFA+CLA cows, indicating that EFA supplementation may influence changes in mammary gland FA metabolism achieved by CLA.

Automatic recording of individual oestrus vocalisation in group-housed dairy cattle: development of a cattle call monitor.

Oestrus detection remains a problem in the dairy cattle industry. Therefore, automatic detection systems have been developed to detect specific behavioural changes at oestrus. Vocal behaviour has not been considered in such automatic oestrus detection systems in cattle, though the vocalisation rate is known to increase during oestrus. The main challenge in using vocalisation to detect oestrus is correctly identifying the calling individual when animals are moving freely in large groups, as oestrus needs to be detected at an individual level. Therefore, we aimed to automate vocalisation recording and caller identification in group-housed dairy cows. This paper first presents the details of such a system and then presents the results of a pilot study validating its functionality, in which the automatic detection of calls from individual heifers was compared to video-based assessment of these calls by a trained human observer, a technique that has, until now, been considered the 'gold standard'. We developed a collar-based cattle call monitor (CCM) with structure-borne and airborne sound microphones and a recording unit and developed a postprocessing algorithm to identify the caller by matching the information from both microphones. Five group-housed heifers, each in the perioestrus or oestrus period, were equipped with a CCM prototype for 5 days. The recorded audio data were subsequently analysed and compared with audiovisual recordings. Overall, 1404 vocalisations from the focus heifers and 721 vocalisations from group mates were obtained. Vocalisations during collar changes or malfunctions of the CCM were omitted from the evaluation. The results showed that the CCM had a sensitivity of 87% and a specificity of 94%. The negative and positive predictive values were 80% and 96%, respectively. These results show that the detection of individual vocalisations and the correct identification of callers are possible, even in freely moving group-housed cattle. The results are promising for the future use of vocalisation in automatic oestrus detection systems.

Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) supplementation on fatty acid (FA) composition, performance, and systemic and hepatic antioxidative and inflammatory responses in dairy cows. Four cows (126 ± 4 d in milk) were investigated in a 4 × 4 Latin square and were abomasally infused with 1 of the following for 6 wk: (1) coconut oil (control treatment, CTRL; 38.3 g/d; providing saturated FA), (2) linseed and safflower oil (EFA treatment; 39.1 and 1.6 g/d, respectively; providing mainly α-linolenic acid), (3) Lutalin (BASF, Ludwigshafen, Germany; CLA treatment; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d each), (4) or EFA+CLA. The initial dosage was doubled every 2 wk, resulting in 3 dosages (dosage 1, 2, and 3). Cows were fed a corn silage-based total mixed ration with a high n-6/n-3 FA ratio. Dry matter intake and milk yield were recorded daily, and milk composition was measured weekly. The FA compositions of milk fat and blood plasma were analyzed at wk 0, 2, 4, and 6. The plasma concentration and hepatic mRNA abundance of parameters linked to the antioxidative and inflammatory response were analyzed at wk 0 and 6 of each treatment period. Infused FA increased in blood plasma and milk of the respective treatment groups in a dose-dependent manner. The n-6/n-3 FA ratio in milk fat was higher in CTRL and CLA than in EFA and EFA+CLA. The sum of FA

Comparative analyses of estimated and calorimetrically determined energy balance in high-yielding dairy cows.

Our aim was to compare the energy balance estimated (EBest) according to equations published by various energy feeding systems (German Society for Nutrition Physiology, French National Institute for Agricultural Research, and US National Research Council) and the EB calculated by use of calorimetrically measured heat production (EBhp) of 20 high-yielding (≥10,000 kg/305 d) German Holstein cows at -4 (pregnant, nonlactating) and 2 wk (early lactation) relative to parturition. In addition to heat production, feed and water intake, physical activity (including standing-lying behavior), body weight, body condition score, body temperature, plasma concentrations of fatty acids and ß-hydroxybutyrate, milk yield, and milk composition were measured to characterize the metabolic status. The EBhp was balanced [2.74 ± 4.09 MJ of metabolizable energy (ME)/d; ±standard error] before calving, but strongly negative (-84.7 ± 7.48 MJ of ME/d) at wk 2 of lactation. At both time points, EBhp and EBest differed significantly. On average, the equations overestimated the antepartum EB by 33 MJ of ME/d and underestimated the postpartum negative EB by 67 MJ of ME/d, respectively. Because the same ME intake and energy-corrected milk values were used for calculation of EBest and EBhp in our study, we considered that the factors (0.488 to 0.534 MJ of ME/kg0.75) currently used to calculate the ME requirements for maintenance probably underestimate the needs of high-yielding dairy cows, particularly during early lactation. In accord, heat production values determined under standard conditions of thermoneutrality and locomotion restriction amounted to 0.76 ± 0.02 MJ of ME/kg0.75 (4 wk antepartum) and 1.02 ± 0.02 MJ of ME/kg0.75 (2 wk postpartum), respectively. The expected positive correlation between EBhp and DMI was observed in pregnant cows only; however, a bias of 26 MJ of ME/d between mean actual energy intake and ME intake predicted according to German Society for Nutrition Physiology was found in cows at wk 4 antepartum. At both investigated time points, mobilization of tissue energy reserves (reflected by plasma fatty acid concentration) was related to EBhp. In early lactating cows, metabolic body weight (kg0.75) and the percentage of milk fat showed the strongest correlation (correlation coefficient = -0.70 and -0.73) to EBhp. Our findings must be taken into account when experimental data are interpreted because the true energy status might be significantly overestimated when EBest is used.

Effects of ad libitum milk replacer feeding and butyrate supplementation on behavior, immune status, and health of Holstein calves in the postnatal period.

Animal welfare in dairy calf husbandry depends on calf rearing and is probably improved by intensive milk feeding programs. In addition, butyrate supplementation in milk replacer (MR) stimulates postnatal growth and may affect the immune system in calves. We have investigated the combined effects of ad libitum MR feeding and butyrate supplementation on feeding behavior, health, and the immune responses in calves. Holstein calves (n = 64) were examined from birth until wk 11 of age. Calves received MR either ad libitum (Adl) or restrictively (Res) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate supplementation starting on d 4. From wk 9 to 10, all calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Calves were housed in straw-bedded group pens with automatic MR feeders, where feed intake and feeding behavior were documented. Blood was drawn on d 1 before the first colostrum intake; on d 2, 4, and 7; and weekly thereafter until the end of the study to measure plasma concentrations of total protein, albumin, the immunoglobulins IgG1, IgG2, and IgM, and the acute phase proteins fibrinogen, serum amyloid A, and haptoglobin. Liver samples were taken on d 50 and 80 to determine gene expression related to acute phase proteins. Body temperature was measured daily for the first 3 wk, and clinical traits were scored daily. Ad libitum MR feeding resulted in greater MR intake, greater MR intake per meal, slower sucking rate, and greater body weight, but in a lower number of unrewarded visits and lower concentrate intake when compared with Res. Butyrate reduced the sucking rate but increased MR intake per meal. Immunoglobulins in the blood plasma increased after colostrum intake in all calves, with only minor differences among groups throughout the study. Plasma fibrinogen and serum amyloid A increased in the first week of life in all calves, and fibrinogen was greater in Res than in Adl on d 21, 49, and 63. Hepatic gene expression of fibrinogen on d 80 was greater in Adl than in Res. Gene expression of SAA2 was greater on d 50 in Adl than in Res and on d 80 was greater in ResB+ than in ResB-. Body temperature was greater in Adl than in Res during the first 2 wk, but neither MR feeding nor butyrate affected the health status. An improved animal welfare in Adl calves is supported by fewer signs of hunger, but intensive milk feeding and butyrate did not affect the health and immune status of the calves in a consistent manner.

Indices of heart rate variability as potential early markers of metabolic stress and compromised regulatory capacity in dried-off high-yielding dairy cows.

High performing dairy cows experience distinct metabolic stress during periods of negative energy balance. Subclinical disorders of the cow's energy metabolism facilitate failure of adaptational responses resulting in health problems and reduced performance. The autonomic nervous system (ANS) with its sympathetic and parasympathetic branches plays a predominant role in adaption to inadequate energy and/or fuel availability and mediation of the stress response. Therefore, we hypothesize that indices of heart rate variability (HRV) that reflect ANS activity and sympatho-vagal balance could be early markers of metabolic stress, and possibly useful to predict cows with compromised regulatory capacity. In this study we analysed the autonomic regulation and stress level of 10 pregnant dried-off German Holstein cows before, during and after a 10-h fasting period by using a wide range of HRV parameters. In addition heat production (HP), energy balance, feed intake, rumen fermentative activity, physical activity, non-esterified fatty acids, ß-hydroxybutyric acid, cortisol and total ghrelin plasma concentrations, and body temperature (BT) were measured. In all cows fasting induced immediate regulatory adjustments including increased lipolysis (84%) and total ghrelin levels (179%), reduction of HP (-16%), standing time (-38%) and heart rate (-15%). However, by analysing frequency domain parameters of HRV (high-frequency (HF) and low-frequency (LF) components, ratio LF/HF) cows could be retrospectively assigned to groups reacting to food removal with increased or decreased activity of the parasympathetic branch of the ANS. Regression analysis reveals that under control conditions (feeding ad libitum) group differences were best predicted by the nonlinear domain HRV component Maxline (L MAX, R 2=0.76, threshold; TS=258). Compared with cows having L MAX values above TS (>L MAX: 348±17), those with L MAX values below TS (L MAX cows (18.5±0.4 and 47.3 kg/day). From the present study, it seems conceivable that L MAX can be used as a predictive marker to discover alterations in central autonomic regulation that might precede metabolic disturbances.

Assessing immune competence in pigs by immunization with tetanus toxoid.

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs' age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.

Evaluation of electrical broad bandwidth impedance spectroscopy as a tool for body composition measurement in cows in comparison with body measurements and the deuterium oxide dilution method.

Body fatness and degree of body fat mobilization in cows vary enormously during their reproduction cycle and influence energy partitioning and metabolic adaptation. The objective of the study was to test bioelectrical impedance spectroscopy (BIS) as a method for predicting fat depot mass (FDM), in living cows. The FDM is defined as the sum of subcutaneous, omental, mesenteric, retroperitoneal, and carcass fat mass. Bioelectrical impedance spectroscopy is compared with the prediction of FDM from the deuterium oxide (DO) dilution method and from body conformation measurements. Charolais × Holstein Friesian (HF; = 18; 30 d in milk) crossbred cows and 2 HF (lactating and nonlactating) cows were assessed by body conformation measurements, BIS, and the DO dilution method. The BCS of cows was a mean of 3.68 (SE 0.64). For the DO dilution method, a bolus of 0.23 g/kg BW DO (60 atom%) was intravenously injected and deuterium (D) enrichment was analyzed in plasma and whey by stabile isotope mass spectrometry, and total body water content was calculated. Impedance measurement was performed using a 4-electrode interface and time domain-based measurement system consisting of a voltage/current converter for applying current stimulus and an amplifier for monitoring voltage across the sensor electrodes. For the BIS, we used complex impedances over three frequency decades that delivers information on intra- and extracellular water and capacity of cell membranes. Impedance data (resistance of extra- and intracellular space, cell membrane capacity, and phase angle) were extracted 1) by simple curve fit to extract the resistance at direct current and high frequency and 2) by using an electrical equivalent circuit. Cows were slaughtered 7 d after BIS and D enrichment measurements and dissected for the measurement of FDM. Multiple linear regression analyses were performed to predict FDM based on data obtained from body conformation measurements, BIS, and D enrichment, and applied methods were evaluated by cross-validation. The FDM varied widely between cows and was correlated to D enrichment in plasma ( = 0.91, < 0.05). Prediction of FDM by body size measurements was less precise ( = 0.84), but FDM prediction was more accurate using D enrichment in plasma ( = 0.90) and BIS ( = 0.99) data. Therefore, both BIS and D enrichment analysis resulted in similarly good predictions of FDM in cows, and we conclude that BIS could have the potential to predict FDM in dairy cows from 40 to 380 kg.

Phenotype analysis of male transgenic mice overexpressing mutant IGFBP-2 lacking the Cardin-Weintraub sequence motif: Reduced expression of synaptic markers and myelin basic protein in the brain and a lower degree of anxiety-like behaviour.

Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1.

Browning of subcutaneous fat and higher surface temperature in response to phenotype selection for advanced endurance exercise performance in male DUhTP mice.

For the assessment of genetic or conditional factors of fat cell browning, novel and polygenic animal models are required. Therefore, the long-term selected polygenic mouse line DUhTP originally established in Dummerstorf for high treadmill performance is used. DUhTP mice are characterized by increased fat accumulation in the sedentary condition and elevated fat mobilization during mild voluntary physical activity. In the present study, the phenotype of fat cell browning of subcutaneous fat and a potential effect on oral glucose tolerance, an indicator of metabolic health, were addressed in DUhTP mice. Analysis of peripheral fat pads revealed increased brite (brown-in-white) subcutaneous adipose tissues and in subcutaneous fat from DUhTP mice higher levels of irisin and different markers of fat cell browning like T-box transcription factor (Tbx1), PPARα, and uncoupling protein (UCP1) (P < 0.05) when compared to unselected controls. UCP1 was further increased in subcutaneous fat from DUhTP mice in response to mild exercise (fourfold, P < 0.05). In addition, surface temperature of DUhTP mice was increased when compared to controls indicating a physiological effect of increased UCP1 expression. The present study suggests that DUhTP mice exhibit different markers of mitochondrial biogenesis and fat browning without external stimuli. At an age of 43 days, sedentary DUhTP mice have improved metabolic health as judged from lower levels of blood glucose after an oral glucose tolerance test. Consequently, the non-inbred mouse model DUhTP represents a novel model for the identification of fat cell browning mechanisms in white adipose tissues.

Body fat mobilization in early lactation influences methane production of dairy cows.

Long-chain fatty acids mobilized during early lactation of dairy cows are increasingly used as energy substrate at the expense of acetate. As the synthesis of acetate in the rumen is closely linked to methane (CH4) production, we hypothesized that decreased acetate utilization would result in lower ruminal acetate levels and thus CH4 production. Twenty heifers were sampled for blood, rumen fluid and milk, and CH4 production was measured in respiration chambers in week -4, +5, +13 and +42 relative to first parturition. Based on plasma non-esterified fatty acid (NEFA) concentration determined in week +5, animals were grouped to the ten highest (HM; NEFA > 580 µmol) and ten lowest (LM; NEFA < 580 µmol) mobilizing cows. Dry matter intake (DMI), milk yield and ruminal short-chain fatty acids did not differ between groups, but CH4/DMI was lower in HM cows in week +5. There was a negative regression between plasma NEFA and plasma acetate, between plasma NEFA and CH4/DMI and between plasma cholecystokinin and CH4/DMI in week +5. Our data show for the first time that fat mobilization of the host in early lactation is inversely related with ruminal CH4 production and that this effect is not attributed to different DMI.