Short communication: Decreasing the dietary ratio of n-6 to n-3 fatty acids increases the n-3 concentration of peripheral blood mononuclear cells in weaned Holstein heifer calves.

Utilization of nutrients to improve overall heifer health is of interest because of the importance of replacement heifers to the dairy industry. The objective of our study was to compare the effect of supplementation of dietary n-3 and n-6 fatty acids (FA) on FA concentrations in peripheral blood mononuclear cells (PBMC) of Holstein calves. Twenty-seven Holstein heifer calves (107 ± 2.6 d of age; 142.6 ± 6.5 kg of body weight) from the university research and teaching herd were randomly assigned to a common TMR supplemented with 1 of 3 treatments: Ca salts of flaxseed FA (Virtus Nutrition, Corcoran, CA) containing 35% 18:3 n-3 (N3), Ca salts of soybean FA (Virtus Nutrition) containing 50% 18:2 n-6 (N6), or a 50:50 mix of N3 and N6. Treatments were supplemented with FA at 4% of dietary dry matter and fed for 30 d. Feed intake was recorded daily, and body weight, wither height, and body condition score were measured weekly throughout the study. On d 28 heifers were vaccinated with a Pasteurella vaccine and the temperature response to the vaccine was recorded. Blood was collected on d 0 and 28 for PBMC isolation. After total lipid extraction and FA methyl ester preparation, FA composition of PBMC was measured. We observed no effect of treatment on body weight gain, body condition score change, or wither height change. Heifers receiving the N3 diet had a lower temperature response to Pasteurella challenge compared with both the mix and N6 diets. Heifers consuming the N3 diet had a greater content of total n-3 FA, α-linolenic acid, and eicosapentaenoic acid in PBMC compared with heifers fed the N6 and mix diets. Heifers receiving the N3 diet also had a lower content of total n-6 FA, linoleic acid, and arachidonic acid in PBMC than heifers fed the N6 and mix diets. In conclusion, our study determined that feeding weaned female Holstein heifers a diet high in n-3 FA increased concentrations of n-3 FA in PBMC.

Short communication: Characterizing metabolic and oxidant status of pastured dairy cows postpartum in an automatic milking system.

The periparturient period represents a stressful time for dairy cows as they transition from late gestation to early lactation. Undesirable fluctuations in metabolites and impaired immune defense mechanisms near parturition can severely affect cow health and have residual effects on performance and longevity. Metabolic and oxidative stress profiles of multiparous and primiparous dairy cows in traditional parlor and feeding systems are well characterized, but status of these profiles in alternative management systems, such as grazing cows managed with an automatic milking system (AMS), are poorly characterized. Therefore, the objective of this case study was to characterize the metabolic and oxidant status of pastured cows milked with an AMS. It was hypothesized that primiparous and multiparous cows milked with an AMS would experience changes in oxidative and metabolic status after parturition; however, these changes would not impair cow health or production. Blood was collected from 14 multiparous and 8 primiparous Friesian-cross dairy cows at 1, 7, 14, and 21 d relative to calving for concentrations of insulin, glucose, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, reduced glutathione, oxidized glutathione, and antioxidant potential. Milk production and milking frequency data were collected postpartum. Milk production differed on d 7 and 14 between primiparous and multiparous cows and frequency was not affected by parity. Primiparous cows had higher levels of glucose than multiparous cows. No differences in insulin, NEFA, or ß-hydroxybutyrate concentrations were noted between multiparous and primiparous cows postpartum, though days relative to calving significantly affected insulin and NEFA. Primiparous cows also had higher antioxidant potential than multiparous cows during the postpartum period. Results from this study show that, although responses were within expected ranges, periparturient multiparous cows responded differently than periparturient primiparous cows with respect to metabolic and oxidative measures during the postpartum period at this pastured-AMS dairy, suggesting different management strategies may need to be considered with primiparous and multiparous cows.

Comparison of supplementation of n-3 fatty acids from fish and flax oil on cytokine gene expression and growth of milk-fed Holstein calves.

The ability to reduce incidence of disease in calves and improve early vaccination strategies is of particular interest for dairy producers. The n-3 fatty acids have been reported to reduce inflammatory diseases in humans but limited research has been done in calves. The objective of this study was to compare supplementation of n-3 fatty acids from fish and flax oil on gene expression of whole blood cells and growth of milk-fed Holstein calves. Forty-eight Holstein bull calves from a commercial dairy were randomly assigned to 1 of 3 diets beginning at 4d old: (1) control milk replacer (MR) with all pork fat, (2) MR with 2% flax oil, and (3) MR with 2% fish oil. All MR were 17% fat, 27% crude protein on a dry matter (DM) basis, with all protein from whey sources. Calves were each fed 654g DM of MR daily for the first 25d and then 327g/d for d26, 27, and 28. On d28, calves were challenged with a Pasteurella vaccine and the temperature response to the vaccine was recorded. Milk and feed intake and fecal scores were recorded daily, and body weight and hip width were recorded weekly. Blood was collected from all calves on d25. One tube of collected blood was incubated with endotoxin (lipopolysaccharide; LPS) for 2h and frozen with a second tube of control blood. Quantitative real-time PCR was used to assess the effects of LPS stimulation on cytokine gene expression. During the 28 d, calves supplemented with flax oil had a greater growth rate and feed efficiency than calves fed fish oil (0.52±0.02 vs. 0.48±0.02g of gain:g of feed). Fish oil tended to decrease LPS stimulation of tumor necrosis factor-α expression. Flax oil, but not fish oil, decreased the expression of IL-4 and tended to decrease expression of osteopontin and IL-8. Flax oil tended to reduce the increase in rectal temperature in response to a Pasteurella vaccine. In conclusion, our data support the idea that supplementation with n-3 fatty acids affects cytokine gene expression.

Validating the accuracy of activity and rumination monitor data from dairy cows housed in a pasture-based automatic milking system.

Behavioral observations are important in detecting illness, injury, and reproductive status as well as performance of normal behaviors. However, conducting live observations in extensive systems, such as pasture-based dairies, can be difficult and time consuming. Activity monitors, such as those created for use with automatic milking systems (AMS), have been developed to automatically and remotely collect individual behavioral data. Each cow wears a collar transponder for identification by the AMS, which can collect data on individual activity and rumination. The first aim of this study was to examine whether cow activity levels as reported by the AMS activity monitor (ACT) are accurate compared with live observations and previously validated pedometers [IceQube (IQ), IceRobotics, Edinburgh, UK]. The second aim of the study was to determine if the AMS rumination monitors (RUM) provide an accurate account of time spent ruminating compared with live observations. Fifteen lactating Holstein cows with pasture access were fitted with ACT, RUM, and IQ. Continuous focal observations (0600-2000 h) generated data on lying and active behaviors (standing and walking), as well as rumination. Activity recorded by live observation and IQ included walking and standing, whereas IQ steps measured cow movement (i.e., acceleration). Active behaviors were analyzed separately and in combination to ascertain exactly what behavioral components contributed to calculation of ACT "activity." Pearson correlations (rp) were computed between variables related to ACT, RUM, IQ, and live observations of behavior. A linear model was used to assess significance differences in the correlation coefficients of the 4 most relevant groups of variables. Significant but moderate correlations were found between ACT and observations of walking (r(p)=0.61), standing (r(p)=0.46), lying (r(p)=-0.57), and activity (r(p)=0.52), and between ACT and IQ steps (r(p)=0.75) and activity (r(p)=0.58) as well as between RUM and observations of rumination (rp=0.65). These data indicate that ACT and RUM do reflect cow walking and rumination, respectively, but not with a high degree of accuracy, and lying cannot be distinguished from standing.

Short communication: the effect of temperature on performance of milk ketone test strips.

Ketosis is estimated to affect 15% of early lactation dairy cows. A ketone test strip (Keto-Test; Elanco Animal Health, Greenfield, IN) allows producers a method to determine the concentration of ß-hydroxybutyrate (BHBA) in milk to track individual animal and herd incidence of ketosis. The objective of this study was to determine the effect of altering the temperature of milk and the test strips at the time of the test on the reliability of the Keto-Test. A total of 118 Holstein cows, ranging from 5 to 17 DIM, were selected from a commercial Holstein dairy herd in Michigan. A milk sample was collected from the right rear quarter of each cow during the a.m. milking. Each sample was tested under 4 temperature conditions: (1) Keto-Test strips and milk at room temperature (RT; 24.0 ± 0.1°C; control; manufacturer's instructions), (2) cold strips (10.8 ± 0.9°C) and milk at RT, (3) cold strips and fresh milk, and (4) strips at RT and fresh milk. Milk was recorded as negative (0-99 µmol/L), weak positive (100-199 µmol/L), positive (200-499 µmol/L), or highly positive (≥ 500 µmol/L). Blood samples were collected immediately following milk collection and analyzed for BHBA concentration using a ketone test meter. Cows with blood BHBA concentration of ≥ 1,400 µmol/L were considered positive for subclinical ketosis. Accuracy of the Keto-Test strips under the 4 conditions was determined by the κ coefficient of agreement, using the result of condition 1 as the accepted true value. Additionally, sensitivity and specificity were calculated using the blood BHBA concentrations and results of each of the 4 conditions. Using the Keto-Test 60.2% of cows tested negative for milk BHBA, 24.6% tested weak positive, 14.4% tested positive, and 0.8% tested highly positive. The weighted κ coefficient of agreement between the control condition (1) and condition 2, 3, and 4 and 95% lower and upper confidence intervals were as follows: condition 2=0.71 (0.62, 0.80), condition 3=0.69 (0.60, 0.78), and condition 4=0.63 (0.54, 0.73). These results indicate good agreement between the outcome of condition 1 and conditions 2, 3, and 4. The sensitivities/specificities for 1, 2, 3, and 4 were as follows: 0.77/0.79, 0.74/0.75, 0.69/0.88, and 0.69/0.84, indicating that the test in all temperature conditions had a strong ability to detect the presence of BHBA in milk. In conclusion, the reliability of the Keto-Test strips was not dependent on the temperature of the milk or the test strips.

Effect of snack food by-product inclusion on production of laying hens.

The increased interest in becoming green for consumers and companies is driving groups to develop innovative ways to become more efficient and reduce their waste. Foods past their expiration dates are large sources of waste and are causing food-manufacturing companies to develop waste disposal strategies. Integrating by-products from these companies into animal diets, specifically that of laying hens, could be significantly more cost effective for both the human food manufacturers and the agricultural producers. The study's objective is to evaluate laying hen diets containing snack food by-product, consisting mostly of expired potato chips, and the effect on hen performance. In total, 192 White Leghorn laying hens (45 wk old) were selected from the Michigan State University Poultry Farm. Hens were housed in conventional cages (3 birds/cage) and received 1 of 4 diets for 5 wk: 1) industry control corn-soybean meal, 2) control with 3% by-product, 3) control with 6% by-product, and 4) control with 9% by-product. Diets were formulated to be isocaloric, isonitrogenous, and balanced for sodium. Feed intake was measured for 3 consecutive days each week, and no overall differences between treatments were observed. However, during the first week, feed intake was significantly higher in birds fed the 6% and 9% diets compared with those fed control (P < 0.05). Birds fed the 6% had a higher feed intake than that of the control again during the fourth week (P < 0.01). Egg production, egg weight, and specific gravity were measured weekly. Hen BW was measured on d 1, 14, 28, and 35. Egg production, egg weight, specific gravity, and BW were not significantly affected by the addition of snack food by-products to the diet. In conclusion, the addition of expired snack food by-product into poultry diets does not significantly affect laying hen egg production and has the potential to be used as an alternative feed stuff in the future.

Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period.

The incidence and severity of mastitis can be high during the period of transition from pregnancy to lactation when dairy cattle are susceptible to oxidative stress. Oxidative stress may contribute to the pathogenesis of mastitis by modifying the expression of proinflammatory genes. The overall goal of this study was to determine the relationship between critical antioxidant defense mechanisms and proinflammatory markers in normal bovine mammary tissue during the periparturient period. Mammary tissue samples were obtained from 12 cows at 35, 20, and 7 d before expected calving and during early lactation (EL, 15 to 28 d in milk). Enzyme activities for cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase were relatively low during the dry period, but increased during EL, whereas activity of thioredoxin reductase 1 did not change significantly as a function of time. In contrast, gene expression for these antioxidant selenoproteins and for heme oxygenase-1 gradually decreased as parturition approached and then increased during EL. The expression of intercellular vascular adhesion molecule-1 and vascular cell adhesion molecule-1 followed a similar trend where mRNA abundance gradually declined as parturition approached with a slight rebound in EL. Gene expression of the pro-oxidant, 15-lipoxygenase 1, which is known to increase during times of oxidative stress, also increased dramatically in mammary tissue from EL cows. Expression of the proinflammatory cytokines, IL-1beta, IL-6, and IL-8 did not change significantly during the periparturient period. Strong positive correlations were found between several antioxidant enzymes (cytosolic glutathione peroxidase, thioredoxin reductase 1, and heme oxygenase-1) and vascular adhesion molecules (intercellular vascular adhesion molecule-1, vascular cell adhesion molecule-1) suggesting a protective response of these antioxidants to an enhanced proinflammatory state. Ability to control oxidative stress through manipulation of key antioxidant enzymes in the future may modify the proinflammatory state of periparturient cows and reduce incidence and severity of some diseases such as mastitis.

Osteopontin: a novel cytokine involved in the regulation of Mycobacterium avium subspecies paratuberculosis infection in periparturient dairy cattle.

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-gamma and IL-1 alpha for nonstimulated PBMC and IFN-gamma and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-alpha was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-gamma, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.

Osteopontin immunoreactivity in the ileum and ileocecal lymph node of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. The objective of this experiment was to identify Opn in the ileum and ileocecal lymph node (ICN) of dairy cows naturally infected with MAP and to compare the frequency and intensity of staining between noninfected healthy controls, subclinical and clinical cows. Sections from these three groups of animals were selected from a tissue archive. Immunohistochemical analysis was used to determine the location and expression of Opn. The frequency and intensity of staining was also reported. Confirmation of acid-fast bacilli in the tissue sections was achieved by the Ziehl-Neelsen method. Within the ileal tissue, macrophages, lymphocytes, and plasma cells stained positive for Opn. Clinical cows expressed Opn at a greater frequency in the lamina propria. Control and subclinical cows did not have areas of granulomatous inflammation but cells staining for Opn were equally intense for the three groups. The frequency of staining for Opn in the ICN was not affected by MAP infection. Results of this study confirm for the first time, the expression of Opn in the ileum and ICN of MAP-infected cattle.

Modulation of cytokine gene expression and secretion during the periparturient period in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression may contribute to the transition from the subclinical to the clinical stage of infection. Understanding the effects of stressors such as parturition on the escalation of disease may provide information that will help to manage JD. The objective of this study was to characterize cytokine gene expression and secretion in periparturient dairy cows naturally infected with MAP. Blood was collected from the jugular vein of healthy noninfected, and subclinically and clinically infected dairy cows for 3 weeks pre-calving to 4 weeks post-calving. Real-time PCR was performed to evaluate the expression of the following cytokine genes by peripheral blood mononuclear cells: IFN-gamma, TNF-alpha, IL-12p35, IL-10, TGF-beta, and IL-4. To assess the effects of parturient immunosuppression on cytokine gene expression, RT-PCR data were analyzed by using 2(-ddCt) values calibrated to dCt value at +1 day relative to calving for each animal. Overall, cytokine gene expression was not influenced by infection status of the cows in this study. However, significant effects in cytokine gene expression were noted across sampling days within the periparturient period. Expression of IFN-gamma by NS and ConA-stimulated PBMCs declined at calving compared with prepartum values in both control and infected cows. Similarly, a decline in expression of IL-4 and IL-10 was observed for cells isolated from subclinically infected cows after stimulation with ConA. ConA-stimulated PBMCs isolated from infected cows secreted higher concentrations of IFN-gamma compared with the controls. A significant decline in IFN-gamma secretion was noted for MPS-stimulated cells for clinical cows from -21 days to +1 day. Stimulating cells with MPS resulted in greater secretion of IL-10 by infected cows during the postpartum period. A trend was also observed for higher TGF-beta secretion by NS PBMCs isolated from clinical cows in the postpartum period. Cells isolated from clinically infected cows and stimulated with MPS secreted higher levels of nitric oxide throughout the periparturient period when compared to control or subclinically infected cows. These data suggest that parturition is a very dynamic time period for host immunity, with potential for altered immunity to hinder the ability of dairy cows to thwart infectious diseases.

Parturition invokes changes in peripheral blood mononuclear cell populations in Holstein dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.

Effect of dietary carbohydrate and monensin on expression of gluconeogenic enzymes in liver of transition dairy cows.

Thirty-four multiparous Holstein cows were used in a randomized block design to evaluate the effects of feeding nonforage fiber sources (NFFS), monensin, or their combination on expression of gluconeogenic enzymes in the liver during the transition to lactation. The addition of 0 or 300 mg/d of monensin to a conventional (CONV) or NFFS prepartum diet was evaluated in a 2 x 2 factorial arrangement of treatments. The NFFS diet was formulated by replacing 30% of the forage component of the CONV diet with cottonseed hulls and soyhulls. The CONV and NFFS basal diets were fed at dry-off and continued through parturition. Monensin was fed from -28 d relative to calving (DRTC) through parturition. At calving, all cows were placed on the same diet. Liver biopsy samples obtained at -28, -14, +1, +14, and +28 DRTC were used to determine pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Feeding NFFS resulted in greater (P < 0.05) prepartum DMI compared with the CONV diet. There was no effect of prepartum diets on postpartum DMI or average milk production to 56 d of lactation. Expression of PC mRNA was elevated (P < 0.05) at 1 d postpartum, but there was no effect of NFFS or monensin on PC mRNA abundance. Expression of PEPCK-C mRNA at calving was increased (P < 0.05) with prepartum monensin feeding. The data indicate that feeding monensin to transition cows induces hepatic PEPCK-C mRNA expression before calving. The increased expression of hepatic PEPCK-C mRNA with monensin feeding suggests a feed-forward mechanism of metabolic control in ruminants that links molecular control of gluconeogenesis with the profile of rumen fermentation end products.