Unraveling metabolic stress response in dairy cows: genetic control of plasma biomarkers throughout lactation and the transition period.

Breeding animals able to effectively respond to stress could be a long-term, sustainable, and affordable strategy to improve resilience and welfare in livestock systems. In the present study, the concentrations of 29 plasma biomarkers were used as candidate endophenotypes for metabolic stress response in single-SNP, gene- and haplotype-based GWAS using 739 healthy lactating Italian Holstein cows and 88,271 variants. Significant genetic associations were found in all the 3 GWAS approaches for plasma γ-glutamyl transferase concentration on BTA17, for paraoxonase on BTA4, and for alkaline phosphatase and zinc on BTA2. On these chromosomes, single-SNP and gene-based chromosome-wide association studies were performed, confirming GWAS findings. The signals identified for paraoxonase, γ-glutamyl transferase, and alkaline phosphatase were in proximity of the genes coding for them. The heritability of these 4 biomarkers ranged from moderate to high (from 0.39 to 0.54). Plasma biomarkers are known to undergo large changes in concentration during metabolic stress in the transition period, with an inter-individual variability in the rate of change and recovery time. Genetics may account in part for these differences. To assess this, we studied a subset of 139 periparturient cows homozygous at 3 SNPs known to be respectively associated with concentration of plasma ceruloplasmin, paraoxonase and γ-glutamyl transferase. We compared the immune-metabolic profile measured in plasma at -7, +5 and +30 d relative to calving between groups of opposite homozygotes. A significant effect of the genotype was found on paraoxonase and γ-glutamyl transferase plasma concentration at all the 3 time points. No evidence for genotype effect was detected for ceruloplasmin. Understanding the genetic control underlying metabolic stress response may suggest new approaches to foster resilience in dairy cows.

Comparison between indicine and taurine cattle DNA methylation reveals epigenetic variation associated to differences in morphological adaptive traits.

Indicine and taurine subspecies present distinct morphological traits as a consequence of environmental adaptation and artificial selection. Although the two subspecies have been characterized and compared at genome-wide level and at specific loci, their epigenetic diversity has not yet been explored. In this work, Reduced Representation Bisulphite Sequencing (RRBS) profiling of the taurine Angus (A) and indicine Nellore (N) cattle breeds was applied to identify methylation differences between the two subspecies. Genotyping by sequencing (GBS) of the same animals was performed to detect single nucleotide polymorphisms (SNPs) at cytosines in CpG dinucleotides and remove them from the differential methylation analysis. A total of 660,845 methylated cytosines were identified within the CpG context (CpGs) across the 10 animals sequenced (5 N and 5 A). A total of 25,765 of these were differentially methylated (DMCs). Most DMCs clustered in CpG stretches nearby genes involved in cellular and anatomical structure morphogenesis. Also, sequences flanking DMC were enriched in SNPs compared to all other CpGs, either methylated or unmethylated in the two subspecies. Our data suggest a contribution of epigenetics to the regulation and divergence of anatomical morphogenesis in the two subspecies relevant for cattle evolution and sub-species differentiation and adaptation.

Identification of genomic regions associated with total and progressive sperm motility in Italian Holstein bulls.

Sperm motility is directly related to the ability of sperm to move through the female reproductive tract to reach the ovum. Sperm motility is a complex trait that is influenced by environmental and genetic factors and is associated with male fertility, oocyte penetration rate, and reproductive success of cattle. In this study we carried out a GWAS in Italian Holstein bulls to identify candidate regions and genes associated with variations in progressive and total motility (PM and TM, respectively). After quality control, the final data set consisted of 5,960 records from 949 bulls having semen collected in 10 artificial insemination stations and genotyped at 412,737 SNPs (call rate >95%; minor allele frequency >5%). (Co)variance components were estimated using single trait mixed models, and associations between SNPs and phenotypes were assessed using a genomic BLUP approach. Ten windows that explained the greatest percentage of genetic variance were located on Bos taurus autosomes 1, 2, 4, 6, 7, 23, and 26 for TM and Bos taurus autosomes 1, 2, 4, 6, 8, 16, 23, and 26 for PM. A total of 150 genes for TM and 72 genes for PM were identified within these genomic regions. Gene Ontology enrichment analyses identified significant Gene Ontology terms involved with energy homeostasis, membrane functions, sperm-egg interactions, protection against oxidative stress, olfactory receptors, and immune system. There was significant enrichment of quantitative trait loci for fertility, calving ease, immune response, feed intake, and carcass weight within the candidate windows. These results contribute to understanding the architecture of the genetic control of sperm motility and may aid in the development of strategies to identify subfertile bulls and improve reproductive success.

Genomic breed composition of pure registered Brazilian Gir.

INTRODUCTION: Gir is a Bos indicus breed originally from India, first imported to Brazil in 1850. High-performance Dairy Gir has been systematically selected in Brazil from its arrival. Since the major phenotypic difference between Gir in India and Brazil is a higher milk production in the former, it is speculated that Brazilian Gir was strategically crossed with Holstein or another Bos taurus to improve milk yield. This study evaluated the purity of Brazilian Gir breed stocks from BASA Farms in Brazil, trying to identify possible admixture events with other cattle breeds based on DNA analysis. MATERIALS AND METHODS: The population included 1061 pure registered individuals genotyped using two commercial platforms with 37 k and 25 k SNPs. Admixture analysis was performed individually to estimate levels of genomic composition derived from six different reference populations, three indicine and three taurine breeds. RESULTS: A Gir ancestry of 99% or higher was found for 94.2% of the population, while the remaining showed levels of non-Gir ancestry up to 6.8%. Only five individuals were identified with possible taurine ancestry, all of them exhibiting levels lower than 2%. The remaining non-Gir ancestry identified was derived from indicine breeds. The levels of admixture observed in the population were from low to non-detectable. No consistent patterns of admixture were observed indicating sustained introgression of taurine lines as means of genetic improvement. CONCLUSION: According to these results, genetic improvement achieved by Brazilian Gir breeders is the result of within-breed selection methods applied intensively over the past five decades, rather than the result from sustained introgression.

Bos taurus haplotypes segregating in Nellore (Bos indicus) cattle.

Brazil is the largest exporter of beef in the world, and most of that beef derives from Nellore cattle. Although considered a zebu breed (Bos indicus), the history of Nellore cattle in Brazil is marked by the importation of bulls from India, the use of a Creole taurine (Bos taurus) maternal lineage to quickly expand the herds and backcrossing to Nellore bulls to recover zebu ancestry. As a consequence, the current Brazilian Nellore population carries an average taurine ancestry of approximately 1%. Although that percentage seems small, some taurine variants deviate substantially from that average, with the better-known cases being the PLAG1-Q haplotype involved with body size variation and the Guarani (PG ) polled variant producing hornless animals. Here, we report taurine haplotypes in 9074 Nellore animals genotyped for 539 657 imputed SNP markers. Apart from PLAG1-Q and PG , our analysis further revealed common taurine haplotypes (>3%) spanning genes related to immunity, growth, reproduction and hair and skin phenotypes. Using data from 22 economically important traits, we showed that many of the major QTL previously reported in the breed are at least partially driven by taurine haplotypes. As B. taurus and B. indicus haplotypes are highly divergent, presenting widely different sets of functional variants, our results provide promising targets for future scrutiny in Nellore cattle.

Pain distraction during awake low anterior resection and Cuddle Delivery initiative for inpatient: frugal procedural options to support surgery in the COVID-19 era.

OBJECTIVE: Since minimally invasive surgery and general anesthesia are both aerosol-generating procedures, their use became controversial during the outbreak of coronavirus disease 2019 (COVID-19). Moreover, social distancing resulted in serious psychological consequences for inpatients. This case report investigates pain distraction during awake laparotomy, as well as new possibilities for emotional postoperative support to inpatients. PATIENTS AND METHODS: A 72-year-old man affected by middle rectal adenocarcinoma underwent lower anterior resection plus total mesorectal excision under combined spinal-epidural anesthesia. A 3D mobile theatre (3DMT) was intraoperatively used for pain distraction. A postoperative "Cuddle delivery" service was instituted: video-messages from relatives and close friends were delivered daily to the patient through the 3DMT. Emotional correlations were investigated through a clinical interview by the psychologist of our Hospital. RESULTS: Intraoperative, as well as postoperative pain, resulted well-controlled: visual analogue scale (VAS) ≤3. Conversion to general anesthesia and postoperative intensive support/monitoring were unnecessary. The "Cuddle delivery" initiative positively fed our patient's mood and attitude, strengthening his bond to life. CONCLUSIONS: During pandemic, awake laparotomy under loco-regional anesthesia may be a crucial option in delivering acute care surgery to selected patients when intensive care beds are unavailable. Our procedure introduces potential ways to optimize this approach.

Evidence of distinct gene functional patterns in GC-poor and GC-rich isochores in Bos taurus.

Vertebrate genomes are mosaics of megabase-size DNA segments with a fairly homogeneous base composition, called isochores. They are divided into five families characterized by different guanine-cytosine (GC) levels and linked to several functional and structural properties. The increased availability of fully sequenced genomes allows the investigation of isochores in several species, assessing their level of conservation across vertebrate genomes. In this work, we characterized the isochores in Bos taurus using the ARS-UCD1.2 genome version. The comparison of our results with the well-studied human isochores and those of other mammals revealed a large conservation in isochore families, in number, average GC levels and gene density. Exceptions to the established increase in gene density with the increase in isochores (GC%) were observed for the following gene biotypes: tRNA, small nuclear RNA, small nucleolar RNA and pseudogenes that have their maximum number in H2 and H1 isochores. Subsequently, we assessed the ontology of all gene biotypes looking for functional classes that are statistically over- or under-represented in each isochore. Receptor activity and sensory perception pathways were significantly over-represented in L1 and L2 (GC-poor) isochores. This was also validated for the horse genome. Our analysis of housekeeping genes confirmed a preferential localization in GC-rich isochores, as reported in other species. Finally, we assessed the SNP distribution of a bovine high-density SNP chip across the isochores, finding a higher density in the GC-rich families, reflecting a potential bias in the chip, widely used for genetic selection and biodiversity studies.

Association of missense variants in GDF9 with litter size in Entlebucher Mountain dogs.

In the past two decades, average litter size (ALS) in Entlebucher Mountain dogs decreased by approximately 0.8 puppies. We conducted a GWAS for ALS using the single-step methodology to take advantage of 1632 pedigree records, 892 phenotypes and 372 genotypes (173 662 markers) for which only 12% of the dogs had both phenotypes and genotypes available. Our analysis revealed associations towards the growth differentiation factor 9 gene (GDF9), which is known to regulate oocyte maturation. The trait heritability was estimated at 43.1%, from which approximately 15% was accountable by the GDF9 locus alone. Therefore, markers flanking GDF9 explained approximately 6.5% of the variance in ALS. Analysis of WGSs revealed two missense substitutions in GDF9, one of which (g.11:21147009G>A) affected a highly conserved nucleotide in vertebrates. The derived allele A was validated in 111 dogs and shown to be associated with decreased ALS (-0.75 ± 0.22 puppies per litter). The variant was further predicted to cause a proline to serine substitution. The affected residue was immediately followed by a six-residue deletion that is fixed in the canine species but absent in non-canids. We further confirmed that the deletion is prevalent in the Canidae family by sequencing three species of wild canids. Since canids uniquely ovulate oocytes at the prophase stage of the first meiotic division, requiring maturation in the oviduct, we conjecture that the amino acid substitution and the six-residue deletion of GDF9 may serve as a model for insights into the dynamics of oocyte maturation in canids.

Genomic clues of the evolutionary history of Bos indicus cattle.

Together with their sister subspecies Bos taurus, zebu cattle (Bos indicus) have contributed to important socioeconomic changes that have shaped modern civilizations. Zebu cattle were domesticated in the Indus Valley 8000 years before present (YBP). From the domestication site, they expanded to Africa, East Asia, southwestern Asia and Europe between 4000 and 1300 YBP, intercrossing with B. taurus to form clinal variations of zebu ancestry across the landmass of Afro-Eurasia. In the past 150 years, zebu cattle reached the Americas and Oceania, where they have contributed to the prosperity of emerging economies. The zebu genome is characterized by two mitochondrial haplogroups (I1 and I2), one Y chromosome haplogroup (Y3) and three major autosomal ancestral groups (Indian-Pakistani, African and Chinese). Phenotypically, zebu animals are recognized by their hump, large ears and excess skin. They are rustic, resilient to parasites and capable of bearing the hot and humid climates of the tropics. Many resources are available to study the zebu genome, including commercial arrays of SNP, reference assemblies and publicly available genotypes and whole-genome sequences. Nevertheless, many of these resources were initially developed to support research and subsidize industrial applications in B. taurus, and therefore they can produce bias in data analysis. The combination of genomics with precision agriculture holds great promise for the identification of genetic variants affecting economically important traits such as tick resistance and heat tolerance, which were naturally selected for millennia and played a major role in the evolution of B. indicus cattle.

Reference range of liver corrected T1 values in a population at low risk for fatty liver disease-a UK Biobank sub-study, with an appendix of interesting cases.

PURPOSE: Corrected T1 (cT1) value is a novel MRI-based quantitative metric for assessing a composite of liver inflammation and fibrosis. It has been shown to distinguish between non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis. However, these studies were conducted in patients at high risk for liver disease. This study establishes the normal reference range of cT1 values for a large UK population, and assesses interactions of age and gender. METHODS: MR data were acquired on a 1.5 T system as part of the UK Biobank Imaging Enhancement study. Measures for Proton Density Fat Fraction and cT1 were calculated from the MRI data using a multiparametric MRI software application. Data that did not meet quality criteria were excluded from further analysis. Inter and intra-reader variability was estimated in a set of data. A cohort at low risk for NAFL was identified by excluding individuals with BMI ≥ 25 kg/m2 and PDFF ≥ 5%. Of the 2816 participants with data of suitable quality, 1037 (37%) were classified as at low risk. RESULTS: The cT1 values in the low-risk population ranged from 573 to 852 ms with a median of 666 ms and interquartile range from 643 to 694 ms. Iron correction of T1 was necessary in 36.5% of this reference population. Age and gender had minimal effect on cT1 values. CONCLUSION: The majority of cT1 values are tightly clustered in a population at low risk for NAFL, suggesting it has the potential to serve as a new quantitative imaging biomarker for studies of liver health and disease.

Genome-wide association study of milk fatty acid composition in Italian Simmental and Italian Holstein cows using single nucleotide polymorphism arrays.

Bovine milk is important for human nutrition, but its fat content is often criticized as a risk factor in cardiovascular disease. Selective breeding programs could be used to alter the fatty acid (FA) composition of bovine milk to improve the healthiness of dairy products for human consumption. Here, we performed a genome-wide association study (GWAS) on bovine milk to identify genomic regions or specific genes associated with FA profile and to investigate genetic differences between the Italian Simmental (IS) and Italian Holstein (IH) breeds. To achieve this, we first characterized milk samples from 416 IS cows and 436 IH cows for their fat profile by gas chromatography. Subjects were genotyped with single nucleotide polymorphism array and a single-marker regression model for GWAS was performed. Our findings confirm previously reported quantitative trait loci strongly associated with bovine milk fat composition. More specifically, our GWAS results revealed significant signals on chromosomes Bos taurus autosome 19 and 26 for milk FA. Further analysis using a gene-centric approach and pathway meta-analysis identified not only some well-known genes underlying quantitative trait loci for milk FA components, such as FASN, SCD, and DGAT1, but also other significant candidate genes, including some with functional roles in pathways related to "Lipid metabolism." Highlighted genes related to FA profile include ECI2, PCYT2, DCXR, G6PC3, PYCR1, and ALG12 in IS, and CYP17A1, ACO2, PI4K2A, GOT1, GPT, NT5C2, PDE6G, POLR3H, and COX15 in IH. Overall, the breed-specific association outcomes reflect differences in the genetic backgrounds of the IS and IH breeds and their selective breeding histories.

MUGBAS: a species free gene-based programme suite for post-GWAS analysis.

UNLABELLED: Genome Wide Association Studies between molecular markers and phenotypes are now routinely run in model and non-model species. However, tools to estimate the probability of association of functional units (e.g. genes) containing multiple markers are not developed for species other than humans. Here we introduce MUGBAS (MUlti species Gene-Based Association Suite), software that estimates the P-value of a gene using information on annotation, single marker GWA results and genotype. The software is species and annotation independent, fast, highly parallelized and ready for high-density marker studies. AVAILABILITY AND IMPLEMENTATION: https://bitbucket.org/capemaster/mugbas

Imputation accuracy is robust to cattle reference genome updates.

Genotype imputation is routinely applied in a large number of cattle breeds. Imputation has become a need due to the large number of SNP arrays with variable density (currently, from 2900 to 777,962 SNPs). Although many authors have studied the effect of different statistical methods on imputation accuracy, the impact of a (likely) change in the reference genome assembly on imputation from lower to higher density has not been determined so far. In this work, 1021 Italian Simmental SNP genotypes were remapped on the three most recent reference genome assemblies. Four imputation methods were used to assess the impact of an update in the reference genome. As expected, the four methods behaved differently, with large differences in terms of accuracy. Updating SNP coordinates on the three tested cattle reference genome assemblies determined only a slight variation on imputation results within method.

Is there a role of percutaneous drainage in non-parasitic splenic cysts? Case report.

Pathogenesis, classification and treatment of non-parasitic splenic cysts (NPSCs) are controversial. The utility of percutaneous aspiration of the cyst is not well understood. We report a case of a 32 year-old woman with a symptomatic giant epidermoid cyst of the spleen treated with laparoscopic splenectomy. A percutaneous transcatheter drainage was performed under ultrasound guidance before surgical procedure in order to classify the type of cyst and to choose the best treatment.

Noise correlations and SNR in phased-array MRS.

The acquisition of magnetic resonance spectroscopy (MRS) signals by multiple receiver coils can improve the signal-to-noise ratio (SNR) or alternatively can reduce the scan time maintaining a reliable SNR. However, using phased array coils in MRS studies requires efficient data processing and data combination techniques in order to exploit the sensitivity improvement of the phased array coil acquisition method. This paper describes a novel method for the combination of MRS signals acquired by phased array coils, even in presence of correlated noise between the acquisition channels. In fact, although it has been shown that electric and magnetic coupling mechanisms produce correlated noise in the coils, previous algorithms developed for MRS data combination have ignored this effect. The proposed approach takes advantage of a noise decorrelation stage to maximize the SNR of the combined spectra. In particular Principal Component Analysis (PCA) was exploited to project the acquired spectra in a subspace where the noise vectors are orthogonal. In this subspace the SNR weighting method will provide the optimal overall SNR. Performance evaluation of the proposed method is carried out on simulated (1)H-MRS signals and experimental results are obtained on phantom (1)H-MR spectra using a commercially available 8-element phased array coil. Noise correlations between elements were generally low due to the optimal coil design, leading to a fair SNR gain (about 0.5%) in the center of the field of view (FOV). A greater SNR improvement was found in the peripheral FOV regions.

Objective selection of EEG late potentials through residual dependence estimation of independent components.

This paper presents a novel method to objectively select electroencephalographic (EEG) cortical sources estimated by independent component analysis (ICA) in event-related potential (ERP) studies. A proximity measure based on mutual information is employed to estimate residual dependences of the components that are then hierarchically clustered based on these residual dependences. Next, the properties of each group of components are evaluated at each level of the hierarchical tree by two indices that aim to assess both cluster tightness and physiological reliability through a template matching process. These two indices are combined in three different approaches to bring to light the hierarchical structure of the cluster organizations. Our method is tested on a set of experiments with the purpose of enhancing late positive ERPs elicited by emotional picture stimuli. Results suggest that the best way to look for physiologically plausible late positive potential (LPP) sources is to explore in depth the tightness of those clusters that, taken together, best resemble the template. According to our results, after brain sources clustering, LPPs are always identified more accurately than from ensemble-averaged raw data. Since the late components of an ERP involve the same associative areas, regardless of the modality of stimulation or specific tasks administered, the proposed method can be simply adapted to other ERP studies, and extended from psychophysiological studies to pathological or sport training evaluation support.

Independent component analysis applied to the removal of motion artifacts from electrocardiographic signals.

Electrocardiographic (ECG) signals are affected by several kinds of artifacts that may hide vital signs of interest. In this study we apply independent component analysis (ICA) to isolate motion artifacts. Standard or instantaneous ICA, which is currently the most addressed ICA model within the context of artifact removal, is compared to two other ICA techniques. The first technique is a frequency domain approach to convolutive mixture separation. The second is based on temporally constrained ICA, which enables the estimation of only one component close to a particular reference signal. Performance indexes evaluate ECG complex enhancement and relevant heart rate errors. Our results show that both convolutive and constrained ICA implementations perform better than standard ICA, thus opening up a new field of application for these two methods. Moreover, statistical analysis reveals that constrained ICA and convolutive ICA do not significantly differ concerning heart rate estimation, even though the latter overcomes the former in ECG morphology recovery.

Residual dependency estimation of independent components applied to EEG event related potentials associated with emotional processing.

Independent component analysis can be employed as an exploratory method in electroencephalographic (EEG) data analysis. However, the assumption of statistical independence among the estimated components is not always fulfilled by ICA-based numerical methods. Furthermore it may happen that one physiological source can be split in two or more components. As a consequence, the estimated components must be further investigated to assess the existence of reciprocal similarities. In this work a method for finding residual dependency subsets of component is proposed. Firstly a hierarchical clustering stage is carried out to classify ICA results. Then the hierarchical tree is investigated at each level by two indices to evaluate the tightness of all clusters. At the same time clustered scalp projections are compared with a template, which is shaped by applying ensemble ICA to a training dataset. Results are shown on EEG data acquired in event-related brain potentials (ERPs) studies for emotional pictures processing. In this kind of experiment ERPs are measured whilst unpleasant and neutral images are shown to a subject. The clustering procedure and the performance indices succeeded in isolating compact groups of components. These components, taken together, reflect the brain's biopotentials related to emotional processing at different cortical areas.

Multichannel techniques for motion artifacts removal from electrocardiographic signals.

Electrocardiographic (ECG) signals are affected by several kinds of artifacts, that may hide vital signs of interest. Motion artifacts, due to the motion of the electrodes in relation to patient skin, are particularly frequent in bioelectrical signals acquired by wearable systems. In this paper we propose different approaches in order to get rid of motion confounds. The first approach we follow starts from measuring electrode motion provided by an accelerometer placed on the electrode and use this measurement in an adaptive filtering system to remove the noise present in the ECG. The second approach is based on independent component analysis methods applied to multichannel ECG recordings; we propose to use both instantaneous model and a frequency domain implementation of the convolutive model that accounts for different paths of the source signals to the electrodes.