Screening of mtr-miR156a from exosomes of dairy cow blood to milk and its regulatory effect on milk protein synthesis in BMECs.

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows.

Combining pretreatments and co-fermentation as successful approach to improve biohydrogen production from dairy cow manure.

The present paper is focused on enhancing the production of biohydrogen (bioH2) from dairy cow manure (DCM) through dark fermentation (DF). Two enhancement production strategies have been tested: i) the combination of H2O2 with sonification as pretreatment and ii) the co-fermentation with cheese whey as co-substrate. Concerning the pretreatment, the best combination was investigated according to the response surface methodology (RSM) by varying H2O2 dosage between 0.0015 and 0.06 g/gTS and ultrasonic specific energy input (USEI) between 35.48 and 1419.36 J/gTS. The increase of carbohydrates concentration was used as target parameter. Results showed that the combination of 0.06 g/gTS of H2O2 with 1419.36 J/gTS of USEI maximized the concentration of carbohydrates. The optimized conditions were used to pretreat the substrate prior conducting DF tests. The use of pretreatment resulted in obtaining a cumulative bioH2 volume of 51.25 mL/L and enhanced the bioH2 production by 125% compared to the control test conducted using raw DCM. Moreover, the second strategy, i.e. co-fermentation with cheese whey (20% v/v) as co-substrate ended up to enhancing the DF performance as the bioH2 production reached a value of 334.90 mL/L with an increase of 1372% compared to the control DF test. To further improve the process, dark fermentation effluents (DFEs) were valorized via photo fermentation (PF), obtaining an additional hydrogen production aliquot.

Periparturient blood T-lymphocyte PD-1 and CTLA-4 expression as potential predictors of new intramammary infections in dairy cows during early lactation (short communication).

BACKGROUND: The periparturient period in dairy cows is marked by immunosuppression which increases the likelihood of infectious disorders, particularly also mastitis. An in-depth understanding of peripartum leukocyte biology is vital for the implementation of highly successful post-partum disease prevention measures. Immune checkpoint molecules, such as programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), are critical inhibitory receptors expressed on immune cells, particularly T cells, that drive immunosuppressive signaling pathways. However, the potential role of immune checkpoint molecules expression in T-cells on udder health has never been explored. Thus, the association between the occurrence of new postpartum intramammary infections (IMIs) and the expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on blood T-cells during the peripartum period was investigated. RESULTS: In this study, the incidence of IMIs by any pathogen in early lactation was not associated with a higher expression of PD-1 and CTLA-4 in the periparturient period. However, the incidence of IMIs by major pathogens throughout the first month of lactation was significantly associated with higher expression of PD-1 at 14 days before calving (P = 0.03) and CTLA-4 at parturition (P = 0.03) by blood T-cells. Also, the expression of CTLA-4 at D0 (P = 0.012) by T-cells was associated with the occurrence of persistent IMIs during the first month of lactation. CONCLUSIONS: To our knowledge, this is the first report to investigate the expression of PD-1 and CTLA-4 by blood T-lymphocytes during the periparturient period in dairy cows and to explore their relationship with the incidence of new IMIs in the postpartum period. Thus, a comprehensive understanding of leukocyte biology during peripartum would appear to be a prerequisite for the identification of resilient dairy cows or targets innovative (immunological) non-antibiotic approaches in the transition period.

Body conformation traits in early-lactation associated with clinical mastitis and lameness in lactating Chinese holstein cows.

BACKGROUND: Comprehending the correlation between body conformation traits of cows at the early stages of lactation and prevalent lactation diseases might facilitate the execution of selection and feeding strategies that prioritize cow health. This study aimed to evaluate the impact of body conformation traits on the incidence of clinical mastitis and lameness in Chinese Holstein cows. From a pasture herd of 1472 early lactating Chinese Holstein cows, we evaluated 20 body conformation traits. During lactation, this pasture herd was visited weekly to gather clinical mastitis and lameness data. A nine-point scale was used to determine the conformation traits of cows to clarify their linear characters, including frame capacity, rump (RU), feet and leg (FL), mammary system (MS), and dairy character. A longitudinal binary disease (0 = healthy; 1 = diseased) data structure was created by allocating disease records to adjacent official test dates. The impact of body conformation traits on the risk of developing diseases (clinical mastitis and lameness) was analyzed using the logistic regression models. RESULTS: Compared to cows with low total scores (75-79 points), those with high total scores (80-85 points) of body conformation traits had a significantly lower risk of mastitis (P < 0.001). The disease status (0 or 1: binary variable) of clinical mastitis in lactating cows was significantly impacted negatively by age (P < 0.05). The fore udder attachment (FUA), angularity, rear attachment height (RAH), and rear teat placement (RTP) were all significantly associated with clinical mastitis during lactation (P < 0.05). The rear leg-rear view (RLRV) was significantly correlated with correlated considerably (P < 0.05) with lameness during lactation. An ideal score of four points on the lameness risk dimension of the RLRV may indicate a low risk of lameness. Since the risk of mastitis decreased as this trait score increased, the RTP may be an ideal marker for mastitis risk. CONCLUSIONS: According to the study, clinical mastitis and lameness risks in cows can be estimated using their body conformation traits. Cows with more centrally located rear teats have a lower risk of mastitis. These results may help dairy farmers identify cows at high risk of disease early in lactation and aid in breeding for disease resistance in cows.

PI3K/AKT/mTORC1 signalling pathway regulates MMP9 gene activation via transcription factor NF-κB in mammary epithelial cells of dairy cows.

Matrix metalloproteinase 9 (MMP9) plays a pivotal role in mammary ductal morphogenesis, angiogenesis and glandular tissue architecture remodeling. However, the molecular mechanism of MMP9 expression in mammary epithelial cells of dairy cows remains unclear. This study aimed to explore the underlying mechanism of MMP9 expression. In this study, to determine whether the PI3K/AKT/mTORC1/NF-κB signalling pathway participates in the regulation of MMP9 expression, we treated mammary epithelial cells with specific pharmacological inhibitors of PI3K (LY294002), mTORC1 (Rapamycin) or NF-κB (Celastrol), respectively. Western blotting results indicated that LY294002, Rapamycin and Celastrol markedly decreased MMP9 expression and P65 nuclear translocation. Furthermore, we found that NF-κB (P65) overexpression resulted in elevated expression of MMP9 protein and activation of MMP9 promoter. In addition, we observed that Celastrol markedly decreases P65-overexpression-induced MMP9 promoter activity. Moreover, the results of the promoter assay indicated that the core regulation sequence for MMP9 promoter activation may be located at -420 ∼ -80 bp downstream from the transcription start site. These observations indicated that the PI3K/AKT/mTORC1 signalling pathway is involved in MMP9 expression by regulating MMP9 promoter activity via NF-κB in the mammary epithelial cells of dairy cows.

Can lameness prevalence in dairy herds be predicted from farmers' reports of their motivation to control lameness and barriers to doing so? An observational study from New Zealand.

Understanding what motivates and prevents behavioral change in farmers is a critical step in disease control in dairy cattle. A total of 101 New Zealand dairy farmers across 8 regions were randomly enrolled into a cross-sectional study to investigate farmer barriers and motivators to lameness control for cows managed 100% at pasture and the relationship between these responses and the true lameness status on farm. Trained technicians lameness scored all lactating cows on the enrolled farms on 2 occasions during one lactation. Farm-level prevalence proportions were calculated as the mean of the 2 lameness scores. Enrolled farmers were asked their perception of lameness in the current milking season and responded to 26 ordinal Likert-type items with 5 options ranging from not important at all to extremely important. The questions were grouped under 3 categories; barriers to lameness control (n = 9), impacts of lameness (n = 10), and motivators to control lameness (n = 7). The association between farmer perception of lameness and lameness prevalence was reported using linear regression. Multiple-factor analysis was conducted to identify latent variable themes within the responses. Linear discriminant analysis was used to assess whether barriers, impacts, and motivators could be used to predict farmer perception of lameness and lameness prevalence. Lameness prevalence was 0.8% greater on farms where farmers perceived lameness as a moderate or a major problem compared with farms where the farmer perceived lameness as a minor problem or not a problem. Farmers ranked all potential motivators to lameness control as important and declared few barriers to be important at preventing them from controlling lameness. Feeling sorry for lame cows and pride in a healthy herd were the most important motivators, with lack of time and skilled labor the most important barriers. The most important effects of lameness were cow-related factors such as pain and production, with farm and industry impacts of less importance. Farmers place different weightings of importance on barriers to lameness control compared with motivators for lameness control. The impacts and motivators were strongly correlated with the first dimension from the multiple-factor analysis, with only weak correlation between barriers and the first dimension. Linear discriminant analysis identified that the importance that farmers place on barriers, motivators, and impacts of lameness were poor predictors of farmers' belief in regard to their lameness problem or actual lameness prevalence (above or below the median lameness prevalence for the study cohort). Despite relatively low lameness prevalence, many New Zealand dairy farmers believe lameness is a problem on their farm, and they rank welfare effects of lameness of high importance. To investigate how farmer behavior change can be used to manage lameness, future studies should consider theoretical social science frameworks beyond the theory of planned behavior or involve prospective interventional studies investigating farmer actions instead of beliefs.

Longitudinal characterization of the muscle metabolome in dairy cows during the transition from lactation cessation to lactation resumption.

Skeletal muscle is vital in maintaining metabolic homeostasis and adapting to the physiological needs of pregnancy and lactation. Despite advancements in understanding metabolic changes in dairy cows around calving and early lactation, there are still gaps in our knowledge, especially concerning muscle metabolism and the changes associated with drying off. This study aimed to characterize the skeletal muscle metabolome in the context of the dietary and metabolic changes occurring during the transition from the cessation of lactation to the resumption of lactation in dairy cows. Twelve Holstein dairy cows housed in tie stalls were dried off 6 weeks (wk) before the expected calving date. Cows were individually fed ad libitum total mixed rations composed of grass silage, corn silage, and concentrate during lactation and of corn silage, barley straw, and concentrate during the dry period. The metabolome was characterized in skeletal muscle samples (M. longissimus dorsi) collected on wk -7 (9 d before dry-off), -5 (6 d after dry-off), and wk -1, and 1 relative to calving. The targeted metabolomics approach was conducted using the MxP Quant 500 kit (Biocrates Life Sciences AG) with liquid chromatography, flow injection, and electrospray ionization triple quadrupole mass spectrometry. Statistical analysis on the muscle metabolite data was performed using MetaboAnalyst 5.0, which allowed us to conduct various multivariate analyses such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), informative heat map generation, and hierarchical clustering. The statistical analysis revealed a clear separation between pregnancy (wk -7, -5, and -1) and post-calving (wk 1). Starting 5 wk before calving and continuing through the first wk thereafter, the concentration of 3-methylhistidine (3-MH) in the muscle increased. This coincided with an increase in the concentrations of 11 AA (Phe, His, Tyr, Trp, Arg, Asn, Leu, Ile, Gly, Ser, and Thr) in the first wk after calving, whereas Gln decreased. l-arginine pathway metabolites (homoarginine, ornithine, citrulline, and asymmetric dimethylarginine), betaine, and sarcosine followed a similar pattern, increasing from wk -7 to -5, but decreasing from wk -1 to 1. The transition from pregnancy to lactation was associated with an increase in concentrations of the long-chain acylcarnitine species C16, C16:1, C18, and C18:1 in the muscle, whereas the concentrations of phosphatidylcholine and sphingomyelin in the muscle remained stable. The significant changes observed in the metabolome mainly concerned the AA and AA-related metabolites, indicating muscle protein breakdown in the first wk after calving. The metabolites produced by the L-Arg pathway might contribute to regulating skeletal muscle mass and function in periparturient dairy cows. The elevated concentrations of long-chain acylcarnitine species in the muscle in the first wk after calving suggest incomplete fatty acid oxidation, likely due to insufficient metabolic adaptation in response to the fatty acid load around the time of calving.

Effect of automated health monitoring based on rumination, activity, and milk yield alerts versus visual observation on herd health monitoring and performance outcomes.

A primary objective of this randomized trial was to compare the percentage of cows that underwent clinical examination and were diagnosed with clinical health disorders (CHD) with a health monitoring program that relied only on automated monitoring system alerts vs a program that relied only on visual observation of clinical signs of disease to select cows for clinical examination. Another objective was to compare the effects of these health monitoring programs on milk yield, the herd exit dynamics (i.e., cows sold and dead), and first service reproductive outcomes. Lactating Holstein cows (n = 1,204) enrolled in the experiment were fitted with a neck-attached sensor of an automated monitoring system (HR Tags; Merck & Co., Inc.) that generated health alerts based on rumination time and activity. Milk yield was monitored 3 times per day by automated milk meters (MM27BC, DeLaval). Cows were blocked by parity, close-up period diet, and stratified by previous lactation milk yield, and then were randomly assigned within block to different programs for monitoring health from 3 to 21 d in milk (DIM). Cows in the visual observation group (VO; n = 597) were selected for clinical examination exclusively based on visual observation of clinical signs of disease, whereas cows in the automated health monitoring group (AHM; n = 607) were selected for clinical examination based on health alerts consisting of the following: a Health Index Score < 86 arbitrary units, daily rumination < 250 min, or a reduction of > 20% in daily milk yield. Once selected for examination, the clinical exam was the same for both treatment groups. Binary data such as the occurrence of CHD, herd exit, and pregnancies per AI were analyzed with logistic regression. Daily and weekly milk yield were analyzed using ANOVA with repeated measurements. More cows underwent a clinical examination, more cows were diagnosed with at least one CHD, and more cows received treatment in the AHM than the VO treatment group. Cows in the AHM treatment had more accumulated milk than cows in the VO treatment from 2 to 21 DIM. Cows in the AHM treatment diagnosed with at least one CHD produced more milk from 3 to 18 and 20 to 21 DIM than cows diagnosed with a CHD in the VO treatment. Fewer cows left the herd up to 21 DIM for the AHM than the VO treatment. Pregnancies per AI at first service were greater for the VO than the AHM treatment at 30 d but not at 50 d after AI and no difference in pregnancy loss was detected. In conclusion, a health monitoring strategy that used automated health alerts increased the risk of undergoing clinical examination and having CHD diagnosed compared with a program that selected cows for clinical examination based exclusively on visual observation. Cows monitored with the program that relied on automated alerts also had greater milk yield in the first 21 DIM. Thus, monitoring cow health based on automated behavior and milk yield alerts might be a more effective alternative for health monitoring than exclusive use of visual observation of clinical signs of disease.

The effect of selective dry cow therapies based on 2 different algorithms on antimicrobial use, udder health, milk production, and culling in the absence of internal teat sealant use at dry-off.

The objective of this study was to evaluate the effect of selective dry cow therapy (SDCT) strategies based on 2 different algorithms in comparison with blanket dry cow therapy for measures of udder health, milk yield, and culling in herds not using internal teat sealant. Cows from 2 commercial farms in west Texas were randomized into 3 different groups: SDCT algorithm 1 (ALG1; n = 455) cows treated with an intramammary antimicrobial at dry-off if SCC >200,000 cells/mL at any Dairy Herd Improvement Association test date or if the cow had 2 or more cases of clinical mastitis (CM) during the enrollment lactation; SDCT algorithm 2 (ALG2; n = 458) cows treated with an intramammary antimicrobial at dry-off if SCC >200,000 cell/mL at last test date or any case of CM during the enrollment lactation; and control cows (CON = 447) received blanket dry cow therapy. None of the cows enrolled in the study received an internal or external teat sealant. Data related to milk and SCC linear score (LSCC) were collected monthly. Milk yield and LSCC during the first 6 mo of lactation were analyzed using repeated measures ANOVA models, while Cox proportional hazards models were fitted to culling and CM data. The farm was fitted as a random effect in all models. The percentage of cows receiving antimicrobials at dry-off was 51.3%, 24.7%, and 100% for ALG1, ALG2, and CON, respectively. Treatment did not influence the IMI dynamics during the dry period. Additionally, no statistical differences related to treatment were observed for LSCC and milk yield. The LSCC for ALG1, ALG2, and CON cows was 2.44, 2.41, and 2.26, respectively. The average milk yield for ALG1, ALG2, and CON cows was 43.2, 43.2, and 44.0 kg/d, respectively. Treatment did not affect CM incidence or culling. The cumulative incidence of CM was 19.6%, 19.4%, and 21.4% for ALG1, ALG2, and CON cows, respectively. Additionally, the cumulative risk of death or culling was 18.5%, 17.1%, and 19.5% for ALG1, ALG2, and CON cows, respectively. In conclusion, SDCT strategies led to a decrease in antimicrobial drug use at dry-off, without significantly affecting the incidence of CM, the risk of culling, LSCC, or milk yield of dairy cows. However, numerical differences in LSCC and milk yield were observed between treatment groups.

Invited review: Ketone biology-The shifting paradigm of ketones and ketosis in the dairy cow.

Ketosis is currently regarded as a major metabolic disorder of dairy cows, reflective of the animal's efforts to adapt to energy deficit while transitioning into lactation. Currently viewed as a pathology by some, ketosis is associatively implicated in milk production losses and peripartal health complications that increase the risk of early removal of cows from the herd, thus carrying economic losses for dairy farmers and jeopardizing the sustainability of the dairy industry. Despite decades of intense research in the mitigation of ketosis and its sequelae, our ability to lessen its purported effects remains limited. Moreover, the association of ketosis to reduced milk production and peripartal disease is often erratic and likely mired by concurrent potential confounders. In this review, we discuss the potential reasons for these apparent paradoxes in the light of currently available evidence, with a focus on the limitations of observational research and the necessary steps to unambiguously identify the effects of ketosis on cow health and performance via controlled randomized experimentation. A nuanced perspective is proposed that considers the dissociation of ketosis-as a disease-from healthy hyperketonemia. Furthermore, in consideration of a growing body of evidence that highlights positive roles of ketones in the mitigation of metabolic dysfunction and chronic diseases, we consider the hypothetical functions of ketones as health-promoting metabolites and ponder on their potential usefulness to enhance dairy cow health and productivity.

Feeding nanoparticles of copper oxide coated with lysine with or without added antagonists affects the copper status but not performance of Holstein dairy cows.

The apparent absorption of copper (Cu) in ruminants is low, with between 0.01 and 0.07 g/g absorbed from sources such as copper oxide (CuO) under typical feeding conditions, resulting in high levels of excretion. Improving the bioavailability of Cu could reduce the supplemental amount required to maintain Cu status and reduce excretion, particularly in the presence of dietary antagonists such as sulfur (S) and molybdenum (Mo). The objective of our study was to determine the Cu status of cows when fed nanoparticle CuO coated with Lys compared with conventional CuO when fed without or in combination with antagonists to Cu absorption (S and Mo) in the diet of dairy cows. Fifty-six multiparous Holstein-Friesian cows that were 48 d ± 17.4 (mean ± SD) post calving and yielding 40.6 ± 6.9 kg milk/d were used in a 2 × 2 factorial design. The 4 treatment groups were; CuO (O-), CuO with added antagonists (O+), nano CuO with a lysine coating (N-), and nano CuO with a Lys coating with added antagonists (N+), fed for 16 wks. We formulated the diets to contain approximately 17 mg Cu/kg dry matter (DM) and diets with antagonists contained an additional 1 g S/kg DM and 6 mg Mo/kg DM, with Lys added to O- and O+ to provide the same daily supply as N- and N+. Blood samples were collected at wk 0, 2, 4, 6, 10 and 16, and liver biopsy samples at wk 0 and 16. We found no effect of dietary treatment on DM intake, milk yield, live weight or body condition score, with mean values of 23.3 kg/d, 40.1 kg/d, 646 kg and 2.68, but milk SCC was higher in cows fed conventional compared with non CuO, or with added antagonist. We also found no effect of treatment on blood activity of gamma glutamyl transferase, superoxide dismutase or ceruloplasmin, hematology profile, or plasma Cu and iron concentration. We found that plasma Mo concentration was increased from 0.36 µmol/L in cows fed O- or N- to 0.80 µmol/L in those receiving O+ or N+. Additional dietary antagonists also decreased the concentration of Cu in the liver of cows fed conventional CuO (C+) over the study period by 1.3 mg/kg DM/d, but in cows fed dietary antagonists and nano CuO coated with Lys (N+), liver Cu concentration was increased by 1.1 mg/kg DM/d. Our study is the first to demonstrate that reducing the particle size of CuO into the nano scale with a lysine coating improves the bioavailability of CuO in the presence of dietary antagonists in dairy cattle, and we did not observe any negative effects on performance or health.

The effect of supplementary selenium source on apparent and true absorption, retention, performance, and selenium status in lactating Holstein cows.

Selenium is an essential trace mineral for dairy cattle and can be provided in the diet in various forms that may differ in bioavailability. The objective of this study was to determine how source of Se affects animal performance, Se status, retention, and apparent and true absorption. Multiparous Holstein cows (n = 24; 597 ± 49 kg of BW) were blocked by DIM (161 ± 18) and randomly assigned to receive 0.3 mg Se/kg of DM (100% of National Academies of Sciences, Engineering, and Medicine requirements) of either organic Se (ORG; selenized yeast) or inorganic Se (INO; sodium selenite). The Se premix was top dressed on a common TMR fed daily and mixed into the top 15 cm directly before feeding. Following an 11-wk adaptation period, cows received simultaneous infusions of an intraruminal isotope dose of 77Se in the same chemical form as the premix, and an intravenous dose of 82Se in an inorganic form. Infusions were followed by a 4-d period of blood and rumen fluid sampling, and total collection of feces, urine, and milk. Daily DMI (23 ± 0.6 kg), milk yield (35 ± 1.2 kg), and serum Se (0.11 ± 0.003 µg/g) were not different between treatments during the adaptation period, but milk Se concentrations were greater for ORG compared with INO. Serum 77Se maximum concentration and area under the curve (AUC) were not different between treatments for 72 h following infusion, but rumen fluid 77Se AUC was higher for ORG than INO. Apparent absorption (64% ± 1.4%), and retention (44% ± 1.5%) of the 77Se dose did not differ between treatments. True absorption was calculated using 82Se enrichment in serum and feces and was determined to be 69% ± 1.3% and did not differ between treatments. Fecal excretion of the 77Se dose was not different between treatments (36% ± 1.4%), but ORG had lower urinary excretion and higher milk excretion compared with INO. These results indicate that organic Se resulted in greater Se concentration of milk and lower urinary Se excretion into the environment, but absorption, Se status, and performance of the cow were not affected by Se source at this supplementation level.

Application of behavior data to predictive exploratory models of metritis self-cure and treatment failure in dairy cows.

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Relationships between gastrointestinal permeability, heat stress, and milk production in lactating dairy cows.

Heat stress (HS) is a global issue that decreases farm profits and compromises animal welfare. To distinguish between the direct and indirect effects of HS, 16 multiparous Holstein cows approximately 100 DIM were assigned to one of 2 treatments: pair fed to match HS cow intake, housed in thermoneutral conditions (PFTN, n = 8) or cyclical HS (n = 8). All cows were subjected to 2 experimental periods. Period 1 consisted of a 4 d thermoneutral period with ad libitum intake. During period 2 (P2), the HS cows were housed in cyclical HS conditions with a temperature-humidity index (THI) ranging from 76 to 80 and the PFTN cows were exposed to a constant THI of 64 for 4 d. Dry matter intake of the PFTN cows was intake matched to the HS cows. Milk yield, milk composition, rectal temperature, and respiration rate were recorded twice daily, blood was collected daily via a jugular catheter, and cows were fed twice daily. On d 3 of each period, Cr-EDTA and sucralose were orally administered and recovered via 24 h total urine collection to assess gastrointestinal permeability. All data were analyzed using the GLIMMIX procedure in SAS. The daily data collected in P1 was averaged and used as a covariate if deemed significant in the model. Heat stress decreased voluntary feed intake by 35% and increased rectal temperature and respiration rate (38.4°C vs. 39.4°C and 40 vs. 71 respirations/min, respectively). Heat stress reduced DMI by 35%, which accounted for 66% of the decrease in milk yield. The yields, and not concentrations, of milk protein, fat, and other solids were lower in the HS cows on d 4 of P2. Milk urea nitrogen was higher and plasma urea nitrogen tended to be higher on d 3 and d 4 of HS. Glucose was 7% lower in the HS cows and insulin was 71% higher in the HS cows than the PFTN cows on d 4 of P2. No difference in lipopolysaccharide-binding protein was observed. Heat stress cows produced 7 L/d more urine than PFTN cows. No differences were detected in the urine concentration or percentage of the oral dose recovered for Cr-EDTA or sucralose. In conclusion, HS was responsible for 34% of the reduction of milk yield. The elevated MUN and the tendency for elevated plasma urea nitrogen indicate a whole-body shift in nitrogen metabolism. No differences in gastrointestinal permeability or lipopolysaccharide-binding protein were observed. These results indicate that, under the conditions of this experiment, activation of the immune system by gut-derived lipopolysaccharide was not responsible for the decreased milk yield observed during HS.

Ovarian function and endocrine phenotypes of lactating dairy cows during the estrous cycle are associated with genomic-enhanced predictions of fertility potential.

The objectives of this prospective cohort study were to characterize associations among genomic merit for fertility with ovarian and endocrine function and the estrous behavior of dairy cows during an entire nonhormonally manipulated estrous cycle. Lactating Holstein cows entering their first (n = 82) or second (n = 37) lactation had ear-notch tissue samples collected for genotyping using a commercial genomic test. Based on genomic predicted transmitting ability values for daughter pregnancy rate (gDPR), cows were classified into high (Hi-Fert; gDPR > 0.6, n = 36), medium (Med-Fert; gDPR -1.3 to 0.6, n = 45), and low fertility (Lo-Fert; gDPR < -1.3, n = 38) groups. At 33 to 39 DIM, cohorts of cows were enrolled in the Presynch-Ovsynch protocol for synchronization of ovulation and initiation of a new estrous cycle. Thereafter, the ovarian function and endocrine dynamics were monitored daily until the next ovulation by transrectal ultrasonography and concentrations of progesterone (P4), estradiol, and FSH. Estrous behavior was monitored with an ear-attached automated estrus detection system that recorded physical activity and rumination time. Overall, we observed an association between fertility group and the ovarian and hormonal phenotype of dairy cows during the estrous cycle. Cows in the Hi-Fert group had greater circulating concentrations of P4 than cows in the Lo-Fert group from d 4 to 13 after induction of ovulation and from day -3 to -1 before the onset of luteolysis. The frequency of atypical estrous cycles was 3-fold greater for cows in the Lo-Fert than the Hi-Fert group. We also observed other modest associations between genomic merit for fertility with the follicular dynamics and estrous behavior. We found several associations between milk yield and parity with ovarian, endocrine, and estrous behavior phenotypes as cows with greater milk yield and in the second lactation were more likely to have unfavorable phenotypes. These results demonstrate that differences in reproductive performance between cows of different genomic merit for fertility classified based on gDPR may be partially associated with circulating concentrations of P4, the incidence of atypical phenotypes during the estrous cycles, and, to a lesser extent, the follicular wave dynamics. The observed physiological and endocrine phenotypes might help explain part of the differences in reproductive performance between cows of superior and inferior genomic merit for fertility.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line by impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy.

Reproductive physiological outcomes of dairy cows with different genomic merit for fertility: Biomarkers, uterine health, endocrine status, estrus features, and response to ovarian synchronization.

Our overarching objective was to characterize associations between genomic merit for fertility and the reproductive function of lactating dairy cows in a prospective cohort study. In this manuscript, we present results of the association between genomic merit for fertility and indicators of metabolic status and inflammation, uterine health, endocrine status, response to synchronization, and estrous behavior in dairy cows. Lactating Holstein cows entering their first (n = 82) or second (n = 37) lactation were enrolled at parturition and fitted with an ear-attached sensor for automated detection of estrus. Ear-notch tissue samples were collected from all cows and submitted for genotyping using a commercial genomic test. Based on genomic PTA values for daughter pregnancy rate (gDPR), cows were classified into a high (Hi-Fert; gDPR >0.6; n = 36), medium (Med-Fert; gDPR -1.3 to 0.6; n = 45), or low (Lo-Fert; gDPR <-1.3; n = 38) group. At 33 to 39 DIM, cohorts of cows were enrolled in the Presynch-Ovsynch protocol for synchronization of estrus and ovulation. Body weights, BCS, and uterine health measurements (i.e., vaginal discharge, uterine cytology) were collected from parturition to 60 DIM, and milk yield was collected through 90 DIM. Blood samples were collected weekly through 3 wk of lactation for analysis of BHB, nonesterified fatty acids (NEFA), and haptoglobin plasma concentrations. Body weight, BCS, NEFA, BHB, and haptoglobin were not associated with fertility groups from 1 to 9 wk after parturition. The proportion of cows classified as having endometritis at 33 to 36 DIM tended to be greater for the Lo-Fert than the Hi-Fert group. The proportion of cows that resumed cyclicity did not differ at any time point evaluated, and there were no significant associations between probability or duration and intensity of estrus with fertility group. Cows of superior genetic merit for fertility were more likely to ovulate, have a functional corpus luteum, have greater circulating progesterone, and have larger ovulatory size than cows of inferior fertility potential at key time points during synchronization of estrus and ovulation. Despite observing numerical differences with potential performance consequences for the proportion of cows that responded to synchronization of ovulation and were both cyclic and responded to the Ovsynch portion of the synchronization protocol, we did not observe significant differences between fertility groups. Although not consistent and modest in magnitude, the collective physiological and endocrine differences observed suggested that cows of superior genetic fertility potential might have improved reproductive performance, at least in part, because of modestly improved endocrine status, uterine health, and ability to ovulate.

Graduate Student Literature Review: System, plant, and animal factors controlling dietary pasture inclusion and their impact on ration formulation for dairy cows.

Diet formulation in a pasture-based dairy system is a challenge as the quality and quantity of available pasture, which generally constitutes the base diet, is constantly changing. The objective of this paper is to cover a more in-depth review of the nutritional characteristics of pasture-based diets, identifying potential system, plant, and animal factors that condition pasture dietary inclusion in dairy cows. In practice, there is a wide diversity of pasture-based systems with predominant to minimal use of pasture requiring a more specific classification that potentially considers the amount and time of access to pasture, access to housing, length of grazing season, seasonality of calving, and level and method of supplementation. There are important differences in the nutritional quality between pasture species and even cultivars. However, under management practices that promote maintenance of pasture in a vegetative state as well as controlling the availability of pasture, it is possible to achieve high dry matter intakes (∼2.9%-3.4% of live weight) of pasture with moderate to high diet energy density, protein supply, and digestibility. The amount of pasture to include in the diet will depend on several factors, such as the type of production system, the cost of supplementary feeds, and the farmer's objectives, but inclusions of ∼40% to 50% of the diet seem to potentially reduce costs while apparently not limiting voluntary feed intake. Considering that there seems to be a continuum of intermediate management systems, a better understanding of the factors inherent to the feed ingredients used, as well as the use of nutrients by cows, and potential interactions between animal × system should be addressed in greater depth. This requires a meta-analysis approach, but given the diversity of the pasture-based system in practice, the existing information is highly fragmented. A clear definition of "subsystems" is necessary to direct the future research and development of mechanistic models.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Effect of a blend of cinnamaldehyde, eugenol, and Capsicum oleoresin on methane emission and lactation performance of Holstein-Friesian dairy cows.

This study aimed to investigate the effect of administering a standardized blend of cinnamaldehyde, eugenol, and Capsicum oleoresin (CEC) to lactating dairy cattle for 84 d (i.e., 12 wk) on enteric CH4 emission, feed intake, milk yield and composition, and body weight. The experiment involved 56 Holstein-Friesian dairy cows (145 ± 31.1 d in milk at the start of the trial; mean ± standard deviation) in a randomized complete block design. Cows were blocked in pairs according to parity, lactation stage, and current milk yield, and randomly allocated to 1 of the 2 dietary treatments: a diet including 54.5 mg of CEC/kg of DM or a control diet without CEC. Diets were provided as partial mixed rations in feed bins, which automatically recorded individual feed intake. Additional concentrate was fed in the GreenFeed system that was used to measure emissions of CO2, CH4, and H2. Feeding CEC decreased CH4 yield (g/kg DMI) by on average 3.4% over the complete 12-wk period and by on average 3.9% from 6 wk after the start of supplementation onward. Feeding CEC simultaneously increased feed intake and body weight, and tended to increase milk protein content, whereas no negative responses were observed. These results must be further investigated and confirmed in longer-term in vivo experiments.