Genome-wide epigenetic modifications in sports horses during training as an adaptation phenomenon.

With his bicentennial breeding history based on athletic performance, the Thoroughbred horse can be considered the equine sport breed. Although genomic and transcriptomic tools and knowledge are at the state of the art in equine species, the epigenome and its modifications in response to environmental stimuli, such as training, are less studied. One of the major epigenetic modifications is cytosine methylation at 5' of DNA molecules. This crucial biochemical modification directly mediates biological processes and, to some extent, determines the organisms' phenotypic plasticity. Exercise indeed affects the epigenomic state, both in humans and in horses. In this study, we highlight, with a genome-wide analysis of methylation, how the adaptation to training in the Thoroughbred can modify the methylation pattern throughout the genome. Twenty untrained horses, kept under the same environmental conditions and sprint training regimen, were recruited, collecting peripheral blood at the start of the training and after 30 and 90 days. Extracted leukocyte DNA was analyzed with the methylation content sensitive enzyme ddRAD (MCSeEd) technique for the first time applied to animal cells. Approximately one thousand differently methylated genomic regions (DMRs) and nearby genes were called, revealing that methylation changes can be found in a large part of the genome and, therefore, referable to the physiological adaptation to training. Functional analysis via GO enrichment was also performed. We observed significant differences in methylation patterns throughout the training stages: we hypothesize that the methylation profile of some genes can be affected early by training, while others require a more persistent stimulus.

Y-Chromosome Haplotype Report among Eight Italian Horse Breeds.

Horse domestication and breed selection processes have profoundly influenced the development and transformation of human society and civilization over time. Therefore, their origin and history have always attracted much attention. In Italy, several local breeds have won prestigious awards thanks to their unique traits and socio-cultural peculiarities. Here, for the first time, we report the genetic variation of three loci of the male-specific region of the Y chromosome (MSY) of four local breeds and another one (Lipizzan, UNESCO) well-represented in the Italian Peninsula. The analysis also includes data from three Sardinian breeds and another forty-eight Eurasian and Mediterranean horse breeds retrieved from GenBank for comparison. Three haplotypes (HT1, HT2, and HT3) were found in Italian stallions, with different spatial distributions between breeds. HT1 (the ancestral haplotype) was frequent, especially in Bardigiano and Monterufolino, HT2 (Neapolitan/Oriental wave) was found in almost all local breeds, and HT3 (Thoroughbred wave) was detected in Maremmano and two Sardinian breeds (Sardinian Anglo-Arab and Sarcidano). This differential distribution is due to three paternal introgressions of imported stallions from foreign countries to improve local herds; however, further genetic analyses are essential to reconstruct the genetic history of native horse breeds, evaluate the impact of selection events, and enable conservation strategies.

Exploring the Italian equine gene pool via high-throughput genotyping.

Introduction: The Italian peninsula is in the center of the Mediterranean area, and historically it has been a hub for numerous human populations, cultures, and also animal species that enriched the hosted biodiversity. Horses are no exception to this phenomenon, with the peculiarity that the gene pool has been impacted by warfare and subsequent "colonization". In this study, using a comprehensive dataset for almost the entire Italian equine population, in addition to the most influential cosmopolitan breeds, we describe the current status of the modern Italian gene pool. Materials and Methods: The Italian dataset comprised 1,308 individuals and 22 breeds genotyped at a 70 k density that was merged with publicly available data to facilitate comparison with the global equine diversity. After quality control and supervised subsampling to ensure consistency among breeds, the merged dataset with the global equine diversity contained data for 1,333 individuals from 54 populations. Multidimensional scaling, admixture, gene flow, and effective population size were analyzed. Results and Discussion: The results show that some of the native Italian breeds preserve distinct gene pools, potentially because of adaptation to the different geographical contexts of the peninsula. Nevertheless, the comparison with international breeds highlights the presence of strong gene flow from renowned breeds into several Italian breeds, probably due to historical introgression. Coldblood breeds with stronger genetic identity were indeed well differentiated from warmblood breeds, which are highly admixed. Other breeds showed further peculiarities due to their breeding history. Finally, we observed some breeds that exist more on cultural, traditional, and geographical point of view than due to actual genetic distinctiveness.

Unlocking Horse Y Chromosome Diversity.

The present equine genetic variation mirrors the deep influence of intensive breeding programs during the last 200 years. Here, we provide a comprehensive current state of knowledge on the trends and prospects on the variation in the equine male-specific region of the Y chromosome (MSY), which was assembled for the first time in 2018. In comparison with the other 12 mammalian species, horses are now the most represented, with 56 documented MSY genes. However, in contrast to the high variability in mitochondrial DNA observed in many horse breeds from different geographic areas, modern horse populations demonstrate extremely low genetic Y-chromosome diversity. The selective pressures employed by breeders using pedigree data (which are not always error-free) as a predictive tool represent the main cause of this lack of variation in the Y-chromosome. Nevertheless, the detailed phylogenies obtained by recent fine-scaled Y-chromosomal genotyping in many horse breeds worldwide have contributed to addressing the genealogical, forensic, and population questions leading to the reappraisal of the Y-chromosome as a powerful genetic marker to avoid the loss of biodiversity as a result of selective breeding practices, and to better understand the historical development of horse breeds.

The Use of Ascophyllum nodosum and Bacillus subtilis C-3102 in the Management of Canine Chronic Inflammatory Enteropathy: A Pilot Study.

The aim was to assess the effects of Ascophyllum nodosum (AN) with/without Bacillus subtilis C-3102 as alternative treatments for Chronic Inflammatory Enteropathy (CIE) of dogs. Fourteen CIE patients, which had received the same control (CTR) diet, were enrolled to serially receive three diets: (1) hydrolysed protein (HP) diet; (2) 4.0% AN supplemented HP (HPA) food, (3) HPA diet fortified with 125 billion B. subtilis C-3102 spores/10 kg body weight (HPAB diet). Clinical outcome was assessed by Canine Inflammatory Bowel Disease Activity Index (CIBDAI), whereas gut microbiota compositional variations were investigated via 16S rRNA gene analysis, and faecal fermentation end-products by liquid chromatography. Higher abundances of the Ruminococcaceae and Rikenellaceae families were shown in HPA relative to CTR treatment, with Bacillus genus being differentially abundant on HPAB diet. Concentrations of acetate were higher (p < 0.05) in dogs fed HPA compared to CTR diet, and amounts of isovalerate and isobutyrate were greater (p < 0.05) in HPA compared to HP food. A tendency for higher amounts of faecal butyrate was found for the HPAB treatment (p = 0.06). Comprehensively, while displaying potentially positive effects on faecal fermentations, the tested substances failed to improve CIBDAI scores and microbial richness in CIE dogs.

Methadone or Butorphanol as Pre-Anaesthetic Agents Combined with Romifidine in Horses Undergoing Elective Surgery: Qualitative Assessment of Sedation and Induction.

While butorphanol is the most commonly used opioid in horses, methadone is not licensed in most countries. Our aim was to compare the effects of both drugs, combined with romifidine, regarding the quality of sedation and induction in horses undergoing elective surgery. Results indicate the suitability of both methadone and butorphanol in this patient population. Animals were scored 10 min after intravenous injection of sedatives. Despite lower overall sedation (OS) score in horses receiving methadone (p = 0.002), the quality and time of induction and intubation remained unchanged. None of the horses had the lowest OS score (no sedation), nor the highest score for ataxia (horse falling). Methadone induced a tendency for minor noise reaction yet minor head lowering scores, the latter being probably the most influencing parameter when scoring OS. Measured physiological parameters decreased in both groups, with greater bradycardia recorded after methadone (p = 0.017), including a higher incidence of atrioventricular blocks that resolved during general anaesthesia. The quality of induction was good-excellent in most of the animals. While comparisons between the degree of antinociception were beyond the scope of this study, analgesic potency might influence the choice when considering opioids as pre-anaesthetic drugs in combination with romifidine before surgery in equines.

A Genetic Window on Sardinian Native Horse Breeds through Uniparental Molecular Systems.

Sardinia, an island located to the west of Italy in the Mediterranean Sea, boasts three native horse breeds: Giara, Sarcidano, and Sardinian Anglo-Arab. Here, we have investigated for the first time three loci of the non-recombining region of the Y chromosome (NRY) in 34 stallions from these breeds and performed a phylogenetic analysis of the maternal relationships among 178 previously published mitochondrial control regions. We found that the current NRY diversity of Sardinian horse breeds is linked to three haplotypes (HT), all identified within Sarcidano. Each breed showed a typical HT: HT1 (ancestral) was the most represented in Sarcidano, HT2 (Neapolitan/Oriental wave) in Giara, and HT3 (Thoroughbred wave) in Sardinian Anglo-Arab. The specificity of each haplotype suggests the influence of independent breeding strategies and the effect of genetic drift in each Sardinian population. The female counterpart, extended to 178 horses, showed a low genetic variability and a common maternal origin for Giara and Sarcidano. The higher variability of the Sardinian Anglo-Arab indicates multiple mare lineages in its current population. Further genetic analyses will be crucial to understand the paternal history of male horses, preserve the endangered mares' and stallions' lineages, and improve the enhancement of autochthonous genetic resources on this island.

Elo Method and Race Traits: A New Integrated System for Sport Horse Genetic Evaluation.

This first survey on Sardinian Anglo-Arab horse (SAA) race traits highlights important aspects for the breeding purpose of this population. The heritability of the race traits were estimated through a trivariate model; the estimates were 0.39, 0.33, and 0.30 for the number of placings, total earnings and Elo rating, respectively. The genetic progress could be improved by using an MT genetic evaluation of stallions and mares, combining information from competition traits.

Genetic Variability and Population Structure in the Sardinian Anglo-Arab Horse.

The Sardinian Anglo Arab (SAA) is a famous horse breed in Italy, with a significant historical background in the island of Sardinia. The aim of the study is to perform an evaluation of genetic variability in SAA using pedigree and mitochondrial data. In the current population, pedigree completeness was observed to be close to 100%, while the inbreeding coefficient and the average relatedness were lower than 3%. The ratio of effective founders/numbers of ancestors was 3.68 for the whole pedigree. The effective population size (Ne) computed by an individual increase in inbreeding (Ne_1) was 456.86, the Ne on equivalent generations (Ne_2) was 184.75, and this value slightly increased to 209.31 when computed by log-regression on equivalent generations (Ne_3). These results suggest the presence of crossbreeding and bottleneck phenomena, and they were compared with other Italian horses (reported in bibliography) to present the SAA among the Italian horse breeds scenario. Furthermore, the noteworthy mitochondrial variability reflects the use of a considerable number of founder mares; the contribution of L lineage was very important, probably because of the re-colonization from the Iberian Peninsula after the Last Glacial Maximum.

Mitochondrial DNA variation in the Italian Heavy Draught Horse.

BACKGROUND: In the last decades, Italy as well as other developed countries have registered a decrease in the population size of many local horse breeds. The continuous crossbreeding has determined the dilution of genetic heritage of several native breeds. The Italian Heavy Draught Horse (IHD) is the only autochthonous Italian coldblooded horse among these breeds; therefore, it represents a resource to be preserved. In 1927, the first generation of this breed was officially created by crossing different Heavy Draught horses with local mares and recorded in a Studbook. METHODOLOGY: To provide the first comprehensive overview of the genetic diversity of Italian Heavy Draught horses from Central Italy, we produced and phylogenetically analysed 52 mitochondrial DNA (mtDNA) control-region sequences. Furthermore, we evaluated data available from GenBank (N = 568) to have a more complete scenario and to understand the relationships with other European Heavy Draught horse breeds. RESULTS: Among the IHD samples that were analysed, we identified ten of the 17 haplogroups described in modern horses. Most of these sequences fell into L, G, and M lineages, thus showing the overall mtDNA legacy of the ancestral mares that were probably used at the initial stages of breeding selections a long time ago. The high mitochondrial haplotype diversity (Hd = 0.969) found in our samples reflected the multiple maternal origins of the horses. Our results highlighted a considerable percentage of haplotypes shared especially with Bardigiano and Hungarian Heavy Draught breeds. Furthermore, both the presence of four unique haplotypes detected in our samples and their absence among all equine mitochondrial published data demonstrate a mitochondrial peculiarity that needs to be further investigated and preserved with careful breeding practices.

Genetic Parameters and Inbreeding Effect of Morphological Traits in Sardinian Anglo Arab Horse.

The purpose of this study was to estimate the heritability and genetic correlations of four biometric measurements and an overall score (OS) in the Sardinian Anglo-Arab horse (SAA); moreover, the effect of inbreeding on these traits was investigated. A dataset with 43,624 horses (27,052 females and 16,572 males) was provided by the Agricultural Research Agency of Sardinia (AGRIS). Cannon bone circumference (BC), chest girth (CG), shoulder length (SL), and withers height (WH) were measured on 6033 SAA horses born in Sardinia between 1967 and 2005; beside the measurements, an overall score (OS) was taken comparing the morphology of each horse to an "ideal type" that is scored out of 100. The mean value is 20.5 cm for BC, 185.9 cm for CG, 67.6 cm for SL, 160.8 cm for WH, and 73.2 for the OS. The heritability estimates ranged from 0.78 to 0.23. The results allow to foresee high genetic progress through the breeding programs. The most affected trait by the inbreeding rate seems to only be the withers height.

Mitochondrial DNA Survey Reveals the Lack of Accuracy in Maremmano Horse Studbook Records.

* Correspondence: andrea [...].

Gallop Racing Shifts Mature mRNA towards Introns: Does Exercise-Induced Stress Enhance Genome Plasticity?

Physical exercise is universally recognized as stressful. Among the "sport species", the horse is probably the most appropriate model for investigating the genomic response to stress due to the homogeneity of its genetic background. The aim of this work is to dissect the whole transcription modulation in Peripheral Blood Mononuclear Cells (PBMCs) after exercise with a time course framework focusing on unexplored regions related to introns and intergenic portions. PBMCs NGS from five 3 year old Sardinian Anglo-Arab racehorses collected at rest and after a 2000 m race was performed. Apart from differential gene expression ascertainment between the two time points the complexity of transcription for alternative transcripts was identified. Interestingly, we noted a transcription shift from the coding to the non-coding regions. We further investigated the possible causes of this phenomenon focusing on genomic repeats, using a differential expression approach and finding a strong general up-regulation of repetitive elements such as LINE. Since their modulation is also associated with the "exonization", the recruitment of repeats that act with regulatory functions, suggesting that there might be an active regulation of this transcriptional shift. Thanks to an innovative bioinformatic approach, our study could represent a model for the transcriptomic investigation of stress.

Serum microRNAs in buffalo cows: Potential biomarkers of pregnancy.

MiRNAs (microRNA) constitute a large family of single-stranded, non-coding small RNAs. Although the functions and target genes of most miRNAs are still unknown, it has been well established that they are involved in embryogenesis, organogenesis and neonatal birth. In recent decades, interest in buffalo breeding has largely increased worldwide thus focusing the attention on this species as a dairy purpose animal. Problems related to long calving interval, late puberty and seasonal anestrus hamper reproductive efficiency in this species. Early pregnancy diagnosis is important to shorten the calving interval and increase lifetime production on dairy animals. MicroRNAs have recently emerged as key molecules in fertilization of several species even though in buffalo, few previous studies have investigated miRNAs. The aim of this research was to identify the best miRNA reference in serum among miR-191, miR-25-3p, SNORD44, and SNORD48. Consequently, assess the expression levels of miR-103, miR-200b, miR-301a, miR-423-5p, miR-375, miR-451 and miR-452 involved in buffalo progesterone-maturation oocyte and pregnancy. Interestingly, we found that all the miRNAs analyzed at 40days after artificial insemination were differentially expressed among pregnant and non-pregnant buffalo. Our research may be a first step for gain further insight in the biological function of circulating miRNAs in this species.

An Overview of Ten Italian Horse Breeds through Mitochondrial DNA.

BACKGROUND: The climatic and cultural diversity of the Italian Peninsula triggered, over time, the development of a great variety of horse breeds, whose origin and history are still unclear. To clarify this issue, analyses on phenotypic traits and genealogical data were recently coupled with molecular screening. METHODOLOGY: To provide a comprehensive overview of the horse genetic variability in Italy, we produced and phylogenetically analyzed 407 mitochondrial DNA (mtDNA) control-region sequences from ten of the most important Italian riding horse and pony breeds: Bardigiano, Esperia, Giara, Lipizzan, Maremmano, Monterufolino, Murgese, Sarcidano, Sardinian Anglo-Arab, and Tolfetano. A collection of 36 Arabian horses was also evaluated to assess the genetic consequences of their common use for the improvement of some local breeds. CONCLUSIONS: In Italian horses, all previously described domestic mtDNA haplogroups were detected as well as a high haplotype diversity. These findings indicate that the ancestral local mares harbored an extensive genetic diversity. Moreover, the limited haplotype sharing (11%) with the Arabian horse reveals that its impact on the autochthonous mitochondrial gene pools during the final establishment of pure breeds was marginal, if any. The only significant signs of genetic structure and differentiation were detected in the geographically most isolated contexts (i.e. Monterufolino and Sardinian breeds). Such a geographic effect was also confirmed in a wider breed setting, where the Italian pool stands in an intermediate position together with most of the other Mediterranean stocks. However, some notable exceptions and peculiar genetic proximities lend genetic support to historical theories about the origin of specific Italian breeds.

Powder, capsule and device: An imperative ménage à trois for respirable dry powders.

OBJECTIVES: The development of inhaled products to treat or to prevent lung infection is a very active research field in drug delivery. The pulmonary route is extremely attractive but very challenging. This paper reports the study of excipient, capsule brand and device influence on the aerodynamic behavior of dry powder formulations to treat tuberculosis. METHODS: A capreomycin hydrophobic salt was powdered using spray-drying and formulated using lactose (added after spray-drying, external excipient) or L-leucine (added before spray-drying, internal excipient). Aerosolization performances were investigated loading the formulations in 2 different capsule brands and aerosolizing them with 3 different devices. RESULTS: Capreomycin oleate and capreomycin oleate/l-leucine powders were produced with a yield around 70%. Capreomycin oleate powder was composed of particles with an irregular surface. Particles of capreomycin oleate/l-leucine were roundish and wrinkled on the surface. Capreomycin oleate/l-leucine formulation gave the highest values of respirable fraction in all cases. Statistical analysis asserted the significant effect on the respirable fraction of the powder (p≤0.001), the capsule brand (p≤0.01) and the device (p≤0.05). CONCLUSIONS: The use of L-leucine as internal excipient allows one to avoid the use of lactose, obtaining a carrier-free formulation. Even though differences are not very large, to obtain the highest RFE, the best choice between capsule and device is Quali-V(®) and model 8.

Feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling.

BACKGROUND: Feline eosinophilic dermatoses (FEDs) are common diseases of cats with an unknown pathogenesis. They are histologically characterized by an eosinophilic infiltration and often by the presence of flame figures (FFs) and/or areas of loss of tissue architecture, here termed necrotic foci (NF). It has been postulated that an alteration in the degradation of the extracellular matrix could be responsible for these histological features. Matrix metalloproteinases (MMPs) are a group of proteases that are fundamental in extracellular matrix remodelling. HYPOTHESIS/OBJECTIVES: The aim of the study was to investigate retrospectively the expression of a subgroup of MMPs, in particular MMP-2 and MMP-9 gelatinases, in FEDs. The expression of one of their inhibitors, TIMP-2, was also investigated in order to establish the role of these molecules in the pathogenesis of FEDs. The ultrastructural characteristics of extracellular matrix in FFs and NF were subsequently assessed. METHODS: Fifty-one formalin-fixed, paraffin-embedded specimens from cutaneous and mucosal biopsies diagnosed as FEDs were investigated immunohistochemically. Two selected samples were processed for electron microscopy. RESULTS: This study revealed an increased expression of MMP-2 in NF and a decreased expression of this gelatinase in FFs. An imbalance between MMP-2 and TIMP-2 was evident using immunohistochemistry. No significative results were observed for MMP-9 expression. Electron microscopy confirmed the lack of normal collagen fibres in NF, whereas in FFs only occasional, amorphous material was observed among normal collagen fibres. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that an imbalance in the expression of matrix metalloproteinases could be responsible for different morphological findings in FEDs. Further studies are needed to assess the role of matrix metalloproteinases in the pathogenesis of FEDs.

Efficacy of tigecycline pleurodesis: a comparative experimental study.

BACKGROUND: We investigated whether tigecycline (TIGE) is more effective than talc in inducing pleurodesis in rabbits. METHODS: Fifty-six New Zealand rabbits were utilized in a two-phase study: Effects at 14 d (phase I) and at 28 d (phase II) were assessed. Saline solution (SAL n = 3), talc slurry (TALC 200 mg/kg, n = 5), and TIGE at different concentrations (mg/kg): TIGE0.5 (n = 5); TIGE1 (n = 5); TIGE3 (n = 5); TIGE25 (n = 5); TIGE50 (n = 5) were randomly injected, for each phase, through a right chest drainage. TIGE0.5 and TIGE1 were ineffective during phase I and were thus excluded from further investigation. At post mortem examination, pleurodesis was graded grossly and microscopically by three observers blinded to treatment groups. RESULTS: Phase I: pleurodesis was more effective in TIGE25 and TIGE50 (P < 0.001); TALC was better than TIGE0.5 (P < 0.001), and TIGE1 (P = 0.49), macroscopically. Pleural thickness was significantly higher in TIGE25 compared with SAL, TALC, TIGE0.5, TIGE1, and TIGE3 (P < 0.01). No significant differences were evident between TALC and TIGE3, both macroscopically (P = 0.90) and microscopically (inflammation P = 0.99, fibrosis P = 0.96, pleural thickness P = 0.99). Phase II: better effectiveness of TIGE50 compared with all other groups (P < 0.001) except TIGE 25 (P = 0.29); results similar to phase I for TALC and TIGE3 (P = 0.99), macroscopically. Microscopically greater inflammation in TALC compared with TIGE3 (P < 0.05) and in TIGE50 to TIGE3 (P = 0.05). Significant complications occurred in all TIGE50 group. One of TIGE25 and one of TIGE50 died of respiratory distress and of right hemothorax+ascites, respectively. CONCLUSIONS: Intrapleural TIGE3 mg/kg is as effective as talc in inducing pleurodesis in rabbits. The intrapleural TIGE toxicity threshold was reached at TIGE25 mg/kg concentration.

Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract.

The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction. Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer. Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control.