Association between serum mineral levels and reproductive performance in primiparous dairy cows during the peripartum period.

To the best of the authors' knowledge, no study has previously investigated whether the concentration of minerals is related to reproductive outcomes in primiparous cows. For this reason, two objectives were set in the present study: (i) to assess serum mineral levels, macrominerals, and trace elements during the transition period (period of high nutritional requirements) in primiparous cows, considering reproductive efficiency, and (ii) to address if the serum mineral levels of primiparous cows are related to reproductive efficiency. Blood samples were taken (i) one month before calving, (ii) one week before calving, (iii) one week postpartum, and (iv) one month postpartum. At the beginning and the end of the study, a body condition score (BCS) was assigned to each lactating cow with no clinical signs of disease. The difference between one month before and one month after calving was the body condition loss (ΔBCS). Optimal prepartum concentrations of K and Cl were associated with fewer days open and a shorter interval calving. Furthermore, macrominerals in the serum decreased immediately after calving (one week) but recovered at one month postpartum. In contrast, the highest concentration of trace elements was found at one week postpartum. Primiparous cows with higher postpartum Se, Mn, Co, and Mo concentrations exhibited better reproductive efficiency, and the concentrations of trace elements in serum were correlated with interval calving and the number of inseminations. Finally, primiparous cows with a greater ΔBCS (at least one point) in period 4 exhibited both a longer calving interval and a greater number of days open. In summary, this study showed, for the first time in primiparous cows, that the concentration of some serum minerals not only plays a crucial role during the transition period but is also related to crucial reproductive parameters, such as interval calving and days open.

A randomized controlled trial investigating the effect of transport duration and age at transport on surplus dairy calves: Part II. Impact on hematological variables.

Surplus dairy calves often arrive at veal and dairy-beef rearing facilities with health and blood metabolite level abnormalities, which can affect their welfare and performance, predisposing them to future health challenges. The objective of this randomized controlled trial was to investigate the effects of transport duration and age at the time of transport on blood parameters in surplus dairy calves following 6, 12, or 16 h of continuous road transportation. All surplus calves from 5 commercial dairy farms in Ontario were enrolled and examined daily before transport (n = 175). On the day of transportation, calves were weighed, blood sampled, and randomly assigned to 6, 12, or 16 h of transportation. Blood samples were then collected immediately after transportation, as well as 24, 48, and 72 h thereafter. Serum was analyzed at a provincial diagnostic laboratory for nonesterified fatty acids (NEFA), ß-hydroxybutyric acid (BHBA), creatine kinase (CK), cholesterol, and haptoglobin. In addition, blood gas and electrolyte values were also assessed at the time of sample collection. Mixed models with repeated measures were used to assess the effects of transport duration, breed, sex, transfer of passive immunity status, weight before transportation, and age at transportation on blood parameters. Immediately following transportation, NEFA and BHBA were greater for calves transported for 12 h (Δ = 0.22 mmol/L NEFA, 95% CI = 0.15 to 0.30; Δ = 0.04 mmol/L BHBA, 95% CI = 0.02 to 0.06) and 16 h (Δ = 0.35 mmol/L NEFA, 95% CI = 0.27 to 0.42; Δ = 0.10 mmol/L BHBA, 95% CI = 0.08 to 0.11) compared with calves transported for 6 h. Glucose was lower immediately following transportation in calves transported for 16 h compared with 6 h (Δ = -15.54 mg/dL, 95% CI = -21.54 to -9.54). In addition, pH and HCO3- were lower in calves transported for 12 (Δ = -0.09 pH, 95% CI = -0.13 to -0.05; Δ = -1.59 mmol/L HCO3-, 95% CI = -2.61 to -0.56) and 16 h (Δ = -0.07 pH, 95% CI = -0.12 to -0.03; Δ = -1.95 mmol/L HCO3-, 95% CI = -2.95 to -0.95) compared with calves transported for 6 h. Calves transported between 15 and 19 d of age had a higher concentration of cholesterol and CK (Δ = 0.27 mmol/L cholesterol; 37.18 U/L CK) compared with 2- to 6-d-old calves, and calves 12 to 14 d old had greater reduction in HCO3- (Δ = -0.92 mmol/L) compared with 2- to 6-d-old calves. These findings show that transporting calves for long distances results in lower glucose concentration and suboptimal energy status, and that this effect varies based on the calf's age.

Standardized Whole Blood Assay and Bead-Based Cytokine Profiling Reveal Commonalities and Diversity of the Response to Bacteria and TLR Ligands in Cattle.

Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli, Staphylococcus aureus, and Streptococcus uberis conditions, and that IFN-γ, IL-1ß, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures.

In-line near-infrared analysis of milk coupled with machine learning methods for the daily prediction of blood metabolic profile in dairy cattle.

Precision livestock farming technologies are used to monitor animal health and welfare parameters continuously and in real time in order to optimize nutrition and productivity and to detect health issues at an early stage. The possibility of predicting blood metabolites from milk samples obtained during routine milking by means of infrared spectroscopy has become increasingly attractive. We developed, for the first time, prediction equations for a set of blood metabolites using diverse machine learning methods and milk near-infrared spectra collected by the AfiLab instrument. Our dataset was obtained from 385 Holstein Friesian dairy cows. Stacking ensemble and multi-layer feedforward artificial neural network outperformed the other machine learning methods tested, with a reduction in the root mean square error of between 3 and 6% in most blood parameters. We obtained moderate correlations (r) between the observed and predicted phenotypes for γ-glutamyl transferase (r = 0.58), alkaline phosphatase (0.54), haptoglobin (0.66), globulins (0.61), total reactive oxygen metabolites (0.60) and thiol groups (0.57). The AfiLab instrument has strong potential but may not yet be ready to predict the metabolic stress of dairy cows in practice. Further research is needed to find out methods that allow an improvement in accuracy of prediction equations.

A physiologically based pharmacokinetic (PBPK) model exploring the blood-milk barrier in lactating species - A case study with oxytetracycline administered to dairy cows and goats.

Antibiotic excretion into milk depends on several factors such as the compound's physicochemical properties, the animal physiology, and the milk composition. The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model describing the passage of drugs into the milk of lactating species. The udder is described as a permeability limited compartment, divided into vascular, extracellular water (EW), intracellular water (IW) and milk, which was stored in alveolar and cistern compartments. The pH and ionization in each compartment and the binding to IW components and to milk fat, casein, whey protein, calcium, and magnesium were considered. Bidirectional passive diffusion across the blood-milk barrier was implemented, based on in vitro permeability studies. The model application used to predict the distribution of oxytetracycline in cow and goat milk, after different doses and routes of administration, was successful. By integrating inter-individual variability and uncertainty, the model also allowed a suitable estimation of the withdrawal periods. Further work is in progress to evaluate the predictive ability of the PBPK model for compounds with different physico-chemical properties that are potentially actively transported in order to extrapolate the excretion of xenobiotics in milk of various animal species including humans.

Differences in the serum metabolome profile of dairy cows according to the BHB concentration revealed by proton nuclear magnetic resonance spectroscopy (1H-NMR).

The mobilization of body reserves during the transition from pregnancy to lactation might predispose dairy cows to develop metabolic disorders such as subclinical ketosis or hyperketonemia. These conditions are not easily identifiable and are frequently related to other diseases that cause economic loss. The aim of this study was to evaluate the serum metabolome differences according to the ß-hydroxybutyrate (BHB) concentration. Forty-nine Holstein Friesian dairy cows were enrolled between 15 and 30 days in milk. According to their serum BHB concentration, the animals were divided into three groups: Group 0 (G0; 12 healthy animals; BHB ≤ 0.50 mmol/L); Group 1 (G1; 19 healthy animals; 0.51 ≤ BHB < 1.0 mmol/L); and Group 2 (G2; 18 hyperketonemic animals; BHB ≥ 1.0 mmol/L). Animal data and biochemical parameters were examined with one-way ANOVA, and metabolite significant differences were examined by t-tests. Fifty-seven metabolites were identified in the serum samples. Thirteen metabolites showed significant effects and seemed to be related to the mobilization of body reserves, lipids, amino acid and carbohydrate metabolism, and ruminal fermentation.

Predicting ketosis during the transition period in Holstein Friesian cows using hematological and serum biochemical parameters on the calving date.

Ketosis often occurs during the postpartum transition period in dairy cows, leading to economic and welfare problems. Previously, ketosis was reported to be associated with hematological and serum biochemical parameters. However, the association between the parameters on the calving date and ketosis during the postpartum transition period remains unclear. This study aimed to investigate this association. Blood samples were collected from the jugular vein of Holstein cows on the calving date and ß-hydroxybutyrate was tested once every 3 days (8 times in 21 days). The cows were divided into three groups: non-ketosis, subclinical ketosis, and clinical ketosis. The clinical ketosis group significantly had the highest values of mean corpuscular volume, mean corpuscular hemoglobin, ß-hydroxybutyrate, non-esterified fatty acids, and total bilirubin, but the lowest values of red cell distribution width, the counts of white blood cell, monocyte, and eosinophil, albumin, alanine transaminase, lactate dehydrogenase, and amylase. In contrast, the non-ketosis group showed the opposite results (p < 0.05). In conclusion, these parameters are associated with the development and severity of ketosis. Our findings suggest that these parameters on the calving date may be useful indicators to identify dairy Holstein cow susceptible to ketosis during the transition period.

How long does the mRNA remains stable in untreated whole bovine blood?

BACKGROUND: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. METHODS AND RESULTS: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage. CONCLUSION: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

Virome Characterization in Commercial Bovine Serum Batches-A Potentially Needed Testing Strategy for Biological Products.

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies' established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.

Characterization of the Bacterial Community of Rumen in Dairy Cows with Laminitis.

Laminitis is the inflammation of the lamella, and it has caused great economic loss to the dairy industry and attracted wide attention around the world. In recent years, microbiota are considered to play a significant role in various diseases processes. Therefore, our study aimed to explore the characteristics of ruminal microbiota in laminitis cows. The serum of bovines with or without laminitis was collected to detect concentrations of lipopolysaccharide (LPS), lactic acid, and histamine, and ruminal fluid was collected for 16S rDNA sequence analysis. The results showed a significant increase in LPS and lactic acid levels in the laminitis group compared to the control group cows. In addition, a higher abundance of Candidatus Saccharimonas, Saccharofermentans, Erysipelotrichaceae UCG-009 genus, Acetobacter pasteurianus, Clostridium papyrosolvens, Ruminococcaceae bacterium AE2021, Porphyromonas crevioricanis, Pseudomonas boreopolis, Pseudomonas psychrotolerans, Rothia nasimurium, and Rothia pickettii was detected in the rumen fluid of laminitis bovines. In conclusion, this article confirms that there are differences in rumen microbiota between healthy and laminitis bovines. The elevated abundance of bacteria that enrich acid-enhancing metabolites, as well as increase the concentration of lactic acid and LPS, could be harmful factors to bovines and increase the risk of laminitis.

Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Changes in metabolic and hormonal profiles during transition period in dairy cattle - the role of spexin.

BACKGROUND: This study aimed to evaluate spexin as a novel blood marker and to describe the relationship of this peptide with selected biochemical metabolites measured during the transition period in dairy cows. Additionally, mRNA expression of the spexin gene as well as spexin receptors - galanin receptor type 2 and galanin receptor type 3, was investigated in several bovine tissues. Blood samples were collected at weekly intervals starting at 21 days before the estimated parturition day until 21 days in milk to determine concentrations of spexin, nonesterified fatty acids, ß-hydroxybutyrate acid, total and active ghrelin, progesterone, glucose, insulin, IGF-I, triglycerides, cholesterol, leptin, corticosterone and 17-ß-estradiol as well as the activity of aspartate transaminase, alkaline phosphatase and gamma-glutamyl transferase. RESULTS: Spexin concentration decreased from 21 d before parturition to calving day and next it rose during the first 14 d of lactation. The lowest concentration of spexin was recorded on the calving day and it differed from the mean level of this peptide before parturition as well as postpartum. Moreover, differences were observed between mean spexin concentrations before and after calving. Spexin levels were moderately negatively correlated with NEFA (r = - 0.39) and total ghrelin contents (r = - 0.41), weakly correlated with BHBA (r = - 0.35) while they showed a moderate positive relationship with progesterone concentrations (r = 0.42). Moreover, we detected that mRNA expression of GALR2, GALR3 and SPX is present in various bovine tissues (kidney, bowel, rumen, spinal cord, lung, skeletal muscle, liver, heart, fat and spleen). CONCLUSION: A negative correlation between spexin concentration and NEFA, BHBA and total ghrelin contents as well as a positive relationship with levels of progesterone, metabolites and hormones, which are key players in the dairy cow transition period, may confirm an important function of this peptide in metabolism regulation. Thus measurement of spexin concentration could provide useful supplementary information for dairy cow herd health monitoring.

Plasma concentrations of branched-chain amino acids differ with Holstein genetic strain in pasture-based dairy systems.

In pasture-based systems, there are nutritional and climatic challenges exacerbated across lactation; thus, dairy cows require an enhanced adaptive capacity compared with cows in confined systems. We aimed to evaluate the effect of lactation stage (21 vs. 180 days in milk, DIM) and Holstein genetic strain (North American Holstein, NAH, n = 8; New Zealand Holstein, NZH, n = 8) on metabolic adaptations of grazing dairy cows through plasma metabolomic profiling and its association with classical metabolites. Although 67 metabolites were affected (FDR < 0.05) by DIM, no metabolite was observed to differ between genetic strains while only alanine was affected (FDR = 0.02) by the interaction between genetic strain and DIM. However, complementary tools for time-series analysis (ASCA analysis, MEBA ranking) indicated that alanine and the branched-chain amino acids (BCAA) differed between genetic strains in a lactation-stage dependent manner. Indeed, NZH cows had lower (P-Tukey < 0.05) plasma concentrations of leucine, isoleucine and valine than NAH cows at 21 DIM, probably signaling for greater insulin sensitivity. Metabolic pathway analysis also revealed that, independently of genetic strains, AA metabolism might be structurally involved in homeorhetic changes as 40% (19/46) of metabolic pathways differentially expressed (FDR < 0.05) between 21 and 180 DIM belonged to AA metabolism.

Low serum vitamin D concentrations in Spring-born dairy calves are associated with elevated peripheral leukocytes.

A role for vitamin D in the immune system is emerging from human research but data in the bovine is limited. In the current study, 48 Holstein-Friesian calves were randomly assigned to one of 4 groups designed to expose calves to divergent vitamin D levels for a 7 month period and to determine its effects on circulating immunity in young calves. Concentrations of circulating 25-hydroxyvitamin D (25OHD) was measured in serum using a commercial ELISA with validated bovine standards. Results showed that mean circulating concentrations of 25OHD at birth was 7.64 ± 3.21 ng/ml indicating vitamin D deficiency. Neither the injection of Vit D3 at birth nor the elevated levels in milk replacer yield discernible changes to pre-weaning circulating concentration of 25OHD. No calf reached the recommended level of vitamin D immune sufficiencyof 30 ng/ml of 25OHD until at least 3 months of age (T4). Increasing dietary Vit D3 via ration in the post-weaning period significantly elevated 25OHD concentrations in serum in VitD-In calves. Maximal levels of circulating 25OHD were achieved in VitD-Out calves, reaching 60.86 ± 7.32 ng/ml at 5 months of age (T7). Greatest divergence in haematology profile was observed between Ctl-In vs VitD-In groups with Ctl-In calves showing an elevated count of neutrophils, eosinophils, and basophils associated with reduced 25OHD concentrations. Neither IL-8 expression nor ROS production in serum were significantly different between calves with high and low 25OHD, indicating that other vitamin D-dependent mechanisms may contribute to the divergent circulating cellular profiles observed. This novel data on the vitamin D status of neonatal calves identifies a significant window of vitamin D insufficiency which is associated with significant differences in circulating immune cell profiles. Vitamin D insufficiency may therefore exacerbate pre-weaning disease susceptibility, and further work in now warranted.

Responses of beef calves to long-term heat stress exposure by evaluating growth performance, physiological, blood and behavioral parameters.

The objective of this study was to explore the responses of beef calves to long-term heat stress (HS) exposure at various levels in comparison with the animals under thermoneutral conditions by evaluating growth performance, physiological, blood, and behavioural parameters. Data were collected from sixteen beef calves (BW: 136.9 ± 6.23 kg; age: 169.6 ± 4.60 d) kept at four stress levels of designated temperature humidity index (THI): threshold (22-24 °C, 60%; THI = 70 to 73), mild (26-28 °C, 60%; THI = 74 to 76), moderate (29-31 °C, 80%; THI = 81 to 83), and severe (32-34 °C, 80%; THI = 89 to 91) stress levels in climatic controlled chambers. Feed and water intake were recorded daily, and body weight was measured once a week. Blood was sampled every three days to analyse metabolite parameters. Dry matter intake (DMI) (p = 0.069, tendency) and blood glucose levels (p = 0.028) were decreased after sudden exposure to HS conditions (severe THI levels). Also, blood cortisol (p = 0.002), glutamic-oxaloacetic transaminase (GOT) (p = 0.009), blood urea nitrogen (BUN) (p = 0.004) and standing time (p = 0.009) were increased in moderate and severe THI levels compared with threshold after exposure to HS conditions. However, in the severe THI group, blood cortisol (p < 0.05), glucose (p < 0.05), GOT (p < 0.05) and BUN (p < 0.05) levels were returned to normal range after 6-13 days of continuous HS exposure. In conclusion, DMI, blood cortisol, GOT, glucose, BUN, and standing time were closely associated with long-term HS condition in beef calves. In addition, calves exposed to HS modulated their physiological responses that resulted in the regulation of the pertinent blood metabolites in the blood to maintain homeostasis during the long-term HS.

Effects of parity order on performance, metabolic, and hormonal parameters of grazing beef cows during pre-calving and lactation periods.

BACKGROUND: Metabolic profile evaluation is a tool widely used in ruminant nutrition as metabolic cues that relate nutrition to physiology. Metabolic and hormonal traits change during pre-partum and lactation according to parity in dairy cows, but studies of beef cows under grazing are scarce. The present study aimed to evaluate how metabolic and hormonal traits change over time, their relationship to performance, and determine if these factors differ according to the parity order of grazing beef cows. Thirty-six pregnant Nellore cows (12 nulliparous, 12 primiparous, and 12 multiparous) were used. The study started at 60 d prepartum until 203 d of lactation. RESULTS: The initial body weight (BW) and final BW were higher for multiparous cows (P > 0.05). An interaction occurred between parity and day (P < .0001) for body condition score. Nulliparous and primiparous body condition score were reduced from - 60 prepartum to 30 postpartum, then stabilized 60 postpartum (P < 0.05), while multiparous maintained body condition score from - 60 days until 60 days postpartum (P > 0.05). Calf BW, final BW, and average daily gain did not differ between parities (P > 0.05). Effect of day (P < 0.05) was detected for non-esterified fatty acids, triglycerides, total cholesterol, LDL, VLDL, progesterone, and insulin. An interaction was observed between parity and days for glucose, HDL, ß-hydroxybutyrate, creatinine and IGF-1 (P < 0.05). Parity affected serum urea nitrogen, total proteins, albumin, and globulins (P < 0.05). Parity and day relative to calving did not impact total T3 and T4 (P > 0.05). CONCLUSIONS: Hormonal and metabolic profile is strongly influenced by the stage of lactation. Time effects (pre-partum and lactation period) were more pronounced in nulliparous since they displayed more unbalanced metabolic and hormonal traits and lowered BCS pre- and postpartum.

Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows.

The aim of this study was to determine the association between concentrations of progesterone (P4) during previous the estrous cycle with the intensity of spontaneous or estrogen-induced estrous expression and pregnancy per artificial insemination (P/AI). A total of 1,953 AI events from lactating Holstein cows were used, consisting of 1,289 timed AI events from experiment 1 (Exp. 1) and 664 AI events from experiment 2 (Exp. 2). In Exp. 1, cows were bred after a timed AI protocol based on estradiol and P4. In Exp. 2 animals were bred upon spontaneous estrus detection. In both experiments cows were continuously monitored by an automated activity monitor (AAM), in Exp.1 a relative increase of activity was calculated (i.e., percentage of increase activity at estrus compared to cow's baseline activity) and in Exp.2, activity data from each cow were computed into an index value that ranged from 0 to 100. In Exp.2 duration (hours) of estrus were calculated and defined as the total time above the threshold (35 index). The intensity of estrous expression was determined for each event and classified as either high or low intensity using the median of each experiment. Blood samples were collected for P4 analysis in Exp. 1 at -4 d, 0 d, and 7 d relative to timed AI, and in Exp. 2 immediately following estrus (0 d), 7 d, 14 d, and 21 d post-AI. Concentration of P4 was classified as greater or lower according to the median value in each experiment. Cows with lower concentrations of P4 at AI had greater estrous expression in Exp. 1 (363.6 ± 5.2 vs. 275.9 ± 8.0% relative increase) and Exp. 2 (76.7 ± 1.9 vs. 67.4 ± 4.7 index; and 12.5 ± 0.5 vs. 9.3 ± 1.8 hours). Cows with a greater intensity of estrous expression at timed AI had greater concentrations of P4 at -4 d than cows with lower intensity estrus or no estrous expression (4.6 ± 0.2 vs. 3.6 ± 0.2 vs. 3.7 ± 0.2 ng/mL). Cows with greater concentrations of P4 at -4 d had greater P/AI (32.8 ± 4.4 vs. 22.4 ± 4.5%), whereas cows with lesser concentrations of P4 at d0 for either timed AI (35.2 ± 3.4 vs. 19.6 ± 4.6%) or spontaneous estrus (31.8 ± 2.8 vs. 23.4 ± 3.2%) had greater P/AI. Cows with greater concentrations of P4 7 d post-timed AI had greater P/AI compared with cows that had lower concentration of P4 (39.1 ± 2.9 vs. 24.7 ± 2.6%). Similarly, cows that had lower concentrations of P4 at 7 d, 14 d and 21 d post-spontaneous estrus tended to have lower P/AI when compared with cows with greater concentrations of P4. Overall, concentrations of P4 prior to and at AI were associated with greater estrous intensity and P/AI at both spontaneous and timed AI events.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.