Effects of sodium salicylate and time postpartum on mammary tissue proliferation, gene transcript profile, and DNA methylation.

Previous studies have demonstrated nonsteroidal antiinflammatory drug treatment in early lactation had a positive impact on whole-lactation milk production in older cows. The objective of this study was to evaluate proliferative, transcriptional, and epigenetic changes in the mammary gland that could explain increased production responses due to nonsteroidal antiinflammatory drug treatment. Sodium salicylate (SAL; 125 g/d) or water (CON) were administered via oral drench to multiparous Holstein cows (n = 8/treatment) once daily for 3 d beginning approximately 24 h after parturition, and mammary tissue was collected on d 1, 4, and 45 postpartum. Day 1 tissue was collected immediately preceding the initial drench, and d 4 tissue was collected 24 h following the final drench. Blood was collected twice weekly and analyzed for plasma glucose, insulin, ß-hydroxybutyrate, free fatty acids, and prolactin. Cows were milked twice daily until d 7 of lactation, and thrice daily for the remainder of the study. Total RNA extracted from tissue was deep-sequenced and analyzed for differential gene expression using DESeq2. We detected no treatment effect on milk yield or plasma metabolites through 45 d of lactation; additionally, no change in mammary epithelial cell proliferation was detected when assessed by Ki67 labeling. Comparison of SAL versus CON revealed that only 16 of 18,286 genes were differentially expressed (false discovery rate <0.1) in mammary tissue collected on d 45, whereas no differentially expressed genes due to treatment were detected on d 1 or 4. Analysis of transcriptional differences over time showed downregulation of pathways related to immune cell recruitment and differentiation, and extensive overlap with pathways related to cholesterol synthesis and liver X receptor signaling. Global DNA methylation of mammary tissue was decreased for CON compared with SAL. Transcriptome analysis emphasized extensive involvement of immune-related signaling pathways in the switch from lactogenesis to galactopoiesis, and changes in methylation with SAL treatment merit future investigation into epigenetic effects on milk production.

Behavior, health, and productivity of early-lactation dairy cows supplemented with molasses in automated milking systems.

The objective of this study was to determine the effect of molasses-based liquid feed (LF) supplementation within automated milking systems (AMS) on the behavior, health, and production of early-lactation dairy cows. In 6 commercial AMS dairy herds, 390 dairy cows were randomly assigned before calving to 1 of 2 treatments, within farm, balanced by parity: (1) control group (CON) receiving a standard AMS pellet [mean = 3.9 kg/d on a dry matter (DM) basis, n = 188], or (2) same amount of standard AMS pellet (mean = 3.6 kg/d on a DM basis, n = 202) plus 1 kg/d DM of LF for multiparous cows (1.6 kg/d as fed) and 0.88 kg/d DM for primiparous cows (1.4 kg/d as fed). Across farms, cows were fed partial mixed rations similar in ingredient and nutrient composition. Cows on the LF treatment received supplementation for the first 60 d in milk (DIM). Blood samples were taken 2 times/wk for the first 3 wk postcalving to assess ß-hydroxybutyrate (BHB). Samples with BHB ≥1.2 mmol/L were classified as a positive case of subclinical ketosis (SCK). Cow body condition and lameness were scored every 10 d postcalving until 60 DIM. No differences were detected between treatments for milk yield (average milk yield = 37.4 ± 1.98 kg/d) and milking frequency (average milking frequency = 3.2 ± 0.01 times/d). Similarly, no difference between treatments in daily AMS visits was detected (average AMS visits = 5.3 ± 0.3 times/d). However, CON cows experienced more AMS refusals/d than LF cows (LF = 1.6 refusals/d, CON = 2.1 refusals/d). No difference between treatments in daily rumination time was detected (average rumination time = 476 ± 10.8 min/d). Treatment was associated with the number of times cows tested positive for SCK; cows on LF had fewer repeated diagnoses of SCK, such that 15% of cows on LF had ≥3 diagnoses of SCK out of 5 tests, compared with 27% of CON cows. Cows receiving molasses tended to have lower blood BHB values at ∼15 DIM. Molasses supplementation also allowed cows to maintain a more stable body condition score, as LF cows tended to lose less body condition over the first 60 DIM compared with CON cows. Overall, the results of this study demonstrate that supplementing a molasses-based LF to AMS cows may help support the energy demands of milk production in early lactation and, thus, reduce the incidence of repeat positive SCK tests during that time period.

Effects of molasses-based liquid feed supplementation to a high-straw dry cow diet on feed intake, health, and performance of dairy cows across the transition period.

The objective of this study was to determine the effects of adding molasses-based liquid feed to a high-straw dry cow diet on intake, behavior, health, and performance of dairy cows across the transition period. Holstein cows (n = 40) entering their second lactation or greater were enrolled at dry-off (∼45 d before expected calving) and assigned to 1 of 2 treatments: a high-straw dry cow total mixed ration (TMR) with either (1) no molasses [CON; n = 20; 10.7% crude protein (CP), 1.45 Mcal/kg of net energy for lactation (NEL)] or (2) supplemented with 1 kg/cow daily [dry matter (DM) basis] of molasses-based liquid feed (LF; n = 20; 11.2% CP, 1.49 Mcal/kg of NEL). At calving all cows were fed the same lactating TMR (14.9% CP, 1.68 Mcal/kg of NEL) and followed for 28 d. During the dry period, cows fed the LF diet had greater DM intake (15.3 ± 0.24 vs. 13.4 ± 0.24 kg/d) and tended to have a shorter interval between meals (192.8 ± 8.38 vs. 216.5 ± 7.97 min) and more frequent meals (6.3 ± 0.27 vs. 5.6 ± 0.27 meals/d), while consuming their feed faster (0.09 ± 0.001 vs. 0.08 ± 0.001 kg of DM/min) compared with cows fed the CON diet. Regardless of treatment, cows sorted [100% × (actual intake/predicted intake)] against the longest ration particles (>19 mm), with cows fed the CON diet sorting more against these (81.1 ± 2.6 vs. 93.9 ± 2.6%). Cows fed the CON diet did not sort for or against short particles (<8, >4 mm; 100.2 ± 0.31%), but cows fed the LF diet tended to sort against these (99.2 ± 0.31%). Post-calving, cows fed the LF diet did not sort for long particles (98.5 ± 1.1%), but cows fed the CON diet continued to sort against these (96.3 ± 1.1%). Cows fed the LF diet had higher mean reticulorumen pH in the dry period (6.4 ± 0.05 vs. 6.2 ± 0.06 pH), in the first week after calving (CON = 5.7 ± 0.06 pH, LF = 5.8 ± 0.06 pH), and tended to have higher mean reticulorumen pH in the second week after calving (CON = 5.7 ± 0.06 pH, LF = 5.9 ± 0.06 pH). The results suggest that supplementing a molasses-based liquid feed in high-straw dry cow diets may improve intake and consistency in nutrients consumed during the dry period and in early lactation, as well as possibly promoting better rumen health across the transition period.

Effects of wheat straw chop length in high-straw dry cow diets on intake, health, and performance of dairy cows across the transition period.

The study investigated the effect of chop length of wheat straw in a high-straw dry cow diet on the feeding behavior, health, and performance of Holstein dairy cows across the transition period. Forty cows were enrolled at dry off, approximately 45 d before expected calving, and assigned to 1 of 2 dietary treatments containing (on a dry matter basis) 36% corn silage, 35% dry cow pellet, and 29% wheat straw. Diets were formulated to target an energy consumption of 19 Mcal/d of net energy for lactation per cow. The diets differed in the chop length of wheat straw: (1) long straw chopped with a 10.16-cm screen (LDD; n = 20), or (2) short straw chopped with a 2.54-cm screen (SDD; n = 20). Dietary treatments were fed throughout the dry period. Feeding behavior, rumination time, and reticulorumen pH were monitored electronically, and feed samples were collected to determine feed sorting. Blood samples were taken weekly, and body condition scores and body weights were taken every 2 wk. Postcalving, all cows were fed the same lactating cow total mixed ration for 28 d; the same measurements were recorded with the addition of daily milk yield and twice weekly milk components. During the dry period, cows fed the SDD treatment consumed more dry matter (15.6 vs. 15.0 kg/d), had more consistent dry matter intake in the week leading up to calving, had a faster feeding rate, consumed 0.4 kg/meal more, and had 0.6 fewer meals per day. No differences were observed in rumination time or mean reticulorumen pH between treatments in both the dry and lactating period. Regardless of treatment during the dry period, cows sorted against the long particles (>19 mm) and in favor of the short particles (<8, >4 mm), but cows fed the LDD treatment sorted to a greater extent than cows fed the SDD treatment. Dietary treatment in the dry period did not influence feed sorting behavior postcalving. Cows fed the LDD treatment had a greater daily change (decline) in rumen pH in the first week postcalving and had a greater blood ß-hydroxybutyrate concentration in the third week postcalving. Overall, the results from this study indicate that feeding shorter chopped wheat straw in a high-straw dry cow diet improved intake, reduced sorting during the dry period, and resulted in greater metabolic health and rumen stability in early lactation.

Moisture content of high-straw dry cow diets affects intake, health, and performance of transition dairy cows.

The objective of this study was to determine the effect of adding water to a high-straw dry cow diet on feeding behavior, health, and performance of dairy cows. Holstein cows (n = 40) entering their second or greater lactation were enrolled at dry off (∼45 d before expected calving) and assigned to 1 of 2 dietary treatments, consisting of a high-straw dry cow total mixed ration [TMR; 36% wheat straw, 41% corn silage, 23% pellet, on a dry matter (DM) basis, formulated for 11.6% crude protein, 1.35 Mcal/kg net energy for lactation] with (1) no water (CON; n = 20; DM = 53.4%) or (2) water added to decrease the DM by ∼10 percentage points (wet diet, WD; n = 20; DM = 45.4%). Upon calving, all cows were fed the same lactating TMR (45.2% DM, 14.9% crude protein, 1.68 Mcal/kg net energy for lactation) and followed for 28 d. Dry matter intake, feeding behavior, and rumination activity were recorded automatically. Fresh TMR and orts samples were collected 2×/wk to determine differences in sorting. A particle separator was used to separate feed samples into 4 fractions: long (>19 mm), medium (<19, >8 mm), short (<8, >4 mm), and fine (<4 mm) particles. Feed sorting was calculated as actual intake of each particle fraction expressed as a % of predicted intake. Cows fed WD had greater DM intake during the dry period than cows fed CON (CON = 13.4 ± 0.24, WD = 14.2 ± 0.24 kg/d), but rumination did not differ (CON = 522.2 ± 8.48, WD = 518.5 ± 8.69 min/d) between treatments. Cows fed WD tended to consume their feed faster (CON = 0.08 ± 0.001, WD = 0.09 ± 0.001 kg of DM/min) compared with CON cows. Regardless of treatment, cows sorted against the longest ration particles, with cows fed CON sorting more against these (CON = 81.1 ± 2.51%, WD = 94.9 ± 2.58%). Cows fed CON tended to sort more in favor of the medium particles than those fed WD (CON = 105.0 ± 0.36%, WD = 104.2 ± 0.37%). Postcalving DM intake and feeding behavior were not affected by dry diet treatment. Cows fed WD experienced a less rapid daily decline in rumen pH during the first 7 d after calving compared with those fed the CON diet. The results suggest that increasing the moisture content of high-straw dry cow diets, by adding water, may improve intake and reduce sorting against the longest ration particles, which may help promote consistency in targeted nutrients consumed during the dry period and greater rumen health in early lactation.

Effects of early postpartum sodium salicylate treatment on long-term milk, intake, and blood parameters of dairy cows.

Previous research has shown that cows who receive treatment with nonsteroidal anti-inflammatory drugs after calving may have increased milk yield beginning near peak lactation, resulting in greater 305-d milk production. It has not been demonstrated whether this response is associated with greater feed intake following the first 3 wk of lactation. Dry matter intake (DMI) and milk yield were measured daily for 56 cows over the first 120 d in milk. Cows in their second parity and greater were blocked by parity and alternately enrolled 12 to 36 h after calving into 1 of 2 treatments: either 3 daily drenches of water or 3 daily drenches of a similar volume of water containing 125 g of sodium salicylate (SAL) beginning 12 to 36 h after calving. Cows were housed in individual stalls to monitor DMI. Blood samples were collected before calving and on the last day of treatment, as well as at 7, 11, 14, 18, 21, 35, 49, 63, 77, 91, 105, and 120 d in milk. The SAL treatment did not affect estimated 305-d milk, fat, or protein yields (from monthly test days), daily milk yield or components, energy-corrected milk, fat-corrected milk, or DMI; however, an interaction between parity and treatment was observed for DMI, where second-parity SAL cows had decreased intake with no differences observed in older cows. This resulted in a parity by treatment interaction for the ratio of energy-corrected milk to DMI. Similarly, no main effects of treatment were observed for plasma glucose, ß-hydroxybutyrate (BHB), or fatty acid concentrations, but we noted interactions between treatment and parity for glucose, BHB, and insulin. Older cows had greater plasma glucose and insulin concentrations and decreased plasma BHB following SAL but no differences were observed in second parity animals. Alterations in glucose and insulin resulted in a tendency for a treatment by time interaction for the revised quantitative insulin sensitivity check index. Feeding behavior was also altered following SAL administration, resulting in fewer but longer meals, as well as a tendency for greater meal weight. A tendency for a treatment by week interaction for inter-meal interval was observed, as well as a parity by treatment interaction for meal weight. Despite the lack of a milk yield response, SAL had a prolonged programming effect on feeding behavior and blood variables over the first 120 DIM, with responses largely dependent on parity.

Short communication: Sodium salicylate negatively affects rumen fermentation in vitro and in situ.

Administration of sodium salicylate (SS) to cows in early lactation has a positive effect on whole-lactation milk production but a negative effect on metabolism in some cases. The objective of this trial was to determine whether SS directly affects rumen fermentation. Experiment 1 was designed to investigate the effects of direct inclusion of SS in a 24-h batch culture, and experiment 2 was designed to test the fermentative ability of rumen fluid from heifers who had received SS. In experiment 1, we combined strained and pooled rumen fluid from 3 heifers in a 2:1 ratio with McDougall's buffer, and added 150 mL of the inoculum to each flask (n = 5/treatment) with 2.5 g of fermentation substrate similar to a lactating cow ration, ground to 1 mm. We then added premixed treatments (1-mL volume) to achieve the desired final amount of SS (CON1 = 0 mg, LOW = 125 mg, MED = 250 mg, HI = 375 mg). In experiment 2, 6 heifers (n = 3/treatment) were drenched daily for 3 d, either with 62.5 g of SS dissolved in water (SAL) or an equal volume of water (CON2). Rumen fluid was collected from each heifer and was not pooled. After the fluid was mixed 2:1 with McDougall's buffer, 150 mL of inoculum was added to the fermentation flasks (n = 4/heifer) with 2.5 g of fermentation substrate. This experiment was performed the day before SS treatment began and repeated 1, 13, and 35 d after the end of the treatment period. We also performed an in situ experiment at each of these time points. In the first experiment, inclusion of SS resulted in a decrease in dry matter disappearance (DMD) over 24 h, as well as an increase in final pH. We detected no difference between treatments for gas production asymptotic volume, rate, or lag. In the second experiment, we detected a significant treatment × day interaction for DMD: we observed no difference between groups during a 24-h batch culture on the day following treatment, but SAL resulted in lower DMD on d 13 and d 35. We detected no treatment effect on the final pH of the batch culture or on any gas-production parameters. We observed a tendency for SAL to decrease the DMD rate in situ on the day after treatment. These results indicate that SS administration has a negative effect on rumen microorganisms.

Effects of bismuth subsalicylate and dietary sulfur level on fermentation by ruminal microbes in continuous culture.

In ruminants, excess dietary sulfur can be associated with a reduction in DM intake, poor feedlot performance and sulfur-associated polioencephalomalacia. Bismuth subsalicylate (BSS) has been shown to decrease hydrogen sulfide in vitro. The objective of this experiment was to evaluate effects of BSS inclusion (0 or 0.5% of diet DM) and dietary sulfur (0.21 or 0.42% of diet DM) on microbial fermentation in continuous culture. Treatments were arranged in a 2 × 2 factorial design. Eight dual-flow continuous culture fermenters were used during 2 consecutive 10-d periods consisting of 7 d for stabilization followed by 3 d of sampling. A pelleted feedlot diet containing 39% dry rolled corn, 32% earlage, 21% wet distillers grains, 3.2% corn silage, 1.5% soybean meal, 0.6% urea and 2.7% mineral premix (DM basis) was provided as substrate for microbes at a rate of 75 g of DM × fermenter-1 × d-1. Effluents from sampling days were composited by fermenter within period, resulting in 4 replicates/treatment. Bismuth subsalicylate inclusion decreased (P < 0.01) true OM digestion, while no effects were observed for NDF and ADF digestion. Total VFA concentrations, molar proportions of acetic, propionic, and branched-chained VFA decreased (P < 0.01) with BSS addition. The ratio of acetic to propionic acid and the molar proportion of butyric acid increased (P < 0.01) with BSS addition. In regard to nitrogen metabolism, BSS increased NH3-N concentration, NH3-N and dietary-N flows (P < 0.01), and decreased non-NH3-N flow, microbial-N flow, CP degradation, and efficiency of microbial protein synthesis (P < 0.01). Inclusion of BSS increased mean, minimum, and maximum fermentation pH (P < 0.01). Amount of dietary sulfur and BSS inclusion influenced flows of amino acids and fatty acids from fermenters. Influences on fatty acid biohydrogenation and amino acid flows demonstrated an overall suppression of microbial fermentation. Results from this experiment indicate that BSS inclusion at 0.5% of diet DM has detrimental effects on in vitro rumen fermentation in continuous culture.

Effects of dietary roughage and sulfur in diets containing corn dried distillers grains with solubles on hydrogen sulfide production and fermentation by rumen microbes in vitro.

Dried distillers grains with solubles (DDGS) have been used in production animal diets; however, overuse of DDGS can cause toxic concentrations of ruminal hydrogen sulfide gas (HS), resulting in polioencephalomalacia, a deleterious brain disease. Because HS gas requires an acidic rumen environment and diet can influence ruminal pH, it has been postulated that dietary manipulation could help mitigate HS production. The objective of this study was to assess the effect of dietary roughage and sulfur concentrations on HS production and rumen fermentation. In Exp. 1, 7 dual-flow continuous culture fermenters were used in 4 consecutive 9-d periods consisting of 6 d of adaptation followed by 3 d of sampling. At the conclusion of each 9-d continuous culture period, adapted rumen fluid was used for inoculation of 24-h batch culture incubations for Exp. 2. For both experiments, 6 dietary treatments were formulated to consist of 0.3%, 0.4%, or 0.5% dietary sulfur (LS, MS, and HS, respectively) and 3% or 9% dietary roughage (LR and MR, respectively), using grass hay as the roughage source. A corn-based diet without DDGS was used as a control diet. Headspace gas was sampled to determine HS production and concentration. In Exp. 1, greater dietary roughage had no effect ( = 0.14) on HS production but did create a less acidic environment because of an increase ( < 0.01) in the in vitro pH. In Exp. 2, an increase in dietary sulfur caused an increase ( = 0.04) in ruminal HS production, but there was no direct effect ( = 0.25) of dietary roughage on HS production. Greater dietary roughage resulted in a less ( = 0.01) acidic final batch culture pH but a lower ( < 0.01) total VFA concentration. Further investigation is needed to determine a more effective way to mitigate ruminal HS production using dietary manipulation, which could include greater inclusion of dietary roughage or the use of different roughage sources.

Effect of dietary sugar concentration and sunflower seed supplementation on lactation performance, ruminal fermentation, milk fatty acid profile, and blood metabolites of dairy cows.

Previous research has shown that both sunflower seed (SF) and sucrose (SC) supplementation can result in variation in milk fat concentration and composition, possibly due to altered fermentation patterns and biohydrogenation of fatty acids in the rumen. The objective of this study was to determine the effects of different sugar concentrations with or without SF supplementation on lactation performance, ruminal fermentation, and milk fatty acid profile in lactating dairy cows. Eight multiparous Holstein dairy cows (body weight=620±15kg, 60±10 d in milk, mean ± standard deviation) were randomly assigned to treatments in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of treatments. Each 21-d period consisted of a 14-d diet adaptation period and 7-d collection period. Dairy cows were fed 1 of the following 4 diets: (1) no additional SC without SF supplementation (NSC-SF), (2) no additional SC with SF supplementation (NSC+SF), (3) SC without SF supplementation (SC-SF), and (4) SC with SF supplementation (SC+SF). The diets contained the same amount of forages (corn silage and alfalfa hay). Four isonitrogenous and isoenergetic diets were formulated by replacing corn grain with SC and SF and balanced using change in proportions of canola meal and sugar beet pulp. No interaction was detected between SC and SF supplementation with respect to dry matter intake, milk yield, and composition. A tendency was found for an interaction between inclusion of SC and SF on energy-corrected milk with the highest amount in the SC-SF diet. Ruminal pH and the molar proportion of acetate were affected by SC inclusion, with an increase related to the SC-SF diet. Diets containing SF decreased the concentrations of short-chain fatty acids (4:0 to 10:0) and medium-chain fatty acids (12:0 to 16:0) in milk fat. The addition of SC tended to decrease the concentration of total trans-18:1. These data provide evidence that exchanging SC for corn at 4% of dietary dry matter influenced milk fat content and rumen pH, resulting in a tendency for decreased concentration of trans-18:1 in milk fat. Sucrose alone did not alter the milk fatty acid profile when cows were fed a combination of unsaturated fat and sugar, although several significant interactions between sugar and unsaturated fat were observed.

Hot topic: Early postpartum treatment of commercial dairy cows with nonsteroidal antiinflammatory drugs increases whole-lactation milk yield.

Previous research has shown that postpartum administration of the nonsteroidal antiinflammatory drug (NSAID) sodium salicylate can increase 305-d milk yield in older dairy cattle (parity 3 and greater). However, in this prior work, sodium salicylate was delivered to cows via the drinking water, a method that does not align well with current grouping strategies on commercial dairy farms. The objective of the current study was to replicate these results on a commercial dairy farm with a simplified treatment protocol and to compare sodium salicylate with another NSAID, meloxicam. Dairy cattle in their second lactation and greater (n=51/treatment) were alternately assigned to 1 of 3 treatments at parturition, with treatments lasting for 3d. Experimental treatments began 12 to 36 h after parturition and were (1) 1 placebo bolus on the first day and 3 consecutive daily drenches of sodium salicylate (125 g/cow per day; SAL); (2) 1 bolus of meloxicam (675 mg/cow) and 3 drenches of an equal volume of water (MEL); or (3) 1 placebo bolus and 3 drenches of water (CON). Blood samples were collected on the first day of treatment, immediately following the last day of treatment, and 7d after the last day of treatment; plasma was analyzed for glucose, ß-hydroxybutyrate (BHB), free fatty acids, haptoglobin, and paraoxonase. Milk production, body condition score, reproductive status, and retention in the herd were monitored for 365 d posttreatment, and effects of treatment, parity, days in milk, and interactions were evaluated in mixed effects models. Significance was declared at P<0.05. Whole-lactation milk and protein yields were greater in NSAID-treated cows, although 305-d fat production was not affected. There was a significant interaction of treatment and parity for plasma glucose concentration; MEL increased plasma glucose concentrations compared with CON and SAL in older cows. Sodium salicylate decreased plasma BHB concentration compared with MEL at 7d posttreatment, although no difference was detected immediately following treatment. Haptoglobin concentrations were elevated in SAL cows compared with CON. There was a tendency for CON cows to be removed from the herd more quickly than MEL cows (42 vs. 26% at 365 d posttreatment). Body condition score, concentrations of plasma free fatty acids and paraoxonase, and time to pregnancy were not affected by treatment. These results indicate that NSAID administration in postpartum cows has the potential to be a viable way to improve productivity and potentially longevity in commercial dairies, although further research is necessary to optimize recommendations for producers.

Invited review: Inflammation during the transition to lactation: New adventures with an old flame.

For dairy cattle, the first several weeks of lactation represent the highest-risk period in their lives after their own neonatal period. Although more than 50% of cows during this period are estimated to suffer from at least one subclinical disorder, the complicated admixture of normal adaptations to lactation, infectious challenges, and metabolic disorders has made it difficult to determine which physiological processes are adaptive and which are pathological during this time. Subacute inflammation, a condition that has been well documented in obesity, has been a subject of great interest among dairy cattle physiologists in the past decade. Many studies have now clearly shown that essentially all cows experience some degree of systemic inflammation in the several days after parturition. The magnitude and likely persistence of the inflammatory state varies widely among cows, and several studies have linked the degree of postpartum inflammation to increased disease risk and decreased whole-lactation milk production. In addition to these associations, enhancing postpartum inflammation with repeated subacute administration of cytokines has impaired productivity and markers of health, whereas targeted use of nonsteroidal anti-inflammatory drugs during this window of time has enhanced whole-lactation productivity in several studies. Despite these findings, many questions remain about postpartum inflammation, including which organs are key initiators of this state and what signaling molecules are responsible for systemic and tissue-specific inflammatory states. Continued in vivo work should help clarify the degree to which mild postpartum inflammation is adaptive and whether the targeted use of anti-inflammatory drugs or nutrients can improve the health and productivity of dairy cows.

Impact of oral meloxicam on circulating physiological biomarkers of stress and inflammation in beef steers after long-distance transportation.

Transportation stress can result in significant economic losses to producers due to decreased animal productivity and increased medication costs associated with sickness such as bovine respiratory disease (BRD). Meloxicam (MEL) provides pain relief and anti-inflammatory effects in cattle for several days after a single oral treatment. Our hypothesis was that MEL administration before shipping would reduce the impact of long-distance transportation on circulating physiological biomarkers of stress and inflammation in beef steers. Ninety-seven beef steers were blood sampled for baseline biomarker determination and then randomly assigned to receive either 1 mg/kg MEL (n = 49) or a placebo (CONT; n = 48) per os before a 1,316-km transportation event lasting approximately 16 h. Calves were then blood sampled on arrival and 5 d later. Changes in the hemogram, circulating plasma proteins, total carbon dioxide (TCO2), fibrinogen, substance P (SP), cortisol, haptoglobin (Hp)-matrix metalloproteinase-9 (MMP-9) complexes, and tumor necrosis factor α (TNFα) between treatments over time were compared using a mixed effects model with statistical significance designated as P < 0.05. Analysis of covariance was conducted to assess the relationship between circulating MEL concentrations and biomarker changes over time. An increase in neutrophil, platelet, monocyte, white blood cell, and red blood cell counts occurred after transportation (P < 0.0001) and a decrease in lymphocyte count were observed (P < 0.0001). Meloxicam treatment reduced the stress-induced neutrophilia (P = 0.0072) and circulating monocyte count (P = 0.013) on arrival. Mean corpuscle hemoglobin (P = 0.05), mean corpuscle volume (P = 0.05), and lymphocyte count (P = 0.05) were also greater in the CONT calves compared with MEL calves after transportation. Furthermore, Hp-MMP-9 complexes, TCO2, TNFα, plasma proteins, and SP increased and cortisol decreased after shipping (P < 0.01). Meloxicam treatment tended to reduce serum cortisol concentrations (P = 0.08) and there was evidence of a time × treatment interaction (P = 0.04). An inverse relationship between plasma MEL concentrations and circulation cortisol concentrations (P = 0.002) and neutrophil (P = 0.04) and basophil counts (P = 0.03) was also observed. The results suggest that MEL administration may reduce the impact of long-distance transportation on circulating physiological biomarkers of stress and inflammation in beef calves.

Analysis of rumen microbial populations in lactating dairy cattle fed diets varying in carbohydrate profiles and Saccharomyces cerevisiae fermentation product.

The rumen microbial ecosystem is a critical factor that links diets to bovine physiology and productivity; however, information about dietary effects on microbial populations has generally been limited to small numbers of samples and qualitative assessment. To assess whether consistent shifts in microbial populations occur in response to common dietary manipulations in dairy cattle, samples of rumen contents were collected from 2 studies for analysis by quantitative real-time PCR (qPCR). In one study, lactating Holstein cows (n=8) were fed diets in which a nonforage fiber source replaced an increasing proportion of forages and concentrates in a 4×4 Latin square design, and samples of ruminal digesta were collected at 9-h intervals over 3 d at the end of each period. In the second study, lactating Holstein cows (n=15) were fed diets with or without the inclusion of a Saccharomyces cerevisiae fermentation product (SCFP) in a crossover design. In this study, rumen liquid and solid samples were collected during total rumen evacuations before and after feeding in a 42-h period. In total, 146 samples of ruminal digesta were used for microbial DNA isolation and analysis by qPCR. Validated primer sets were used to quantify total bacterial and anaerobic fungal populations as well as 12 well-studied bacterial taxa. The relative abundance of the target populations was similar to those previously reported. No significant treatment effects were observed for any target population. A significant interaction of treatment and dry matter intake was observed, however, for the abundance of Eubacterium ruminantium. Increasing dry matter intake was associated with a quadratic decrease in E. ruminantium populations in control animals but with a quadratic increase in E.ruminantium populations in cows fed SCFP. Analysis of sample time effects revealed that Fibrobacter succinogenes and fungal populations were more abundant postfeeding, whereas Ruminococcus albus tended to be more abundant prefeeding. Seven of the target taxa were more abundant in either the liquid or solid fractions of ruminal digesta. By accounting for the total mass of liquid and solid fractions in the rumen and the relative abundance of total bacteria in each fraction, it was estimated that 92% of total bacteria were found in the solid digesta fraction.

A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.

Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.

Repression of CDK1 and other genes with CDE and CHR promoter elements during DNA damage-induced G(2)/M arrest in human cells.

Entry into mitosis is controlled by the cyclin-dependent kinase CDK1 and can be delayed in response to DNA damage. In some systems, such G(2)/M arrest has been shown to reflect the stabilization of inhibitory phosphorylation sites on CDK1. In human cells, full G(2) arrest appears to involve additional mechanisms. We describe here the prolonged (>6 day) downregulation of CDK1 protein and mRNA levels following DNA damage in human cells. This silencing of gene expression is observed in primary human fibroblasts and in two cell lines with functional p53 but not in HeLa cells, where p53 is inactive. Silencing is accompanied by the accumulation of cells in G(2), when CDK1 expression is normally maximal. The response is impaired by mutations in cis-acting elements (CDE and CHR) in the CDK1 promoter, indicating that silencing occurs at the transcriptional level. These elements have previously been implicated in the repression of transcription during G(1) that is normally lifted as cells progress into S and G(2). Interestingly, we find that other genes, including those for CDC25C, cyclin A2, cyclin B1, CENP-A, and topoisomerase IIalpha, that are normally expressed preferentially in G(2) and whose promoter regions include putative CDE and CHR elements are also downregulated in response to DNA damage. These data, together with those of other groups, support the existence of a p53-dependent, DNA damage-activated pathway leading to CHR- and CDE-mediated transcriptional repression of various G(2)-specific genes. This pathway may be required for sustained periods of G(2) arrest following DNA damage.

Simultaneous antegrade and retrograde reperfusion after cardioplegic arrest for coronary artery bypass.

Retrograde coronary sinus reperfusion with warm blood during proximal anastomoses permits completion of myocardial revascularization under a single cross-clamp application. Reperfusion with both antegrade (via arterial and vein grafts) and retrograde (via coronary sinus catheter) warm blood has raised concerns about maldistribution of perfusate or overpressurization of capillary beds. This prospective, randomized design compares postcardioplegic myocardial recovery among patients receiving retrograde reperfusion only and patients receiving simultaneous antegrade/retrograde reperfusion. Twenty-four patients were selected among all presenting as outpatients for elective coronary artery bypass (CAB). Each patient underwent CAB with cardioplegic arrest and single cross-clamp technique. During proximal anastomoses the heart was reperfused with warm blood from the cardiopulmonary bypass (CPB) circuit. Twelve received retrograde reperfusion only, and 12 received simultaneous antegrade/retrograde reperfusion via an internal mammary artery (IMA) graft, all vein grafts, and the coronary sinus catheter. Vein graft perfusion was interrupted in each vein as the proximal anastomosis was performed. Myocardial recovery time (interval from initiating reperfusion until electrical and mechanical activity), cardioversion incidence, requirement for inotropic support, and Swan-Ganz hemodynamic parameters measured immediately 6 and 24 hours postoperatively were compared between groups. There were no differences between groups in age, ventricular function, number of grafts, or CPB time. Also, there were no differences in cardioversion, inotropic need, or postoperative hemodynamic performance. Myocardial recovery time was reduced in patients receiving simultaneous antegrade/retrograde reperfusion (13.9+/-7.0 vs 2.6+/-2.1 minutes). Simultaneous reperfusion of warm blood antegrade and retrograde is not deleterious to the myocardium. More rapid recovery of myocardial function may represent a shorter period of warm ischemia but does not appear to translate to improved postoperative myocardial performance.

Systemic cytokine response to hepatic resections under total vascular exclusion.

OBJECTIVE: To investigate the systemic cytokine response to major liver surgery as the basis for assessing potential new treatments. DESIGN: Open prospective study. SETTING: University hospital, UK. SUBJECTS: Thirteen patients undergoing elective hepatic resections that involved total vascular exclusion of the liver. INTERVENTIONS: Blood samples were taken preoperatively, during the operation, and during the first four postoperative days. Concentrations of endotoxin, interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNFalpha), interleukin-1 (IL-1), and interleukin-6 (IL-6) were measured. RESULTS: Endotoxin concentrations were raised in 3/13 patients before operation and in 6 patients during the postoperative period. TNFalpha concentrations were undetectable. IFN-gamma and IL-1 responses followed a low and inconclusive pattern. IL-6 was significantly increased from 6 hours after operation to the third postoperative day, peaking at 699 (+/-277) pg/ml at 24 hours (p < 0.01). The two patients who died had the highest postoperative concentrations of IL-6. CONCLUSIONS: There is a pronounced systemic response to hepatic resection under total vascular exclusion that is reflected by the increase in IL-6 concentration and correlates with the operative blood loss and postoperative outcome. This might be used as an indicator of the response to specific treatments in this type of surgery. Treatments that minimise the IL-6 response to major hepatic resection may be of value.