Does inbreeding contribute to pregnancy loss in Thoroughbred horses?

BACKGROUND: Excessive inbreeding increases the probability of uncovering homozygous recessive genotypes and has been associated with an increased risk of retained placenta and lower semen quality. No genomic analysis has investigated the association between inbreeding levels and pregnancy loss. OBJECTIVES: To compare genetic inbreeding coefficients (F) of naturally occurring Thoroughbred Early Pregnancy Loss (EPLs), Mid and Late term Pregnancy Loss (MLPL) and Controls. The F value was hypothesised to be higher in cases of pregnancy loss (EPLs and MLPLs) than Controls. STUDY DESIGN: Observational case-control study. METHODS: Allantochorion and fetal DNA from EPL (n = 37, gestation age 14-65 days), MLPL (n = 94, gestational age 70 days-24 h post parturition) and Controls (n = 58) were genotyped on the Axiom Equine 670K SNP Genotyping Array. Inbreeding coefficients using Runs of Homozygosity (FROH) were calculated using PLINK software. ROHs were split into size categories to investigate the recency of inbreeding. RESULTS: MLPLs had significantly higher median number of ROH (188 interquartile range [IQR], 180.8-197.3), length of ROH (3.10, IQR 2.93-3.33), and total number of ROH (590.8, IQR 537.3-632.3), and FROH (0.26, IQR 0.24-0.28) when compared with the Controls and the EPLs (p < 0.05). There was no significant difference in any of the inbreeding indices between the EPLs and Controls. The MLPLs had a significantly higher proportion of long (>10 Mb) ROH (2.5%, IQR 1.6-3.6) than the Controls (1.7%, IQR 0.6-2.5), p = 0.001. No unique ROHs were found in the EPL or MLPL populations. MAIN LIMITATIONS: SNP-array data does not allow analysis of every base in the sequence. CONCLUSIONS: This first study of the effect of genomic inbreeding levels on pregnancy loss showed that inbreeding is a contributor to MLPL, but not EPL in the UK Thoroughbred population. Mating choices remain critical, because inbreeding may predispose to MLPL by increasing the risk of homozygosity for specific lethal allele(s).

An organism-wide ATAC-seq peak catalog for the bovine and its use to identify regulatory variants.

We report the generation of an organism-wide catalog of 976,813 cis-acting regulatory elements for the bovine detected by the assay for transposase accessible chromatin using sequencing (ATAC-seq). We regroup these regulatory elements in 16 components by nonnegative matrix factorization. Correlation between the genome-wide density of peaks and transcription start sites, correlation between peak accessibility and expression of neighboring genes, and enrichment in transcription factor binding motifs support their regulatory potential. Using a previously established catalog of 12,736,643 variants, we show that the proportion of single-nucleotide polymorphisms mapping to ATAC-seq peaks is higher than expected and that this is owing to an approximately 1.3-fold higher mutation rate within peaks. Their site frequency spectrum indicates that variants in ATAC-seq peaks are subject to purifying selection. We generate eQTL data sets for liver and blood and show that variants that drive eQTL fall into liver- and blood-specific ATAC-seq peaks more often than expected by chance. We combine ATAC-seq and eQTL data to estimate that the proportion of regulatory variants mapping to ATAC-seq peaks is approximately one in three and that the proportion of variants mapping to ATAC-seq peaks that are regulatory is approximately one in 25. We discuss the implication of these findings on the utility of ATAC-seq information to improve the accuracy of genomic selection.

Effects of Individual and Pair Housing of Calves on Short-Term Health and Behaviour on a UK Commercial Dairy Farm.

Social pair housing of calves has previously demonstrated positive impacts for calves, so this study aimed to compare the health and behaviour of calves kept in individual compared to pair housing on a single commercial UK dairy farm. A total of 457 Holstein and Jersey heifer calves were recruited and systematically allocated to individual and pair housing. Weekly visits were conducted up to 8 weeks of age, with weight and presence of clinical disease measured using both a standardized scoring system and thoracic ultrasonography. A subset of calves (n = 90) had accelerometers attached to monitor activity, with CCTV placed above a further 16 pens to allow behavioural assessments to be made via continuous focal sampling at 1 and 5 weeks of age. During the study, there was a mortality rate of 2.8%, and an average daily liveweight gain (ADLG) of 0.72 kg/day, with no significant effect of housing group (p = 0.76). However, individually housed calves had increased odds of developing disease (OR = 1.88, p = 0.014). Accelerometer data showed that housing group had no effect on lying times, with a mean of 18 h 11 min per day (SD 39 min) spent lying down. The motion index was significantly higher in pair-housed calves (F1,83 = 440.3, p < 0.01), potentially due to more social play behaviour. The total time engaged in non-nutritive oral behaviours (NNOBs) was not impacted by housing group (p = 0.72). Pair-housed calves split their time conducting NNOBs equally between inanimate objects and on their pen mates' body. Individually housed calves spent significantly more time with their head out of the front of the pen (p = 0.006), and also engaged in more self-grooming than pair-housed calves (p = 0.017), possibly due to a lack of socialization. The overall findings of this study indicate that within a UK commercial dairy management system, pair-housed calves were healthier and more active than individually housed calves, while housing group did not influence ADLG or the occurrence of NNOBs.

Hepatic Global Transcriptomic Profiles of Holstein Cows According to Parity Reveal Age-Related Changes in Early Lactation.

Cows can live for over 20 years, but their productive lifespan averages only around 3 years after first calving. Liver dysfunction can reduce lifespan by increasing the risk of metabolic and infectious disease. This study investigated the changes in hepatic global transcriptomic profiles in early lactation Holstein cows in different lactations. Cows from five herds were grouped as primiparous (lactation number 1, PP, 534.7 ± 6.9 kg, n = 41), or multiparous with lactation numbers 2-3 (MP2-3, 634.5 ± 7.5 kg, n = 87) or 4-7 (MP4-7, 686.6 ± 11.4 kg, n = 40). Liver biopsies were collected at around 14 days after calving for RNA sequencing. Blood metabolites and milk yields were measured, and energy balance was calculated. There were extensive differences in hepatic gene expression between MP and PP cows, with 568 differentially expressed genes (DEGs) between MP2-3 and PP cows, and 719 DEGs between MP4-7 and PP cows, with downregulated DEGs predominating in MP cows. The differences between the two age groups of MP cows were moderate (82 DEGs). The gene expression differences suggested that MP cows had reduced immune functions compared with the PP cows. MP cows had increased gluconeogenesis but also evidence of impaired liver functionality. The MP cows had dysregulated protein synthesis and glycerophospholipid metabolism, and impaired genome and RNA stability and nutrient transport (22 differentially expressed solute carrier transporters). The genes associated with cell cycle arrest, apoptosis, and the production of antimicrobial peptides were upregulated. More surprisingly, evidence of hepatic inflammation leading to fibrosis was present in the primiparous cows as they started their first lactation. This study has therefore shown that the ageing process in the livers of dairy cows is accelerated by successive lactations and increasing milk yields. This was associated with evidence of metabolic and immune disorders together with hepatic dysfunction. These problems are likely to increase involuntary culling, thus reducing the average longevity in dairy herds.

Comparison of environment quality measurements between 3 types of calf housing in the United Kingdom.

Preweaning calves are kept in a range of housing types that offer variable protection against the weather and provide differing internal environments. This cross-sectional observational study assessed the effect of housing type (shed, polytunnel, or hutches) on internal environmental parameters, using 2 blocks of 8-wk measurements from 10 commercial dairy farms in the south of England, covering both summer and winter periods. Continuous measurements for internal and external temperature and humidity were recorded by data logger placed within the calf housing and used to calculate the temperature-humidity index (THI). Weekly point readings were also taken for temperature, humidity, light, air speed, ammonia level, and airborne particulate matter. Airborne bacterial levels were determined at wk 2, 5, and 8 by incubating air samples at 35°C for 24 h in aerobic conditions. Data were analyzed using linear mixed models. Housing type influenced THI significantly in both seasons. In summer, calves were exposed to heat stress conditions (THI ≥72) for 39, 31, and 14 of 46 d in polytunnel housing, hutches, and sheds, respectively. The maximum summer temperature (37.0°C) was recorded in both hutch and polytunnel housing, with sheds remaining consistently cooler (maximum 31.0°C). In winter, the lowest minimum internal temperature recorded was in hutches at -4.5°C, with both the sheds and polytunnel, but not hutches, providing a significant increase in temperature compared with the external environment. Hutches remained ≤ 10°C for 86% of the winter study period. Light levels were reduced in all housing types compared with the external environment. The particulate matter in air that is capable of reaching the lungs (particulate matter <10 µm) was highest in sheds, intermediate in hutches, and lowest in polytunnel housing (0.97 ± 3.75, 0.37 ± 0.44, and 0.20 ± 0.24 mg/m3, respectively). This was mirrored by airborne bacterial numbers, which were also highest in sheds (8,017 ± 2,141 cfu/m3), intermediate in hutches (6,870 ± 2,084 cfu/m3), and lowest in the polytunnel (3,357 ± 2,572 cfu/m3). Round, white, catalase-positive, and oxidase-negative colonies were most prevalent, likely indicating Staphylococcus species. This study demonstrated that UK calves are routinely exposed to either heat or cold stress, especially when housed in hutches or polytunnels. Sheds had the highest levels of particulate matter and airborne bacteria, both known contributory factors for respiratory disease. These findings demonstrate that all calf housing systems result in environmental compromises that could have long-term impacts on calf health and growth; therefore, further studies should identify husbandry and housing modifications to mitigate these factors.

Effects of the Individual and Pair Housing of Calves on Long-Term Heifer Production on a UK Commercial Dairy Farm.

Pair housing of pre-weaning dairy calves has previously demonstrated positive impacts on their growth, health and behaviour, but longer-term effects on production are still relatively unknown. This study followed a cohort of 431 Holstein heifers, recruited from a single UK commercial dairy farm, from weaning until either culling or the end of their first lactation. All animals were allocated to either individual or pair housing as a pre-weaning calf. Following weaning, all heifers were similarly managed through group housing, feeding with total mixed rations, the use of automatic heat detection for artificial insemination and weighing every two months until conception. Farm staff identified disease occurrences, which were treated following standard operating procedures. First-lactation monthly milk recording was used to measure milk yields and somatic cell counts. Overall mortality (voluntary and involuntary) was 26.6%, with a decreased hazard of exiting the herd if the heifer was pair housed as a calf (HR 0.70; p = 0.067). The voluntary cull rate was highest in the post-insemination period (13.0%) due to poor fertility. Heifers that were pair housed as calves had significantly increased odds of developing udder health issues as a primiparous cow (OR = 1.93, p = 0.022). Despite this, the 305-day milk yields were not associated with the housing group. However, the total milk produced per calf recruited into the original study was greater for pair-housed compared with individually housed calves (8088 kg vs. 7115 kg; p = 0.071), which is likely due to the significantly higher hazard of individually housed calves exiting the herd prematurely.

Developmental Programming of Fertility in Cattle-Is It a Cause for Concern?

Cattle fertility remains sub-optimal despite recent improvements in genetic selection. The extent to which an individual heifer fulfils her genetic potential can be influenced by fetal programming during pregnancy. This paper reviews the evidence that a dam's age, milk yield, health, nutrition and environment during pregnancy may programme permanent structural and physiological modifications in the fetus. These can alter the morphology and body composition of the calf, postnatal growth rates, organ structure, metabolic function, endocrine function and immunity. Potentially important organs which can be affected include the ovaries, liver, pancreas, lungs, spleen and thymus. Insulin/glucose homeostasis, the somatotropic axis and the hypothalamo-pituitary-adrenal axis can all be permanently reprogrammed by the pre-natal environment. These changes may act directly at the level of the ovary to influence fertility, but most actions are indirect. For example, calf health, the timing of puberty, the age and body structure at first calving, and the ability to balance milk production with metabolic health and fertility after calving can all have an impact on reproductive potential. Definitive experiments to quantify the extent to which any of these effects do alter fertility are particularly challenging in cattle, as individual animals and their management are both very variable and lifetime fertility takes many years to assess. Nevertheless, the evidence is compelling that the fertility of some animals is compromised by events happening before they are born. Calf phenotype at birth and their conception data as a nulliparous heifer should therefore both be assessed to avoid such animals being used as herd replacements.

Locating a novel autosomal recessive genetic variant in the cattle glucokinase gene using only WGS data from three cases and six carriers.

New Mendelian genetic conditions, which adversely affect livestock, arise all the time. To manage them effectively, some methods need to be devised that are quick and accurate. Until recently, finding the causal genomic site of a new autosomal recessive genetic disease has required a two-stage approach using single-nucleotide polymorphism (SNP) chip genotyping to locate the region containing the new variant. This region is then explored using fine-mapping methods to locate the actual site of the new variant. This study explores bioinformatic methods that can be used to identify the causative variants of recessive genetic disorders with full penetrance with just nine whole genome-sequenced animals to simplify and expedite the process to a one-step procedure. Using whole genome sequencing of only three cases and six carriers, the site of a novel variant causing perinatal mortality in Irish moiled calves was located. Four methods were used to interrogate the variant call format (VCF) data file of these nine animals, they are genotype criteria (GCR), autozygosity-by-difference (ABD), variant prediction scoring, and registered SNP information. From more than nine million variants in the VCF file, only one site was identified by all four methods (Chr4: g.77173487A>T (ARS-UCD1.2 (GCF_002263795.1)). This site was a splice acceptor variant located in the glucokinase gene (GCK). It was verified on an independent sample of animals from the breed using genotyping by polymerase chain reaction at the candidate site and autozygosity-by-difference using SNP-chips. Both methods confirmed the candidate site. Investigation of the GCR method found that sites meeting the GCR were not evenly spread across the genome but concentrated in regions of long runs of homozygosity. Locating GCR sites was best performed using two carriers to every case, and the carriers should be distantly related to the cases, within the breed concerned. Fewer than 20 animals need to be sequenced when using the GCR and ABD methods together. The genomic site of novel autosomal recessive Mendelian genetic diseases can be located using fewer than 20 animals combined with two bioinformatic methods, autozygosity-by-difference, and genotype criteria. In many instances it may also be confirmed with variant prediction scoring. This should speed-up and simplify the management of new genetic diseases to a single-step process.

Transcriptomic Analysis of Circulating Leukocytes Obtained during the Recovery from Clinical Mastitis Caused by Escherichia coli in Holstein Dairy Cows.

The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts' phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection.

Proportion of Concentrate in the Diet of Early Lactation Dairy Cows Has Contrasting Effects on Circulating Leukocyte Global Transcriptomic Profiles, Health and Fertility According to Parity.

The functionality of circulating leukocytes in dairy cows is suppressed after calving, with negative energy balance as a risk factor. Leukocyte transcriptomic profiles were compared separately in 44 multiparous (MP) and 18 primiparous (PP) Holstein-Friesian cows receiving diets differing in concentrate proportion to test whether immune dysfunction could be mitigated by appropriate nutrition. After calving, cows were offered either (1) low concentrate (LC); (2) medium concentrate (MC) or (3) high concentrate (HC) diets with proportions of concentrate to grass silage of 30%:70%, 50%:50% and 70%:30%, respectively. Cow phenotype data collected included circulating metabolites, milk yield and health and fertility records. RNA sequencing of circulating leukocytes at 14 days in milk was performed. The HC diet improved energy balance in both age groups. There were more differentially expressed genes in PP than MP cows (460 vs. 173, HC vs. LC comparison) with few overlaps. The MP cows on the LC diet showed upregulation of the complement and coagulation cascade and innate immune defence mechanisms against pathogens and had a trend of more cases of mastitis and poorer fertility. In contrast, the PP cows on the HC diet showed greater immune responses based on both gene expression and phenotypic data and longer interval of calving to conception. The leukocytes of MP and PP cows therefore responded differentially to the diets between age, nutrient supply and immunity affecting their health and subsequent fertility.

Global transcriptomic profiles of circulating leucocytes in early lactation cows with clinical or subclinical mastitis.

Bovine mastitis, an inflammatory disease of the mammary gland, is classified as subclinical or clinical. Circulating neutrophils are recruited to the udder to combat infection. We compared the transcriptomic profiles in circulating leukocytes between healthy cows and those with naturally occurring subclinical or clinical mastitis. Holstein Friesian dairy cows from six farms in EU countries were recruited. Based on milk somatic cell count and clinical records, cows were classified as healthy (n = 147), subclinically (n = 45) or clinically mastitic (n = 22). Circulating leukocyte RNA was sequenced with Illumina NextSeq single end reads (30 M). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21, followed by GO enrichment analysis. Both subclinical and clinical mastitis caused significant changes in the leukocyte transcriptome, with more intensive changes attributed to clinical mastitis. We detected 769 DEGs between clinical and healthy groups, 258 DEGs between subclinical and healthy groups and 193 DEGs between clinical and subclinical groups. Most DEGs were associated with cell killing and immune processes. Many upregulated DEGs in clinical mastitis encoded antimicrobial peptides (AZU1, BCL3, CAMP, CATHL1, CATHL2, CATHL4,CATHL5, CATHL6, CCL1, CXCL2, CXCL13, DEFB1, DEFB10, DEFB4A, DEFB7, LCN2, PGLYRP1, PRTN3, PTX3, S100A8, S100A9, S100A12, SLC11A1, TF and LTF) which were not upregulated in subclinical mastitis. The use of transcriptomic profiles has identified a much greater up-regulation of genes encoding antimicrobial peptides in circulating leukocytes of cows with naturally occurring clinical compared with subclinical mastitis. These could play a key role in combatting disease organisms.

Comparison of the Ability of High and Low Virulence Strains of Non-cytopathic Bovine Viral Diarrhea Virus-1 to Modulate Expression of Interferon Tau Stimulated Genes in Bovine Endometrium.

Bovine Viral Diarrhea virus (BVDV) is a pestivirus with a single-stranded, positive sense RNA genome. It is endemic in many cattle populations, causing major economic losses in part due to reduced fertility. BVDV exhibits great genetic diversity and is classified as type 1 or 2 (BVDV-1, BVDV-2) with either non-cytopathogenic (ncp) or cytopathogenic (cp) biotypes. Differing strains of ncpBVDV differ in virulence, affecting clinical outcome. BVDV replicates in the reproductive tract, affecting host immunity and embryo survival. This study used an in vitro model of primary bovine endometrial cell cultures to compare the effects of two BVDV ncp type 1a strains of differing virulence (termed HO and KY) on endometrial transcription of candidate interferon stimulated genes (ISG) using qPCR. Half the cultures were stimulated with interferon tau (IFNT, the conceptus produced pregnancy recognition factor) in the presence or absence of viral infection. Cultures were replicated on cells from 10 BVDV-free cows. IFNT treatment stimulated transcription of 10 candidate ISGs, whereas both ncpBVDV-1 strains alone inhibited transcription of 8/10 ISGs. In combined BVDV-1+IFNT cultures, the stimulatory effect of IFNT on expression of GBP4, ISG15, HERC5, RSAD2, IFIH1, IFIT3, and MX1 was significantly inhibited by HO, but only ISG15, RSAD2, IFI27, and IFIT3 were decreased by KY. Inhibition by HO was generally greater. The IFNT-induced expression of TRIM56 was, however, increased by HO. These data show that HO, the more virulent ncpBVDV-1 strain, has a greater capacity to inhibit key antiviral pathways. These differences need confirmation at the protein level but may influence immune tolerance of the host. They could also reduce fertility by increasing uterine susceptibility to bacterial infection and disrupting IFNT-mediated pregnancy recognition.

The Health and Behavioural Effects of Individual versus Pair Housing of Calves at Different Ages on a UK Commercial Dairy Farm.

Housing management of dairy calves is one of the factors that contributes to a successful rearing outcome. Individual housing of pre-weaned calves is thought to provide enhanced biosecurity and easier monitoring of the individual, and so remains prevalent in the UK. Behavioural studies have, however, found that pair housing is important for social learning, with positive impacts on health and welfare. This study utilised a single UK commercial dairy farm to establish if individual housing, pair housing from birth, or pair housing from three weeks of age affected health and behavioural parameters. Calves were housed in these allocated groups from birth to eight weeks of age, when they were moved into group pens of five calves for weaning at 10 weeks of age. All management routines other than the housing group were the same for enrolled calves. One hundred Holstein calves were recruited over a six-month period, and systematically allocated to a housing group. Weekly visits were conducted up to 10 weeks of age (weaning) for each calf, with weight, solid feed intake, and presence of clinical disease measured. In addition, a novel object approach test was carried out at six weeks, and a thoracic ultrasound was performed at seven weeks. Housing group had no effect on the average daily liveweight gain (ADLG) (p = 0.74), with an average of 0.66 kg/day over the pre-weaning period. However, on group housing at 8-10 weeks of age, there was a numerical increase in ADLG in the pair housed calves compared to the individually housed calves over the weaning period. Housing group had no significant effect on disease prevalence (p = 0.98) or the time taken to approach the novel object (p = 0.29). However, pair housed calves had increased mean total solid feed intakes from weeks 2-8 (p = 0.011), with 6.2 ± 0.67 kg (standard error of the mean-SEM), 12.7 ± 0.73 kg and 13.6 ± 0.70 kg ingested by individually housed, pair housed from birth and pair housed from three weeks of age, respectively. The overall findings of this study indicate that within a UK commercial dairy management system, there is no detrimental effect of housing calves within pairs (either from birth or three weeks of age) compared to individual housing.

A Cohort Study Risk Factor Analysis for Endemic Disease in Pre-Weaned Dairy Heifer Calves.

Dairy heifer calves experience high levels of contagious disease during their preweaning period, which may result in poor welfare, reduced performance or mortality. We determined risk factors for disease in a cohort study of 492 heifers recruited from 11 commercial UK dairy farms. Every animal received a weekly examination by a veterinarian from birth to nine weeks using the Wisconsin scoring system. Multivariable models were constructed using a hierarchical model with calf nested within farm. Outcome variables for each disease included a binary outcome (yes/no), disease duration and a composite disease score (CDS) including both severity and duration. Diarrhoea, bovine respiratory disease (BRD) and umbilical disease were recorded in 48.2%, 45.9% and 28.7% of calves, respectively. A higher heifer calving intensity in the week of birth reduced the CDS for diarrhoea, with a marginal benefit of improved passive transfer (serum immunoglobulin G (IgG) measured at recruitment). The CDS for BRD was reduced by housing in fixed groups, higher mean temperature in month of birth, increasing milk solids fed, increasing IgG, and higher plasma IGF-1 at recruitment. Conversely, higher calving intensity and higher temperature both increased the CDS for umbilical disease, whereas high IGF-1 was again protective. Although good passive transfer reduced the severity of BRD, it was not significant in models for diarrhoea and umbilical disease, emphasising the need to optimise other aspects of management. Measuring IGF-1 in the first week was a useful additional indicator for disease risk.

Fetal morphological features and abnormalities associated with equine early pregnancy loss.

BACKGROUND: Early pregnancy loss (EPL) occurs in approximately 8% of equine pregnancies, although the aetiology is mostly unknown and embryonic/fetal morphological abnormalities associated with EPL are not defined. OBJECTIVES: To compare the morphology of EPL to clinically normal embryos/fetuses and previously described embryonic/fetal developmental milestones. To identify morphological abnormalities associated with equine EPL. STUDY DESIGN: Observational case-control study. METHODS: Embryos/fetuses were obtained from clinically normal Thoroughbred and pony pregnancies (n = 11) and following EPL from Thoroughbred mares (n = 27). The crown-rump length (CRL) of embryos/fetuses was measured and macroscopic morphology and developmental age were determined independently by three blinded examiners. Sagittal sections of EPL (n = 13) and control (n = 6) embryos/fetuses were assessed microscopically. Fisher's exact test was used to determine significance (P < .05) and correlations were expressed by Pearson coefficient. RESULTS: Age and CRL were strongly positively correlated in clinically normal Thoroughbred and reference (n = 15, R = .9 (95% CI: 0.8-1.0), R2  = .9, P < .0001) but not EPL embryos/fetuses (n = 19, R = .1 (95% CI: -0.4 to 0.5), R2  = .01, P = .75). Relative to controls, the CRL of EPL embryos/fetuses was smaller, with evidence of intrauterine growth retardation (IUGR) in 3/8 fetuses assessed. In 9/13 EPL embryos/fetuses, nonspecific neural tissue alterations were identified including disruption of developing pros-, mes- and rhombencephalon and the presence of haemosiderin, indicating premortem haemorrhage. Failed neural tube closure was identified in 1/13 EPL embryos/fetuses. Subcutaneous haemorrhage was present in 14/27 EPL embryos/fetuses. MAIN LIMITATIONS: Autolysis significantly affected 15/27 EPL embryos/fetuses, excluding them from complete assessment. The IUGR reference cut-off values were based on a small number of controls. CONCLUSIONS: Morphological features associated with equine EPL were a mismatch between embryonic/fetal size and age, and alterations of the developing neural tissue and localised subcutaneous haemorrhage. Failed neural tube closure was confirmed as a rare specific abnormality.

Mining the Unmapped Reads in Bovine RNA-Seq Data Reveals the Prevalence of Bovine Herpes Virus-6 in European Dairy Cows and the Associated Changes in Their Phenotype and Leucocyte Transcriptome.

Microbial RNA is detectable in host samples by aligning unmapped reads from RNA sequencing against taxon reference sequences, generating a score proportional to the microbial load. An RNA-Seq data analysis showed that 83.5% of leukocyte samples from six dairy herds in different EU countries contained bovine herpes virus-6 (BoHV-6). Phenotypic data on milk production, metabolic function, and disease collected during their first 50 days in milk (DIM) were compared between cows with low (1-200 and n = 114) or high (201-1175 and n = 24) BoHV-6 scores. There were no differences in milk production parameters, but high score cows had numerically fewer incidences of clinical mastitis (4.2% vs. 12.2%) and uterine disease (54.5% vs. 62.7%). Their metabolic status was worse, based on measurements of IGF-1 and various metabolites in blood and milk. A comparison of the global leukocyte transcriptome between high and low BoHV-6 score cows at around 14 DIM yielded 485 differentially expressed genes (DEGs). The top pathway from Gene Ontology (GO) enrichment analysis was the immune system process. Down-regulated genes in the high BoHV-6 cows included those encoding proteins involved in viral detection (DDX6 and DDX58), interferon response, and E3 ubiquitin ligase activity. This suggested that BoHV-6 may largely evade viral detection and that it does not cause clinical disease in dairy cows.

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse.

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14-65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies.

Importance of Viral Disease in Dairy Cow Fertility.

Many viral diseases are endemic in cattle populations worldwide. The ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. There is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. These effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. This paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. Acute infection with non-cytopathic bovine viral diarrhea virus (BVDV) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. BVDV infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. Fertility may also be reduced by BVDV-induced immunosuppression, which increases the susceptibility to bacterial infections. Bovine herpesvirus (BHV)-1 is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. Previously infected animals subsequently show reduced fertility. Although this may be associated with lung damage, ovarian lesions have also been reported. Both BHV-1 and BHV-4 remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. While BHV-4 infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as Escherichia coli and Trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. Both Schmallenberg virus (SBV) and bluetongue virus (BTV) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. BTV-8 also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. Although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes.

Bovine Viral Diarrhoea Virus Infection Disrupts Uterine Interferon Stimulated Gene Regulatory Pathways During Pregnancy Recognition in Cows.

In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows.

Mechanisms linking bovine viral diarrhea virus (BVDV) infection with infertility in cattle.

Bovine viral diarrhea virus (BVDV) is an important infectious disease agent that causes significant reproductive and economic losses in the cattle industry worldwide. Although BVDV infection is known to cause poor fertility in cattle, a greater part of the underlying mechanisms particularly associated with early reproductive losses are not clearly understood. Previous studies reported viral compromise of reproductive function in infected bulls. In females, BVDV infection is thought to be capable of killing the oocyte, embryo or fetus directly, or to induce lesions that result in fetal abortion or malformation. BVDV infections may also induce immune dysfunction, and predispose cattle to other diseases that cause poor health and fertility. Other reports also suggested BVDV-induced disruption of the reproductive endocrine system, and a disruption of leukocyte and cytokine functions in the reproductive organs. More recent studies have provided evidence of viral-induced suppression of endometrial innate immunity that may predispose to uterine disease. Furthermore, there is new evidence that BVDV may potentially disrupt the maternal recognition of pregnancy or the immune protection of the conceptus. This review brings together the previous reports with the more recent findings, and attempts to explain some of the mechanisms linking this important virus to infertility in cattle.