Genetic architecture and major genes for backfat thickness in pig lines of diverse genetic backgrounds.

BACKGROUND: Backfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds. METHODS: Data comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model. RESULTS: We found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN. CONCLUSIONS: Our results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry.

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts.

Thermal, mechanical and electrical stimuli in antinociceptive studies in standing horses: an update.

OBJECTIVE: To perform a literature review of the thermal and mechanical antinociceptive devices used in pharmacological studies in standing horses published after 2011 (2012-2019). To complete a full literature review about electrical stimulation used for evaluation in similar studies. DATABASES USED: PubMed, Google Scholar and Web of Science. CONCLUSIONS: A high level of standardization has been reached in antinociceptive studies in standing horses using thermal and mechanical stimuli in most recent years. Commercially available testing devices to deliver thermal, mechanical and electrical stimuli, with observation of aversive responses to these stimuli, are reliable, sensitive and specific. For electrical stimulus testing, there is evidence that the resistance between the electrodes should be measured and should not exceed 3 kΩ to guarantee consistent and reproducible stimuli. The specific analysis of electromyographic activity after an electrical stimulus provides more detailed information about the neurons stimulated.

A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens.

As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.

A possible solution to model nonlinearity in elimination and distributional clearances with α2 -adrenergic receptor agonists: Example of the intravenous detomidine and methadone combination in sedated horses.

The alpha(α)2 -agonist detomidine is used for equine sedation with opioids such as methadone. We retrieved the data from two randomized, crossover studies where detomidine and methadone were given intravenously alone or combined as boli (STUDY 1) (Gozalo-Marcilla et al., 2017, Veterinary Anaesthesia and Analgesia, 2017, 44, 1116) or as 2-hr constant rate infusions (STUDY 2) (Gozalo-Marcilla et al., 2019, Equine Veterinary Journal, 51, 530). Plasma drug concentrations were measured with a validated tandem Mass Spectrometry assay. We used nonlinear mixed effect modelling and took pharmacokinetic (PK) data from both studies to fit simultaneously both drugs and explore their nonlinear kinetics. Two significant improvements over the classical mammillary two-compartment model were identified. First, the inclusion of an effect of detomidine plasma concentration on the elimination clearances (Cls) of both drugs improved the fit of detomidine (Objective Function Value [OFV]: -160) and methadone (OFV: -132) submodels. Second, a detomidine concentration-dependent reduction of distributional Cls of each drug further improved detomidine (OFV: -60) and methadone (OFV: -52) submodel fits. Using the PK data from both studies (a) helped exploring hypotheses on the nonlinearity of the elimination and distributional Cls and (b) allowed inclusion of dynamic effects of detomidine plasma concentration in the model which are compatible with the pharmacology of detomidine (vasoconstriction and reduction in cardiac output).

How to score sedation and adjust the administration rate of sedatives in horses: a literature review and introduction of the Ghent Sedation Algorithm.

OBJECTIVE: To summarize the different methods used to assess sedation and/or adjust the dose or administration rate of alpha-2 agonists in horses and to propose an algorithm to adjust the administration rate of a constant rate infusion of an alpha-2 agonist in horses. DATABASES USED: PubMed and Web of Science; search terms: horse, sedation and score. CONCLUSIONS: Most authors distinguish between sedation depth, sedation quality and degree of ataxia. These three features are evaluated using scoring systems similar to those classically used to assess pain, i.e. simple descriptive scales, numerical rating scales (NRS), visual analogue scales and/or multifactorial sedation scales. In addition, head height above the ground is often used as a measure of the depth of sedation. Very few authors have described how to adjust the administration rate or dose of alpha-2 agonists. Based on the available literature, the Ghent Sedation Algorithm was developed, which assigns scores (NRS) for degree of ataxia, sedation depth and surgical conditions, and uses these to prescribe changes in the administration rate of constant rate infusions of alpha-2 agonists. Studies are needed to validate this algorithm.

Clinical applicability of detomidine and methadone constant rate infusions for surgery in standing horses.

OBJECTIVE: To determine the required rate of a detomidine infusion (loading dose 5 µg kg-1; initial rate 12.5 µg kg-1 hour-1) added to a constant infusion of methadone (0.2 mg kg-1; 0.05 mg kg-1 hour-1) for sedation in standing horses and ponies undergoing elective surgeries with appropriate local anaesthetic techniques. STUDY DESIGN: Prospective, clinical study. ANIMALS: Adult, healthy, client-owned, non-food-producing horses or ponies sedated for elective standing surgeries longer than 45 minutes. METHODS: At baseline (in the stables before administration of sedative agents), at 10 minutes after sedation and every 5 minutes thereafter, ataxia, sedation and surgical condition were evaluated; each scored 0-3. These scores were used to adjust the detomidine administration rate using the Ghent Sedation Algorithm. A 10 cm visual analogue scale (VAS) was used by the main surgeon at the end of the procedure to evaluate the surgical conditions. Heart rate, systolic arterial pressure and respiratory frequency were also recorded at each time point. For statistical analysis, anova for normal, Kruskal-Wallis H-test for non-normal variables, and Mann-Whitney U test for VAS were used. RESULTS: From the 42 horses/ponies included in this study, 28 underwent dental procedures and 14 other types of procedures. Overall, dental procedures required higher mean detomidine rates compared with other types of surgeries (16.9 ± 4.5 versus 9.0 ± 1.9 µg kg-1 hour-1) (p < 0.001). Dental procedures were assigned similar VAS scores, median (range), of 7.8 (5.8-10) with other procedures, 8.7 (2.8-10). Cardiovascular changes were not clinically significant. No signs or behavioural changes of abdominal pain were observed postoperatively. CONCLUSIONS AND CLINICAL RELEVANCE: Satisfactory surgical conditions were achieved using a combination of detomidine and methadone infusions with locoregional anaesthesia, with no adverse effects. Dental procedures required higher detomidine dose rates compared with other surgeries.

Minimal Information About an Immuno-Peptidomics Experiment (MIAIPE).

Minimal information about an immuno-peptidomics experiment (MIAIPE) is an initiative of the members of the Human Immuno-Peptidome Project (HIPP), an international program organized by the Human Proteome Organization (HUPO). The aim of the MIAIPE guidelines is to deliver technical guidelines representing the minimal information required to sufficiently support the evaluation and interpretation of immunopeptidomics experiments. The MIAIPE document has been designed to report essential information about sample preparation, mass spectrometric measurement, and associated mass spectrometry (MS)-related bioinformatics aspects that are unique to immunopeptidomics and may not be covered by the general proteomics MIAPE (minimal information about a proteomics experiment) guidelines.

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

Sedative and antinociceptive effects of different combinations of detomidine and methadone in standing horses.

OBJECTIVE: To evaluate intravenous (IV) detomidine with methadone in horses to identify a combination which provides sedation and antinociception without adverse effects. STUDY DESIGN: Randomized, placebo-controlled, blinded, crossover. ANIMALS: A group of eight adult healthy horses aged (mean ± standard deviation) 7 ± 2 years and 372 ± 27 kg. METHODS: A total of six treatments were administered IV: saline (SAL); detomidine (5 µg kg-1; DET); methadone (0.2 mg kg-1; MET) alone or combined with detomidine [2.5 (MLD), 5 (MMD) or 10 (MHD) µg kg-1]. Thermal, mechanical and electrical nociceptive thresholds were measured, and sedation, head height above ground (HHAG), cardiopulmonary variables and intestinal motility were evaluated at 5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Normal data were analyzed by mixed-model analysis of variance and non-normal by Kruskal-Wallis (p < 0.05). RESULTS: Nociceptive thresholds in horses administered methadone with the higher doses of detomidine (MMD, MHD) were increased above baseline to a greater degree and for longer duration (MMD: 15-30 minutes, MHD: 30-60 minutes) than in horses administered low dose with methadone or detomidine alone (MLD, DET: 5-15 minutes). No increases in nociceptive thresholds were recorded in SAL or MET. Compared with baseline, HHAG was lower for 30 minutes in MMD and DET, and for 45 minutes in MHD. No significant sedation was observed in SAL, MET or MLD. Intestinal motility was reduced for 75 minutes in MHD and for 30 minutes in all other treatments. CONCLUSIONS: Methadone (0.2 mg kg-1) potentiated the antinociception produced by detomidine (5 µg kg-1), with minimal sedative effects. CLINICAL RELEVANCE: Detomidine (5 µg kg-1) with methadone (0.2 mg kg-1) produced antinociception without the adverse effects of higher doses of detomidine.

Comparative Analysis of the Endogenous Peptidomes Displayed by HLA-B*27 and Mamu-B*08: Two MHC Class I Alleles Associated with Elite Control of HIV/SIV Infection.

Indian rhesus macaques are arguably the most reliable animal models in AIDS research. In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV replication. A similar scenario is observed in humans where the expression of HLA-B*27 or HLA-B*57 has been linked to slow or no progression to AIDS after HIV infection. Despite having large differences in their primary structure, it has been reported that HLA-B*27 and Mamu-B*08 display peptides with sequence similarity. To fine-map the Mamu-B*08 binding motif and assess its similarities with that of HLA-B*27, we affinity purified the peptidomes bound to these MHC class I molecules and analyzed them by LC-MS, identifying several thousands of endogenous ligands. Sequence analysis of both sets of peptides revealed a degree of similarity in their binding motifs, especially at peptide position 2 (P2), where arginine was present in the vast majority of ligands of both allotypes. In addition, several differences emerged from this analysis: (i) ligands displayed by Mamu-B*08 tended to be shorter and to have lower molecular weight, (ii) Mamu-B*08 showed a higher preference for glutamine at P2 as a suboptimal binding motif, and (iii) the second major anchor position, found at PΩ, was much more restrictive in Mamu-B*08. In this regard, HLA-B*27 bound efficiently peptides with aliphatic, aromatic (including tyrosine), and basic C-terminal residues while Mamu-B*08 preferred peptides with leucine and phenylalanine in this position. Finally, in silico estimations of binding efficiency and competitive binding assays to Mamu-B*08 of several selected peptides revealed a good correlation between the characterized anchor motif and binding affinity. These results deepen our understanding of the molecular basis of the presentation of peptides by Mamu-B*08 and can contribute to the detection of novel SIV epitopes restricted by this allotype.

Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue.

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Peptide handling by HLA-B27 subtypes influences their biological behavior, association with ankylosing spondylitis and susceptibility to endoplasmic reticulum aminopeptidase 1 (ERAP1).

HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes.

Partial intravenous anaesthesia in the horse: a review of intravenous agents used to supplement equine inhalation anaesthesia. Part 2: opioids and alpha-2 adrenoceptor agonists.

OBJECTIVE: To review the literature with regard to the use of different intravenous agents as supplements to inhalational anaesthesia in horses. The Part 2 of this review will focus in the use of opioids and α2 -agonists. DATABASES USED: Pubmed and Web of Science. Search terms: horse, inhalant anaesthesia, balanced anaesthesia, partial intravenous anaesthesia, opioids, morphine, pethidine, butorphanol, methadone, fentanyl, alfentanil, remifentanil, sufentanil, xylazine, romifidine, detomidine, medetomidine and dexmedetomidine. CONCLUSIONS: Different drugs and their combinations can be administered systemically in anaesthetized horses aiming to reduce the amount of the volatile agent while improving the recovery qualities and providing a multimodal analgesic approach. However, full studies as to whether these techniques improve cardiopulmonary status are not always available and potential disadvantages should also be considered.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Partial intravenous anaesthesia in the horse: a review of intravenous agents used to supplement equine inhalation anaesthesia. Part 1: lidocaine and ketamine.

OBJECTIVE: To review the literature with regard to the use of different intravenous agents as supplements to inhalational anaesthesia in horses. These drugs include lidocaine, ketamine, opioids and α2 -agonists. The Part 1 of this review will focus in the use of lidocaine and ketamine. DATABASES USED: Pubmed & Web of Science. Search terms: horse, inhalant anaesthesia, balanced anaesthesia, partial intravenous anaesthesia, lidocaine, ketamine. CONCLUSIONS: Different drugs and their combinations can be administered systemically in anaesthetized horses, with the aim of reducing the amount of the volatile agent whilst improving the recovery qualities and providing a multimodal analgesic approach. However, full studies as to whether these techniques improve cardiopulmonary status are not always available and potential disadvantages should also be considered.

Minimum end-tidal sevoflurane concentration necessary to prevent movement during a constant rate infusion of morphine, or morphine plus dexmedetomidine in ponies.

OBJECTIVE: To compare the effects of a constant rate infusion (CRI) of dexmedetomidine and morphine to those of morphine alone on the minimum end-tidal sevoflurane concentration necessary to prevent movement (MACNM ) in ponies. STUDY DESIGN: Prospective, randomized, crossover, 'blinded', experimental study. ANIMALS: Five healthy adult gelding ponies were anaesthetized twice with a 3-week washout period. METHODS: After induction of anaesthesia with sevoflurane in oxygen (via nasotracheal tube), the ponies were positioned on a surgical table (T0), and anaesthesia was maintained with sevoflurane (Fe'SEVO 2.5%) in 55% oxygen. Monitoring included pulse oximetry, electrocardiography and measurement of anaesthetic gases, arterial blood pressure and body temperature. The ponies were mechanically ventilated and randomly allocated to receive IV treatment M [morphine 0.15 mg kg⁻¹ (T10-T15) followed by a CRI (0.1 mg kg⁻¹ hour⁻¹)] or treatment DM [dexmedetomidine 3.5 µg kg⁻¹ plus morphine 0.15 mg kg⁻¹ (T10-T15) followed by a CRI of dexmedetomidine 1.75 µg kg⁻¹ hour⁻¹ and morphine 0.1 mg kg⁻¹ hour⁻¹]. At T60, a stepwise MACNM determination was initiated using constant current electrical stimuli at the skin of the lateral pastern region. Triplicate MACNM estimations were obtained and then averaged in each pony. Wilcoxon signed-rank test was used to detect differences in MAC between treatments (α = 0.05). RESULTS: Sevoflurane-morphine MACNM values (median (range) and mean ± SD) were 2.56 (2.01-4.07) and 2.79 ± 0.73%. The addition of a continuous infusion of dexmedetomidine significantly reduced sevoflurane MACNM values to 0.89 (0.62-1.05) and 0.89 ± 0.22% (mean MACNM reduction 67 ± 11%). CONCLUSION AND CLINICAL RELEVANCE: Co-administration of dexmedetomidine and morphine CRIs significantly reduced the MACNM of sevoflurane compared with a CRI of morphine alone at the reported doses.