Effect of nonsteroidal anti-inflammatory drugs on the inflammatory response of bovine endometrial epithelial cells in vitro.

Chronic postpartum uterine infection detrimentally affects subsequent fertility. Nonsteroidal anti-inflammatory drugs (NSAID) are used to alleviate pain and treat inflammatory conditions in transition dairy cows with varying success. To screen the efficacy of NSAID in the absence of animal experiments, we have established an in vitro model to study uterine inflammation. Inflammation was induced in cultured bovine endometrial epithelial cells by challenging cells with an inflammation cocktail: lipopolysaccharide and proinflammatory cytokines, interleukin-1ß (IL1ß) and tumor necrosis factor α (TNFα). Release of the inflammation markers, serum amyloid A (SAA) and α-1-acid glycoprotein (αAGP), was measured by ELISA. Concentration of these markers was used to indicate the effectiveness in dampening inflammation of 5 NSAID: meloxicam, flunixin meglumine, aspirin, ketoprofen, and tolfenamic acid. Three NSAID, meloxicam, flunixin meglumine, and tolfenamic acid, were successful at dampening the release of SAA and αAGP into cell-culture supernatant, and the corresponding treated cells were selected for down-stream mRNA expression analysis. Expression of 192 genes involved in regulation of inflammatory pathways were investigated using Nanostring. Of the genes investigated, 81 were above the mRNA expression-analysis threshold criteria and were included in expression analysis. All SAA genes investigated (SAA2, SAA3, M-SAA3.2) were upregulated in response to the inflammation cocktail, relative to mRNA expression in control cells; however, AGP mRNA expression was below the expression analysis threshold and was, therefore, excluded from analysis. Treatment with NSAID downregulated genes involved in regulating chemokine signaling (e.g., CXCL2, CXCR4, CXCL5, and CXCL16) and genes that regulate the eicosanoid pathway (e.g., LTA4H, PTGS2, PLA2G4A, and PTGDS). Of the 5 NSAID investigated, meloxicam, flunixin meglumine, and tolfenamic acid are recommended for further investigation into treatment of postpartum uterine inflammation. The results from this study confirm the immunomodulatory properties of the endometrial epithelium in response to inflammatory stimuli and suggest that NSAID may be beneficial in alleviating uterine inflammation.

Adaptive immune response ranking is associated with reproductive phenotypes in grazing dairy cows divergent in genetic merit for fertility traits.

A strong adaptive immune response has been reported to have positive effects on fertility; therefore, we investigated antibody- and cell-mediated adaptive immune responses (AMIR and CMIR, respectively) and their associations with reproductive phenotypes using a population of animals that differed in their estimated genetic merit for fertility traits (fertility breeding value; FertBV). Holstein-Friesian heifers (n = 528) grazed on pasture in 4 herds based on age. These herds included 277 heifers of positive (POS) FertBV and 251 of negative (NEG) FertBV. The adaptive immune response (IR) was evaluated before puberty at 7.5 mo of age and used to rank animals as high, average, or low for AMIR, CMIR, and overall IR (combined CMIR and AMIR). The animals were studied from 12 wk of age through to the end of their second lactation to measure growth, puberty, and timing and success of fertility phenotypes, including those related to ovulation and pregnancy. Initial analysis indicated no difference in fertility outcomes between cows ranked as high or average for AMIR (n = 55, n = 407, respectively), CMIR (n = 87, n = 354, respectively), and IR (n = 29, n = 470, respectively), so these groups were pooled as HiAv-IR. Proportions of heifers of POS FertBV were similar within HiAv and low categories across AMIR (0.52 and 0.58, respectively), CMIR (0.51 and 0.59, respectively), and IR (0.53 and 0.48, respectively). Heifers with HiAv-IR had a greater average daily weight gain from 13 to 52 wk of age (661 g, 95% confidence interval 652, 669 vs. 619 g, 95% confidence interval 591, 647) and tended to be younger at puberty (371 d, 95% confidence interval 366, 377 vs. 385 d, 95% confidence interval 369, 401) than low-IR heifers. Low-CMIR cows of a NEG FertBV had a >40 d longer calving to first ovulation interval during their first lactation compared with HiAv-CMIR NEG FertBV cows. Low-CMIR cows also had decreased pregnancy rates at both 3 wk (25% ± 7% vs. 42% ± 3%; least squares means ± standard error) and 6 wk (33% ± 7% vs. 54% ± 3%; least squares means ± standard error) into the seasonal breeding period during their first lactation, compared with HiAv-CMIR cows. In summary, although the number of POS and NEG FertBV cows was similar in each immune group; interaction effects between FertBV and immune ranking on reproductive phenotypes are evident when cows were ranked by the overall IR. There were also associations between dairy cows' CMIR ranking and ability to return to estrus and become pregnant early in the breeding period, which will have benefits in seasonal breeding systems.

Temporal profiles describing markers of inflammation and metabolism during the transition period of pasture-based, seasonal-calving dairy cows.

The physiology of the dairy cow while transitioning from pregnancy to lactation is complex, with multifactorial processes studied extensively for the role they play in manifestation of disease along with associated economic losses and compromised animal welfare. Manuscripts outlining associations among nutrition, production, physiology, and genetics variables and transition cow disorders are common in literature, with blood analytes that are central to energy metabolism (e.g., nonesterified fatty acids; NEFA, ß-hydroxybutyrate; BHB) often reported. Immunity and inflammation have increasingly been explored in the pathogenesis and persistence of disorders, with cytokines and acute phase proteins well documented. However, most of these studies have involved cows fed total mixed rations, which may not always reflect profiles of blood analytes and other physiological indicators of transition cow health in grazing cows consuming fresh pasture. Considering the comparatively lesser characterization of these analytes and markers in pasture-based, seasonal-calving dairy cows, we compiled a database consisting of 2,610 cow lactations that span 20 yr of transition cow research in New Zealand. Using this database, analyte profiles from approximately 28 d precalving to 35 d postcalving were identified in dairy cows with a range of genetics, milk production potentials, and pasture-based farm management systems. These profiles characterize changes in energy reserves and metabolism (NEFA, BHB, glucose, insulin, growth hormone, insulin-like growth factor-1, leptin, body condition score, body weight), liver function (globulin, aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, bilirubin, cholesterol, liver triacylglycerides), protein metabolism (albumin, total protein, albumin:globulin ratio, creatinine, urea, creatine kinase), mineral balance (calcium, magnesium, phosphate, potassium, sodium, chloride, bicarbonate), inflammation (IL-1ß, IL-6, haptoglobin, reactive oxygen species, total antioxidant capacity), and uterine health (polymorphonuclear cells, macrophage cells, vaginal discharge score). Temporal changes are generally consistent with previously characterized homeorhetic changes experienced by the dairy cow during the transition from pregnancy to lactation in both pastoral and housed systems. Some of the profiles had not previously been presented for pastoral systems, or in some cases, presented for either system. Our results indicate that moderate-yielding dairy cows undergo similar homeorhetic changes to high-yielding housed cows; however, differences in diet composition result in greater BHB concentrations than expected, based on their milk production and NEFA concentrations. In addition, most cows were able to transition to a state of higher energy requirement following calving, albeit with an increased metabolic challenge in the liver, and only a small percentage of cows were classified with severe hepatic lipidosis or severe hyperketonemia. Increases in metabolic function of the liver were accompanied by changes in indicators of the immune system and changes in mineral balance that, combined, probably reflect the innate response to the transition from gestation to lactation.

Effect of dose rate and timing of administration of pegbovigrastim on white blood cell responses in grazing dairy cows.

Transition dairy cows experience a decline in immune function that increases the risk of peripartum disease. One strategy to improve peripartum immune function involves the use of a commercially available cytokine: bovine granulocyte-colony stimulating factor, with the addition of polyethylene glycol to increase duration of effectiveness. Treatment with Imrestor (15 mg pegbovigrastim; Elanco) one week before expected calving date (d -7) and again on the day of calving (d 0) was previously reported to increase the neutrophil number and improve neutrophil function; as a result, the incidence of clinical mastitis was reduced. We conducted 2 experiments over consecutive years to investigate the effect of a lower dose rate (half or quarter dose rate) of Imrestor in grazing dairy cattle and reduced administration frequency: one dose instead of the recommended 2. White blood cell counts were measured to determine changes in relative cell populations in response to treatment. Neutrophil function was assessed by measuring myeloperoxidase activity. Imrestor treatment increased the numbers of neutrophils, band cells, lymphocytes, and monocytes until 14 d postcalving in a dose-dependent manner; it also increased neutrophil myeloperoxidase activity. One dose of Imrestor increased white blood cell counts and myeloperoxidase activity, but the timing, degree, and duration of the response were different relative to the recommended 2 doses and were also dependent upon when Imrestor treatment was given. One dose at d -7 relative to expected calving date did not have a lasting effect postcalving, whereas one dose only on d 0 caused a delayed effect relative to cows that received 2 doses. There was no effect of Imrestor on milk yield or on blood indicators of transition cow health. A lower dose rate of Imrestor or a single dose of Imrestor on the day of calving may be sufficient to improve neutrophil function during the early postpartum in grazing dairy cows. Large-scale field studies are required to determine whether the smaller response from lower dose rates or the timing of the immunological response to drug delivery affect animal health in early lactation.

Hepatic one-carbon metabolism enzyme activities and intermediate metabolites are altered by prepartum body condition score and plane of nutrition in grazing Holstein dairy cows.

Precalving feeding level and body condition score (BCS) alter postcalving energy balance and oxidant status of dairy cows. We hypothesized that the reported benefits of a controlled restriction precalving depend on precalving BCS. The objective was to identify alterations in activity and intermediates of the hepatic one-carbon metabolism, transsulfuration, and tricarboxylic acid pathways. Twenty-eight pregnant and nonlactating grazing dairy cows of mixed age and breed (Friesian, Friesian × Jersey) were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial design: 2 prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); 10-point scale], by managing cows in late lactation to achieve the 2 groups at dry-off, and 2 levels of energy intake during the 3 wk preceding calving (75 or 125% of estimated requirements), obtained via allowance (m2/cow) of fresh pasture composed of mostly perennial ryegrass and white cover. Average (± standard deviation) age was 6 ± 2, 6 ± 3, 5 ± 1, and 7 ± 3 yr for BCS4 fed 75 and 125%, and BCS5 fed 75 and 125%, respectively. Breed distribution (average ± standard deviation) for the 4 groups was 79 ± 21, 92 ± 11, 87 ± 31, and 74 ± 23% Friesian, and 17 ± 20, 8 ± 11, 13 ± 31, and 25 ± 23% Jersey. Liver tissue was collected by biopsy at -7, 7, and 28 d relative to calving. Tissue was used for 14C radio-labeling assays to measure betaine-homocysteine S-methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine-ß-synthase (CBS) activity. Liver metabolomics was undertaken using a targeted liquid chromatography with tandem mass spectrometry-based profiling approach. After initial liquid chromatography separation, mass spectra were acquired under both positive and negative ionization, whereas multiple reaction monitoring was used to measure target compound signal response (peak area count). Enzyme activity and metabolite peak area count were normalized with the homogenate protein concentration. Repeated measures analysis of variance via PROC MIXED in SAS (SAS Institute Inc., Cary, NC), with BCS, feeding, and time as fixed effects, and cow as random effect was used. All enzyme activities were affected by time, with betaine-homocysteine S-methyltransferase activity peaking at 7 d, whereas CBS and MTR activity decreased postpartum. Overall, thin cows had greater MTR activity, whereas cows fed 125% requirements had greater CBS activity. An interaction was detected between BCS and feeding for CBS activity, as thin cows fed 125% of requirements had greater overall activity. Compared with liver from BCS4 cows, BCS5 cows had overall greater betaine, glycine, butyrobetaine/acetylcholine, serine, and taurine concentrations. The same metabolites, plus choline and N-N-dimethylglycine, were overall greater in liver of cows fed 75% compared with those fed 125% of requirements. An interaction of BCS and feeding level was detected for the aforementioned metabolites plus methionine, cystathionine, cysteinesulfinate, and hypotaurine, due to greater overall concentrations in BCS5 cows fed 75% of requirements compared with other groups. Overall, differences in hepatic enzyme activity and intermediate metabolites suggest that both BCS and feeding level can alter the internal antioxidant system (e.g., glutathione and taurine) throughout the periparturient period. Further studies are needed to better understand potential mechanisms involved.

Feeding synthetic zeolite to transition dairy cows alters neutrophil gene expression.

Synthetic zeolites are used to control the availability of dietary minerals (e.g., Ca, Mg, and P) in dairy cows. Due to calcium demand increasing with lactation onset, most cows become hypocalcemic immediately postpartum, which likely contributes to poorer immune function because calcium is important for immune cell signaling. To overcome postpartum hypocalcemia, we fed transition cows synthetic zeolite A (sodium aluminosilicate) precalving and hypothesized that it would alter calcium and thus neutrophil function during the transition period. Multiparous Holstein-Friesian cows in late gestation were randomly allocated to an untreated control group (n = 10) or a treatment group in which each cow received 500 g of zeolite A daily (n = 10) for 14 d prior to the expected calving date (actual duration = 17 ± 3 d prepartum). The cows grazed pasture, and each was supplemented with 2 kg/d of maize silage (dry matter basis), with or without zeolite, until calving. Blood samples for neutrophil isolation and analysis of plasma indicators of mineral status, energy status, liver function, and inflammation were collected pretreatment (covariate; d -19); on d -14 and -7 precalving; on the day of calving (d 0); and on d 1, 4, 7, and 28 postcalving. Neutrophils were isolated and gene expression was analyzed using microfluidic gene expression arrays. Neutrophil respiratory burst was assessed using stimulation with phorbol 12-myristate 13-acetate and flow cytometry. Plasma calcium and phosphorus revealed a treatment by time interaction; cows offered zeolite had greater plasma calcium concentrations at d 0, 1, and 4 postcalving and plasma phosphorus concentrations were lower in zeolite-treated cows during the precalving period until d 1 postcalving compared with control animals. Zeolite treatment downregulated neutrophil gene expression of CXCR4 and S100A8 and tended to lower gene expression for other immune mediators (CXCR1, IFNG, S100A12, and S100A9) compared with the control. Zeolite treatment did not affect neutrophil respiratory burst or expression of the other genes investigated. Plasma concentrations of cytokine IL-6 were reduced with zeolite treatment, which was most evident immediately postcalving (d 0, 1, and 7). Overall, feeding zeolite precalving had few effects on neutrophil gene expression and function; however, the lower gene expression of neutrophil inflammatory mediators may be due to altered availability of dietary minerals prepartum and indicates that zeolite A may control inflammation during the transition period.

Transcriptomic analysis of circulating neutrophils in metabolically stressed peripartal grazing dairy cows.

The high metabolic demand during the transition into lactation places cows at greater risk of metabolic and infectious disease than at any other time in their lactation cycle. Additionally, a change occurs in the innate immune response during this period, which contributes to increased risk of disease. In the current study, we compared the transcriptomes of neutrophils from dairy cows divergent in their metabolic health post-calving. Cows (n = 5 per risk group) were selected from a parent experiment (n = 45 cows). Those with high or low concentrations of plasma nonesterified fatty acids, plasma ß-hydroxybutyrate, and liver triacylglycerol in both wk 1 and 2 were deemed to be at "high risk" (HR) or "low risk" (LR) of metabolic dysfunction, respectively. Circulating neutrophils were isolated at 3 time points during the transition period (d 0 and wk 1 and 4 post-calving), and gene expression was analyzed using RNA sequencing. Differential gene expression between the risk groups was determined using edgeR (http://bioconductor.org), and pathway analysis was conducted using Ingenuity Pathway Analysis (Ingenuity Systems, Qiagen, Valencia, CA). Statistical analysis indicated no interaction between risk and week. Therefore, the overall effect of risk was analyzed across all time points. In total, 3,500 genes were differentially expressed between the HR and LR cows (false discovery rate < 0.05). Of these, 2,897 genes were identified by Ingenuity Pathway Analysis and used for pathway analysis. Of the relevant pathways identified, neutrophils isolated from HR cows showed downregulation of genes involved in the recruitment of granulocytes, interferon signaling, and apoptosis, and upregulation of genes involved in cell survival. The results indicate that metabolically stressed cows had reduced neutrophil function during the peripartum period, highlighting a potential relationship between subclinical metabolic disease and innate immune function that suggests that metabolic health negatively affects the innate immune system and may contribute to the state of immunosuppression during the peripartum period. In this way, the metabolic stress among the HR cows may reduce their ability to combat infection during the transition period.

Fertility and the transition dairy cow.

The transition from pregnancy to lactation (i.e. the transition period) is a time of significant metabolic challenge, with a several-fold increase in a cow's requirement for energy, protein and minerals within days of calving. A successful transition involves the initiation and coordination of changes in multiple tissues that facilitate the provision of these nutrients to the cow and, more specifically, to the mammary gland, often at the considerable expense of other tissues. Failure to coordinate the necessary changes effectively results in transition period maladaptation, which can broadly be grouped into three categories: (1) negative energy balance and metabolic diseases associated with energy metabolism; (2) immune dysfunction and inflammation; and (3) metabolic diseases associated with mineral deficiency. Because reinitiation of ovarian activity, follicle recruitment, ovulation, fertilisation and, potentially, even maternal recognition of pregnancy and implantation occur against the backdrop of this metabolic and immunological disturbance in early lactation, the role of nutrition in ensuring a smooth transition between the pregnant and lactating state is important. In this paper we integrate recent research findings with previous knowledge of the interaction between transition cow metabolism and nutrition and reproductive outcomes, and offer new insights into key elements of successful cow management to avoid transition 'maladaptation' and improve pregnancy rates.

Technical note: Evaluation of endogenous control gene expression in bovine neutrophils by reverse-transcription quantitative PCR using microfluidics gene expression arrays.

Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.

Strategies to gain body condition score in pasture-based dairy cows during late lactation and the far-off nonlactating period and their interaction with close-up dry matter intake.

In pasture-based systems, cows are generally thinner at the end of lactation than cows fed total mixed rations and, as a result, over-feeding of metabolizable energy (ME) during the far-off nonlactating period is a standard management policy to achieve optimum calving body condition score (BCS). An alternative would be to manage cows to gain BCS through late lactation, such that cows ended lactation close to optimum calving BCS and maintenance of BCS through to calving. We sought to quantify the effect of moderate or excessive ME intakes during the far-off nonlactating period in cows that had been managed to gain or maintain BCS through late lactation and whether the far-off management strategy interacted with close-up level of feeding. Effects on milk production and circulating indicators of energy balance and metabolic health in early lactation were evaluated. A herd of 150 cows was randomly assigned to 1 of 2 feeding levels in late lactation to achieve a low and high BCS at the time of dry-off (approximately 4.25 and 5.0 on a 10-point scale). Following dry-off, both herds were managed to achieve a BCS of 5.0 one month before calving; this involved controlled feeding (i.e., maintenance) and over-feeding of ME during the far-off dry period. Within each far-off feeding-level treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement (i.e., 25 cows/treatment). Body weight and BCS were measured weekly before and after calving, and milk production was measured weekly until wk 7 postcalving. Blood samples were collected weekly for 4 wk pre-calving and 5 wk postcalving, and on d 0, 1, 2, 3, and 4 relative to calving, and analyzed for indicators of energy balance (e.g., blood fatty acids, ß-hydroxybutyrate), calcium status, and inflammatory state. No interaction was observed between far-off and close-up feeding levels. Over-feeding of ME to low BCS cows during the far-off nonlactating period reduced blood fatty acid and ß-hydroxybutyrate concentrations in early lactation, and increased blood albumin to globulin ratio compared with cows that were dried off close to recommended calving BCS and control-fed during the far-off dry period. Cows consuming 65% of their ME requirements during the close-up period had lower fatty acids and ß-hydroxybutyrate in early lactation, but produced less milk, particularly during the first 21 d of lactation, had more than 3-fold greater concentration of haptoglobin immediately postcalving, and had a lower blood cholesterol concentration and albumin to globulin ratio, when compared with cows offered 90 or 120% of their ME requirements. Collectively, these measurements indicate that a severe restriction (<70% of ME requirements) during the close-up nonlactating period increases the risk of disease in early lactation and reduces milk production. In summary, far-off over-feeding of ME to cows that needed to gain BCS did not influence peripartum metabolic health in grazing dairy cows, but restricting cows below 70% ME requirements during the close-up transition period resulted in a blood profile indicative of greater inflammation.

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows.

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.

Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function.

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of ß-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of ß-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Effects of precalving body condition and prepartum feeding level on gene expression in circulating neutrophils.

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (-1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalving compared with those offered 75% of maintenance requirements. Results indicate that precalving body condition and changes in prepartum energy lead to altered gene expression of circulating neutrophils, highlighting the importance of transition cow nutrition for peripartum health.

Prepartum body condition score and plane of nutrition affect the hepatic transcriptome during the transition period in grazing dairy cows.

BACKGROUND: A transcriptomic approach was used to evaluate potential interactions between prepartum body condition score (BCS) and feeding management in the weeks before calving on hepatic metabolism during the periparturient period. METHODS: Thirty-two mid-lactation grazing dairy cows of mixed age and breed were randomly allocated to one of four treatment groups in a 2 × 2 factorial arrangement: two prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); based on a 10-point scale], and two levels of energy intake during the 3 weeks preceding calving (75 and 125 % of estimated requirements). Liver samples were obtained at -7, 7, and 28 d relative to parturition and subsequent RNA was hybridized to the Agilent 44 K Bovine (V2) Microarray chip. The Dynamic Impact Approach was used for pathway analysis, and Ingenuity Pathway Analysis was used for gene network analysis. RESULTS: The greater number of differentially expressed genes in BCS4 cows in response to prepartum feed allowance (1071 vs 310, over the entire transition period) indicates that these animals were more responsive to prepartum nutrition management than optimally-conditioned cows. However, independent of prepartum BCS, pathway analysis revealed that prepartal feeding level had a marked effect on carbohydrate, amino acid, lipid, and glycan metabolism. Altered carbohydrate and amino acid metabolism suggest a greater and more prolonged negative energy balance postpartum in BCS5 cows overfed prepartum. This is supported by opposite effects of prepartum feeding in BCS4 compared with BCS5 cows in pathways encompassing amino acid, vitamin, and co-factor metabolism. The prepartum feed restriction ameliorates the metabolic adaptation to the onset of lactation in BCS5 cows, while detrimentally affecting BCS4 cows, which seem to better adapt when overfed. Alterations in the glycosaminoglycans synthesis pathway support this idea, indicating better hepatic health status in feed-restricted BCS5 and overfed BCS4 cows. Furthermore, IPA network analysis suggests liver damage in feed-restricted thin cows, likely due to metabolic overload. CONCLUSION: Overall, the data support the hypothesis that overfeeding in late-pregnancy should be limited to underconditioned cows, while cows with optimal degree of body condition should be maintained on an energy-restricted diet.

Epigenetic regulation of pyruvate carboxylase gene expression in the postpartum liver.

Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n=20), or treated with a NSAID during the first 5 d postcalving (n=20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period.

Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils.

Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (-1wk; n=46), day of calving (d 0; n=46), and postcalving at wk 1 (n=46), wk 2 (n=45), and wk 4 (n=43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of functional changes in neutrophils of grazing dairy cows over the transition period and align with studies in cows of conventional total mixed ration systems. This altered function may predispose cows to disease over the transition period and is likely to be a natural change in function due to parturition.

Body condition score and plane of nutrition prepartum affect adipose tissue transcriptome regulators of metabolism and inflammation in grazing dairy cows during the transition period.

Recent studies demonstrating a higher incidence of metabolic disorders after calving have challenged the management practice of increasing dietary energy density during the last ~3 wk prepartum. Despite our knowledge at the whole-animal level, the tissue-level mechanisms that are altered in response to feeding management prepartum remain unclear. Our hypothesis was that prepartum body condition score (BCS), in combination with feeding management, plays a central role in the peripartum changes associated with energy balance and inflammatory state. Twenty-eight mid-lactation grazing dairy cows of mixed age and breed were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial arrangement: 2 prepartum BCS categories (4.0 and 5.0, based on a 10-point scale; BCS4, BCS5) obtained via differential feeding management during late-lactation, and 2 levels of energy intake during the 3 wk preceding calving (75 and 125% of estimated requirements). Subcutaneous adipose tissue was harvested via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative polymerase chain reaction was used to measure mRNA and microRNA (miRNA) expression of targets related to fatty acid metabolism (lipogenesis, lipolysis), adipokine synthesis, and inflammation. Both prepartum BCS and feeding management had a significant effect on mRNA and miRNA expression throughout the peripartum period. Overfed BCS5 cows had the greatest prepartum expression of fatty acid synthase (FASN) and an overall greater expression of leptin (LEP); BCS5 was also associated with greater overall adiponectin (ADIPOQ) and peroxisome proliferator-activated receptor gamma (PPARG), whereas overfeeding upregulated expression of proadipogenic miRNA. Higher postpartum expression of chemokine ligand 5 (CCL5) and the cytokines interleukin 6 (IL6) and tumor necrosis factor (TNF) was detected in overfed BCS5 cows. Feed-restricted BCS4 cows had the highest overall interleukin 1 (IL1B) expression. Prepartum feed restriction resulted in greater chemokine ligand 2 (CCL2) expression. Overall, changes in mRNA expression were consistent with the expression pattern of inflammation-related miRNA. These data shed light on molecular mechanisms underlying the effect of prepartum BCS and feeding management on metabolic and inflammatory status of adipose tissue during the peripartum period. Data support the use of a controlled feed restriction prepartum in optimally conditioned cows, as well as the use of a higher level of dietary energy in under-conditioned cows.

Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows.

Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n=5; high risk) and low (n=5; low risk) concentrations of nonesterified fatty acids, ß-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category; however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows' greater plasma ß-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state.

Effects of precalving body condition score and prepartum feeding level on production, reproduction, and health parameters in pasture-based transition dairy cows.

Precalving feeding level alters postcalving energy balance, dry matter intake, the liver and adipose tissue transcriptome, hepatic lipidosis, and the risk of metabolic diseases in both high-production cows consuming total mixed rations and moderate-production cows grazing pasture. We hypothesized that the reported benefits of a controlled restriction before calving are dependent on precalving body condition score (BCS): low BCS animals would not benefit from reduced feeding levels precalving, but high BCS cows would have metabolic and immunomodulatory profiles indicative of an improved health status. One hundred sixty-one days before calving, 150 cows were allocated randomly to 1 of 6 treatment groups (n = 25) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0; based on a 10-point scale: BCS4 and BCS5, respectively) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Cows in the BCS4 and BCS5 groups were managed through late lactation to ensure that target calving BCS was achieved at dry off. Cows were then fed to maintain this BCS target until 3 wk before expected calving date, at which point they were managed within their allotted precalving energy intake treatments by offering different allowances of fresh pasture/cow per day. Milk production, body weight, and BCS were measured weekly; blood was sampled weekly before and after calving and on d 0, 1, 2, 3, and 4 relative to calving. Aspirated plasma was assayed for nonesterified fatty acids, ß-hydroxybutyrate, total protein, albumin, cholesterol, haptoglobin, IL-1ß, IL-6, total antioxidant capacity, and reactive oxygen species. Liver was sampled wk 1, 2, and 4 postcalving for triacylglycerol analysis. Results confirm that precalving BCS and precalving feeding level have both independent and interdependent effects on production and health characteristics of transition dairy cows. Irrespective of precalving BCS, a controlled restriction precalving reduced the net release of nonesterified fatty acids from adipose tissue postpartum and increased plasma calcium concentrations, reducing the risk of milk fever. Fatter cows produced more milk but lost more BCS postcalving and had greater blood ß-hydroxybutyrate concentrations and increased hepatic lipidosis. In comparison, after calving, indicators of reduced immune competence were accentuated in BCS4 cows subjected to a feed restriction before calving, probably increasing the risk of infectious diseases. It would appear from these results that optimally conditioned cows will benefit from a short-term (2-3 wk) controlled feed restriction (75-90% of requirements), whereas cows in less than optimal condition should be fed to requirements before calving.

Adipose and liver gene expression profiles in response to treatment with a nonsteroidal antiinflammatory drug after calving in grazing dairy cows.

The peripartal or transition period in dairy cattle is often characterized by an inflammatory state that, if not controlled, could be detrimental to production, health, and fertility. Approaches to control the postpartal degree of inflammation include treatments with nonsteroidal antiinflammatory drugs (NSAID) postcalving, which have improved cow production and health. To date, most of the research on NSAID has been conducted in confinement cows that reach milk production levels substantially greater than those on pasture. Furthermore, little data are available on the effect of NSAID on the mRNA expression of inflammation and metabolism-related genes. Transcription regulation is an important mechanism of inflammation and metabolic control. The present study was conducted to examine hepatic and adipose tissue gene expression in response to injections of an NSAID, carprofen, on 1, 3, and 5 d after calving. Grazing Holstein-Friesian cows from a control group and 1 treated with carprofen during the first 5 d postcalving were used. Liver and subcutaneous adipose tissue biopsies were harvested at -1, 1, and 2 wk relative to parturition. More than 30 genes associated with fatty acid oxidation, growth hormone/insulin-like growth factor-1 axis, hepatokines, lipoprotein metabolism, gluconeogenesis, and inflammation were analyzed. After calving, data suggest that both tissues respond to inflammation signals at the onset of lactation. Administration of NSAID led to greater hepatic expression of pyruvate dehydrogenase kinase, isozyme 4 (PDK4), which helps regulate gluconeogenesis, and microsomal triglyceride transfer protein (MTTP), important for the assembly and secretion of very low-density lipoproteins. In adipose tissue, NSAID administration resulted in greater expression of the inflammation-related genes interleukin-1, ß (IL1B), interleukin-6 receptor (IL6R), toll-like receptor 4 (TLR4), and chemokine (C-C motif) ligand 5 (CCL5). The data support the role of inflammation as a normal component of the homeorhetic adaptations to lactation and reveal a possible mechanism of action of carprofen in transition dairy cows, but do not reflect an effect of this NSAID on the extent of the peripartum inflammation.