Udder Ultrasonography of Dairy Cows: Investigating the Relationship between Echotexture, Blood Flow, Somatic Cell Count and Milk Yield during Dry Period and Lactation.

Udder health of dairy cows is related to their productivity and welfare. The period from dry-off to calving and early lactation is crucial. Ultrasonography is a useful and practical tool for the examination of the mammary parenchyma and blood flow. This observational study investigated the relationship between udder echotexture features, blood flow volume (BFVol) in the milk vein, milk somatic cell count (SCC) and daily milk yield (DMY) from late lactation, throughout the dry period and consecutive early lactation. Seventeen repeated measurements were performed on twenty-one Holstein cows. The udder parenchyma was examined with B-mode ultrasonography. Udder echotexture was studied using 15 features: Numerical Pixel Value (NPV), Pixel Standard Deviation (PSD), Skewness, Excess, Contrast, Homogeneity, Correlation, Entropy, Run Percentage, Long-Run Emphasis, Grey Value Distribution, Runlength Distribution, Gradient Mean Value, Gradient Variance and Percentage of Non-zero Gradients. Blood flow in the milk vein was examined with spectral Doppler. Linear mixed-effects models were employed to investigate relationships between BFVol, udder echotexture features, SCC and DMY throughout the study period. Our models showed that a 1 kg increase in DMY was associated with a significant increase of 0.25 L/min in the expected BFVol and that a 1,000,000-cells/mL increase in SCC was associated with a significant BFVol decrease of 0.49 L/min, keeping all other variables constant. Multivariable models showed significant associations between DMY and NPV, between PSD and Long-Run Emphasis, and between SCC and NPV, PSD, Gradient Mean Value, Homogeneity, Gradient Variance and Entropy. In conclusion, udder echotexture and BFVol in the milk vein are related to SCC and milk yield. Ultrasonography can be used for the comprehensive assessment of udder health in support of precision dairy farming.

Identifying novel regulatory effects for clinically relevant genes through the study of the Greek population.

BACKGROUND: Expression quantitative trait loci (eQTL) studies provide insights into regulatory mechanisms underlying disease risk. Expanding studies of gene regulation to underexplored populations and to medically relevant tissues offers potential to reveal yet unknown regulatory variants and to better understand disease mechanisms. Here, we performed eQTL mapping in subcutaneous (S) and visceral (V) adipose tissue from 106 Greek individuals (Greek Metabolic study, GM) and compared our findings to those from the Genotype-Tissue Expression (GTEx) resource. RESULTS: We identified 1,930 and 1,515 eGenes in S and V respectively, over 13% of which are not observed in GTEx adipose tissue, and that do not arise due to different ancestry. We report additional context-specific regulatory effects in genes of clinical interest (e.g. oncogene ST7) and in genes regulating responses to environmental stimuli (e.g. MIR21, SNX33). We suggest that a fraction of the reported differences across populations is due to environmental effects on gene expression, driving context-specific eQTLs, and suggest that environmental effects can determine the penetrance of disease variants thus shaping disease risk. We report that over half of GM eQTLs colocalize with GWAS SNPs and of these colocalizations 41% are not detected in GTEx. We also highlight the clinical relevance of S adipose tissue by revealing that inflammatory processes are upregulated in individuals with obesity, not only in V, but also in S tissue. CONCLUSIONS: By focusing on an understudied population, our results provide further candidate genes for investigation regarding their role in adipose tissue biology and their contribution to disease risk and pathogenesis.

Colour Doppler study of blood flow in the portal vein in relation to blood flow in the milk vein, milk yield and body condition of dairy cows during dry period and lactation.

In dairy cows, the liver supports the increased nutrient demands of the udder for milk production. Blood flow is key for the transport of these nutrients. This study investigated portal vein blood flow volume (PVBFVol) in relation to daily milk yield (DMY), milk vein blood flow volume (MVBFVol) and body condition parameters of high-producing dairy cows, starting from late lactation, throughout dry period, and consecutive early lactation. Seventeen repeated examinations were performed on 19 Holstein cows and 313 measurement days were finally included. Vein morphology and blood flow were examined via B-mode and spectral Doppler (triplex) ultrasonography, respectively. Body condition parameters recorded were body condition score (BCS), backfat thickness (BFT) measurement with ultrasonography, heart girth circumference (HG) and withers height (WH). Longitudinal relationship of PVBFVol with MVBFVol, DMY, BCS, BFT, HG and WH was analyzed with linear mixed models, with random intercept effects, using restricted cubic splines. A significant increase of 8.28% (p < 0.01) in PVBFVol appeared for every 1 L/min increase in MVBFVol in the univariable model. PVBFVol presented a significant negative association with BCS (p < 0.01) and BFT (p = 0.02), while interaction with production stage was significant, too. PVBFVol significantly increased by 0.38% (p = 0.04) for every 1 kg increase in DMY in the multivariable model. In conclusion, the increased PVBFVol during lactation accompanies the escalation in metabolic activity of the liver and the increased blood circulation through the udder, coping with the udder's escalating nutrient demands for milk synthesis.

Mapping interindividual dynamics of innate immune response at single-cell resolution.

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

Genetic Parameters of Serum Total Protein Concentration Measured with a Brix Refractometer in Holstein Newborn Calves and Fresh Cows.

The objective was to estimate the genetic parameters of serum total protein concentration in newborn calves (calfSTP) and post parturient dairy cows (cowSTP). The study included 1013 calves and 989 cows from 10 dairy farms. Calf blood samples were collected 24-48 h after parturition while cow blood and colostrum samples were collected in the first 24 h after calving. Blood serum total protein and colostrum total solids content were determined using a Brix refractometer. Chemical analysis of colostrum was performed with Milkoscan. Univariate mixed linear models were used to estimate the heritability of calfSTP and cowSTP and their genetic and phenotypic correlations with colostrum traits. The heritability estimates of calfSTP and cowSTP were 0.21 and 0.20 (p < 0.05), respectively. Strong genetic correlations (r > 0.90) were detected between calfSTP and colostrum total solids and protein content (p < 0.05). Corresponding phenotypic correlations were 0.31-0.33 (p < 0.05). No genetic or phenotypic correlations were detected with colostrum fat content while the respective correlations with lactose were negative (-0.82 and -0.19, p < 0.05). No genetic correlations were detected between cowSTP and colostrum traits and only a low negative phenotypic one with lactose was detected. The results confirm that genetic selection aiming to improve the passive transfer of immunity in newborn calves and general fresh cow health would be feasible.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

Phenotypical Variation of Ruminal Volatile Fatty Acids and pH during the Peri-Weaning Period in Holstein Calves and Factors Affecting Them.

Two hundred and forty-three clinically healthy Holstein calves from eight commercial dairy farms were used to: (a) describe the evolutionary course of ruminal VFA concentration and pH during the peri-weaning period and (b) assess management factors affecting their phenotypical variation of these parameters. Management practices were recorded individually for each calf as these were not fixed within farms. Samples of ruminal fluid were collected at -7 d, 0 d, and 7 d relative to weaning. Gas chromatography was used to measure ruminal VFAs, and pH was measured on site. Linear mixed models for repeated measurements were used to assess the effects of management factors and their interactions. A large among-calves phenotypical variability was observed. Estimated marginal means showed that concentrations of acetate, butyrate, and total VFAs (but not of propionate) significantly decreased, while acetate propionate increased, from -7 d to 7 d. Age at weaning and body weight at -7 d were positively associated with total and several individual VFA concentrations. Group housing and late forage feeding pre-weaning were associated with higher VFA concentrations; the same factors, as well as step-weaning, were associated with pH values around 6. Feeding 7-8 L of milk replacer daily did not preclude a smooth transition, irrespective of weaning method.

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project.Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p = 3.7 × 10-6), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10-10) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood (p = 6.14 × 10-9). Additionally, ORM1 expression correlated with levels of cfDNA (p = 4.38 × 10-4). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association (ρ g = 0.43, p = 0.089).All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Hematology reference intervals during the prepartum period, first week after calving, and peak lactation in clinically healthy Holstein cows.

Dairy cows face precipitous metabolic adaptations during the first postpartum week. Increasing knowledge of their hematologic profiles during this period could benefit both veterinary clinical pathologists and bovine practitioners. The objectives of this study were: (a) to establish period-specific RIs for Holstein dairy cows and (b) to assess the potential simultaneous effect of several factors on hematology parameters. Blood samples from clinically healthy cows of nine herds were used to establish hematology RIs for these periods. There were 68 cows in the prepartum period (PP), 247 and 218 cows at 2 and 8 days after calving (2 and 8 days in milk [DIM 2 and DIM 8], respectively), and 87 cows in peak lactation (PL). A complete blood count evaluation was performed using the ADVIA 120 analyzer. Linear models estimated the analytes associated with parity. RIs were calculated with Reference Value Advisor. Parity had a significant effect on analytes at all stages. Marked differences were evident between DIM 2 and 8 regarding red blood cell (RBC) counts, hematocrits, hemoglobin concentrations, and white blood cell (WBC), monocyte, and eosinophil counts. Reference intervals of RBC counts, hematocrits, and hemoglobin concentrations at DIM 2 were higher than those at PP and PL. This study provides evidence that period-specific RIs should be used for Holstein cows when making clinical decisions, especially during the critical first week postpartum.

Evaluation of Factors Affecting Colostrum Quality and Quantity in Holstein Dairy Cattle.

The objective of this study was to conduct a large-scale investigation of colostrum composition and yield and an evaluation of factors affecting them. In this study, 1017 clinically healthy Holstein cows from 10 farms were used. The colostrum TS were measured using a digital Brix refractometer. Fat, protein and lactose content were determined using an infrared Milk Analyzer. Statistical analysis was conducted using a series of univariate general linear models. The mean (±SD) percentage of colostrum fat, protein, lactose and TS content were 6.37 (3.33), 17.83 (3.97), 2.15 (0.73) and 25.80 (4.68), respectively. Parity had a significant positive effect on the protein and TS content and a negative one on fat content. The time interval between calving and colostrum collection had a significant negative effect on the fat, protein and TS contents and a positive one on lactose. Colostrum yield had a significant negative effect on the protein and TS content, and it was affected by all factors considered. In addition to TS, the evaluation of the colostrum fat content appears essential when neonates' energy needs are considered. The Brix refractometer, an inexpensive and easy to use devise, can be used effectively in colostrum quality monitoring.

A map of transcriptional heterogeneity and regulatory variation in human microglia.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), play critical roles in immune defense, development and homeostasis. However, isolating microglia from humans in large numbers is challenging. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single-cell and bulk RNA sequencing, we identify how age, sex and clinical pathology influence microglia gene expression and which genetic variants have microglia-specific functions using expression quantitative trait loci (eQTL) mapping. We follow up one of our findings using a human induced pluripotent stem cell-based macrophage model to fine-map a candidate causal variant for Alzheimer's disease at the BIN1 locus. Our study provides a population-scale transcriptional map of a critically important cell for human CNS development and disease.

Coxiella burnetii Shedding in Milk and Molecular Typing of Strains Infecting Dairy Cows in Greece.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.

Association of Body Condition Score with Ultrasound Measurements of Backfat and Longissimus Dorsi Muscle Thickness in Periparturient Holstein Cows.

Most cows experience a period of nutrient deficit during the periparturient period. Body condition scoring (BCS) is widely used on farms to assess body nutrient reserves and mobilization. The aims of this study were to: (i) determine the association of BCS with ultrasound measurements of backfat (BFT) and longissimus dorsi muscle thickness (LDT) during the periparturient period of Holstein cows from different herds, accounting for potential sources of variation, such as herd, parity and period relative to calving and (ii) establish reference intervals (RIs) for BFT and LDT per BCS estimate. Two-hundred and fifty-two cows from six commercial farms were used. Body condition scores, BFT and LDT were assessed at seven time-points during the periparturient period. Assessments of BCS estimates as predictors of BFT and LDT and the contribution of BFT and LDT to BCS estimates were performed with the use of linear mixed models. Reference intervals for BFT and LDT per BCS estimate were established with the Reference Value Advisor. One unit of BCS change was associated with 8.2 mm of BFT and 10.9 mm of LDT pre- and postpartum. Range of BFT and LDT in established RIs per BCS was wide with significant overlap. Both subcutaneous fat and, to a lesser degree, skeletal muscle reserves contribute to BCS estimation. Repeated BCS estimations credibly predict energy balance status in periparturient dairy cows. The metabolic state of muscle tissue should be assessed by repeated ultrasound measurements.

Genetic variant effects on gene expression in human pancreatic islets and their implications for T2D.

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.

Extensive fragmentation and re-organization of transcription in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is the prototype of autoimmune diseases, characterized by extensive gene expression perturbations in peripheral blood immune cells. Circumstantial evidence suggests that these perturbations may be due to altered epigenetic profiles and chromatin accessibility but the relationship between transcriptional deregulation and genome organization remains largely unstudied. In this work we propose a genomic approach that leverages patterns of gene coexpression from genome-wide transcriptome profiles in order to identify statistically robust Domains of Co-ordinated gene Expression (DCEs). Application of this method on a large transcriptome profiling dataset of 148 SLE patients and 52 healthy individuals enabled the identification of significant disease-associated alterations in gene co-regulation patterns, which also correlate with SLE activity status. Low disease activity patient genomes are characterized by extensive fragmentation leading to overall fewer DCEs of smaller size. High disease activity genomes display extensive redistribution of co-expression domains with expanded and newly-appearing (emerged) DCEs. The dynamics of domain fragmentation and redistribution are associated with SLE clinical endophenotypes, with genes of the interferon pathway being highly enriched in DCEs that become disrupted and with functions associated to more generalized symptoms, being located in domains that emerge anew in high disease activity genomes. Our results suggest strong links between the SLE phenotype and the underlying genome structure and underline an important role for genome organization in shaping gene expression in SLE.

Single cell transcriptome in aneuploidies reveals mechanisms of gene dosage imbalance.

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.

Association of bulk tank milk urea nitrogen concentration with elevated individual cow values and investigation of sampling frequency for accurate assessment.

Individual milk urea nitrogen (MUN) levels ≥ 19.63 mg/dL have been recently reported to significantly affect fertility. The objectives of the present study were to (a) predict the percentage of cows with elevated MUN within a herd using bulk tank (BTMUN) levels, in the absence of individual MUN records, and (b) establish a sampling frequency protocol for the assessment of actual BTMUN levels. A database of 17,687 monthly individual MUN and concurrent 229 monthly BTMUN records from 24 dairy herds was used. A ROC analysis was performed to determine the BTMUN threshold over which cows in the herd have elevated MUN concentrations that, based on literature, affect fertility. Moreover, a regression was run to predict the percentage of cows with elevated MUN within a herd from BTMUN values. A second database of 10,687 daily BTMUN records from 29 herds was used to identify an appropriate sampling frequency to assess the actual BTMUN levels. Eleven different sampling frequencies ranging from once to 8 times per month were assessed. A BTMUN value of 15.76 mg/dL was the optimum threshold over which cows with elevated MUN concentrations are included in a herd. The percentage of cows with elevated MUN values can be accurately predicted using BTMUN values (R2 = 0.872; P < 0.001). A bulk tank sampling frequency of once per week seems appropriate for most herds in order to assess the actual BTMUN levels, in case daily BTMUN values are not available from milk processors.

Combined genetic and transcriptome analysis of patients with SLE: distinct, targetable signatures for susceptibility and severity.

OBJECTIVES: Systemic lupus erythematosus (SLE) diagnosis and treatment remain empirical and the molecular basis for its heterogeneity elusive. We explored the genomic basis for disease susceptibility and severity. METHODS: mRNA sequencing and genotyping in blood from 142 patients with SLE and 58 healthy volunteers. Abundances of cell types were assessed by CIBERSORT and cell-specific effects by interaction terms in linear models. Differentially expressed genes (DEGs) were used to train classifiers (linear discriminant analysis) of SLE versus healthy individuals in 80% of the dataset and were validated in the remaining 20% running 1000 iterations. Transcriptome/genotypes were integrated by expression-quantitative trail loci (eQTL) analysis; tissue-specific genetic causality was assessed by regulatory trait concordance (RTC). RESULTS: SLE has a 'susceptibility signature' present in patients in clinical remission, an 'activity signature' linked to genes that regulate immune cell metabolism, protein synthesis and proliferation, and a 'severity signature' best illustrated in active nephritis, enriched in druggable granulocyte and plasmablast/plasma-cell pathways. Patients with SLE have also perturbed mRNA splicing enriched in immune system and interferon signalling genes. A novel transcriptome index distinguished active versus inactive disease-but not low disease activity-and correlated with disease severity. DEGs discriminate SLE versus healthy individuals with median sensitivity 86% and specificity 92% suggesting a potential use in diagnostics. Combined eQTL analysis from the Genotype Tissue Expression (GTEx) project and SLE-associated genetic polymorphisms demonstrates that susceptibility variants may regulate gene expression in the blood but also in other tissues. CONCLUSION: Specific gene networks confer susceptibility to SLE, activity and severity, and may facilitate personalised care.

Hematology reference intervals for neonatal Holstein calves.

Data regarding hematologic reference intervals (RI) for neonatal calves have not been published yet. The aims of this study were: a) to establish hematology RIs for neonatal Holstein calves, b) to compare them with the RIs for lactating cows, and c) to investigate the relationship of age and gender with the hematologic profile of calves. Two-hundred and fifty-four clinically healthy Holstein calves (1-9days old, from 30 farms) and 82 healthy Holstein cows (between 30 and 150days in milk, from 10 farms) were blood sampled once for a complete blood count evaluation, using the ADVIA 120 hematology analyzer. An additional blood sample was collected from each calf for serum total protein concentration measurement. RIs and age-related RIs were calculated with the Reference Value Advisor freeware. Comparisons between calves and cows and between male and female calves were performed with t-test or Mann-Whitney test. Red blood cell count (RBC), white blood cell count (WBC), neutrophil, lymphocyte and platelet counts in calves were higher, while mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were lower than in cows. Lymphocyte and platelets showed a notable increase through age. Finally, female calves had higher RBC, hematocrit and hemoglobin concentration than males. Age-specific RIs should be used for the interpretation of the complete blood count in Holstein calves.