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The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing. Key PointsComparison tests allow laboratories to evaluate their analyses for accuracy and reliability.Two forensic identification, parentage and species determination comparison tests were performed.The study showed that the LCN DNA analysis represented a significant challenge to most laboratories.Lacking familiarity with species tests curbed most laboratories from reporting accurately.A reliance on forensically validated testing standards may have prevented some of the common errors.

Objectives The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. Methods Dental pulp was isolated by explant culture from two cats 2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. Results The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45−, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2–P4. Conclusions and relevance In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.

Simple Summary The main target of mesenchymal stem cell therapy in horses has long been the locomotor system, because these athletic animals commonly suffer from tendon and joint lesions. Originally, mesenchymal stem cells were thought to act by just differentiating into the cells of the injured tissue. However, these cells are also able to regulate and stimulate the body’s own repair mechanisms, opening the door to many applications in inflammatory and immune-mediated disorders in both animals and humans. In horses, beyond their traditional application in the musculoskeletal system, these cells have been studied for ophthalmologic pathologies such as corneal ulcers or immune-mediated processes, and for reproductive disorders such as endometritis/endometrosis. Their potential has been explored for equine pathologies very similar to those affecting people, such as asthma, metabolic syndrome, aberrant wound healing, or endotoxemia, as well as for equine-specific pathologies such as laminitis. Current evidence is still preliminary, and further research is needed to clarify different aspects, although research performed so far shows the promising potential of mesenchymal stem cells to treat a wide variety of equine pathologies, some of which are analogous to human disorders. Therefore, advancements in this path will be beneficial for both animals and people. Abstract The differentiation ability of mesenchymal stem cells (MSCs) initially raised interest for treating musculoskeletal injuries in horses, but MSC paracrine activity has widened their scope for inflammatory and immune-mediated pathologies in both equine and human medicine. Furthermore, the similar etiopathogenesis of some diseases in both species has advanced the concept of “One Medicine, One Health”. This article reviews the current knowledge on the use of MSCs for equine pathologies beyond the locomotor system, highlighting the value of the horse as translational model. Ophthalmologic and reproductive disorders are among the most studied for MSC application. Equine asthma, equine metabolic syndrome, and endotoxemia have been less explored but offer an interesting scenario for human translation. The use of MSCs in wounds also provides a potential model for humans because of the healing particularities in both species. High-burden equine-specific pathologies such as laminitis have been suggested to benefit from MSC-therapy, and MSC application in challenging disorders such as neurologic conditions has been proposed. The available data are preliminary, however, and require further development to translate results into the clinic. Nevertheless, current evidence indicates a significant potential of equine MSCs to enlarge their range of application, with particular interest in pathologies analogous to human conditions.

Mesenchymal stem cells (MSCs) are a promising treatment for equine musculoskeletal injuries because of their ability to regulate the inflammation and to differentiate into other cell types. Since interest in allogeneic therapy is rising, concerns about MSC immunogenicity need to be addressed. Differentiated MSCs from several species increase their expression of immunogenic molecules and induce alloresponses, but equine MSC immunogenic profile after differentiation has not been reported. Therefore, the aim of this study was to assess the gene expression of immunogenic markers in tri-lineage differentiated equine bone marrow derived MSCs (eBM-MSCs). For this purpose, eBM-MSCs (n = 4) were differentiated into osteoblasts, adipocytes and chondrocytes. Differentiation was confirmed by specific staining and gene expression of lineage-related markers. Subsequently, gene expression of MHC-I, MHC-II, CD40 and CD80 was analyzed in undifferentiated (control) and tri-lineage differentiated eBM-MSCs. Osteogenesis and adipogenesis, but not chondrogenesis, significantly upregulated MHC-I; MHC-II expression significantly increased in the three lineages, while CD40 and CD80 expression did not change. Despite this, MHC-I and MHC-II upregulation after differentiation might lead to increased immunogenicity and risk of allorecognition, either eBM-MSCs differentiate in vivo after administration or they are differentiated prior to administration, with potential negative consequences for effectiveness and safety of allogeneic therapy.

The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte-blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.

[Introduction]: Patellar tendon overuse injuries are common in athletes. Imaging may show a change in tissue structure with tendon thickening and disruption of the intratendinous substance. We wish to test the hypothesis that both autologous bone marrow expanded mesenchymal stem cells and autologous leukocyte-poor platelet-rich plasma (LP-PRP) implanted into the area of the disrupted tendinopathic patellar tendon will restore function, but tendon regeneration tissue will only be observed in the subjects treated with autologous bone marrow expanded mesenchymal stem cells., [Methods and analysis]: This is a single-centre, pilot phase I/II, double-blinded clinical trial with randomisation with active control. Twenty patients with a diagnosis of patellar tendinopathy with imaging changes (tendon thickening and disruption of the intratendinous substance at the proximal portion of the patellar tendon) will be randomised in a 1:1 ratio to receive a local injection of either bone-marrow autologous mesenchymal stem cells (MSC), isolated and cultured under GMP at The Institute of Biology and Molecular Genetics (IBGM) (Spain) or P-PRP. The study will have two aims: first, to ascertain whether a clinically relevant improvement after 3, 6 and 12 months according to the visual analogue scale (VAS), Victorian Institute of Sport Assessment for patellar tendons (VISA-P) and dynamometry scales (DYN) will be achieved; and second, to ascertain whether the proposed intervention will restore tendon structure as determined by ultrasonography (US), Doppler ultrasonography (DUS), and innovative MRI and ultrasound techniques: Magnetic Resonance T2 FAT SAT (UTE, Ultrashort Echo TE) sequence and Ultrasound Tissue Characterization (UTC). Patients who are randomised to the P-PRP treatment group but do not achieve a satisfactory primary endpoint after 6 months will be offered treatment with MSC., [Trial registration]: NCT03454737., This work was supported by Institutde Terapia Regenerativa Tissular. Centro Médico Teknon (ITRT). (Institute for Tissue Regeneration Therapy), Barcelona, Spain.

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/μL) or basal PL (BPL; 177 × 103 platelets/μL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P

Chapter 29: et al., Goat farming plays a key role in agricultural activity and in maintaining forest lands in Southwestern Europe. Remarkably, the Iberian Peninsula represents nearly 25% of the European goat census. Goat husbandry is often associated with low input production systems and uses selective breeding programs, which are less advanced than those employed in other livestock. Native goat breeds are very well adapted to produce in marginal areas under extensive conditions. Loss of their genetic diversity could have important economic, ecological and scientific implications as well as social consequences. Several methodologies have been developed to preserve the genetic diversity of single populations, but additional problems arise when a group of breeds, i.e., subpopulations, is considered in conservation programs. The conservation priority of a breed depends on its contribution to the overall genetic diversity of the species, in terms of the intrinsic genetic variation that it harbors and also of its relationship with other breeds. However, the estimation of the contributions of each of these two components to overall genetic diversity cannot be easily assessed. Besides, conservation goals in the short-term (avoidance of inbreeding) and long-term (adaptation to future environmental changes) should be considered when taking conservative decisions. A comprehensive analysis of Iberian goat breeds has been carried out to evaluate conservation priorities based on methodologies that account for within- or between-breed genetic diversity, or combinations of both. Based on genetic distinctiveness, breeds such as Palmera, Formentera, and Blanca Celtibérica were prioritized, whereas the maximum priority was assigned to Florida, Pirenaica, Retinta, and Moncaína breeds when focusing on within-breed diversity. Overall, combined approaches showed very little variation among breeds reflecting a history of extensive gene flow, partly due to transhumance and recent divergence. The main conclusion of our study is that these statistical analyses are useful, but conservation decisions must take into account other factors in addition to strict genetic diversity classification., This study has been partially supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) RZ2004-00037-00-00, RZ2006-00005-C02-00, RZ2007-00005-C02-01 and RZ2007-00005-C02-02 projects. CG was supported by a contract grant from the Fundação para a Ciência e a Tecnologia (project ARADO, IF/00866/2014), Portugal.

International audience; Domestic species such as cattle (Bos taurus taurus and B. t. indicus) represent attractive biological models to characterize the genetic basis of short term evolutionary response to climate pressure induced by their post-domestication history. Here, using newly generated dense SNP genotyping data, we assessed the structuring of genetic diversity of 21 autochtonous cattle breeds from the whole Mediterranean basin and performed genome-wide association analyses with covariables discriminating the different Mediterranean climate sub-types. This provided insights into both the demographic and adaptive histories of Mediterranean cattle. In particular, a detailed functional annotation of genes surrounding variants associated with climate variations highlighted several biological functions involved in Mediterranean climate adaptation such as thermotolerance, UV protection, pathogen resistance or metabolism with strong candidate genes identified (e.g. NDUFB3, FBN1, METTL3, LEF1, ANTXR2 and TCF7). Accordingly, our results suggest that main selective pressures affecting cattle in Mediterranean area may have been related to variation in heat and UV exposure, in food resources availability and in exposure to pathogens, such as anthrax bacteria (Bacillus anthracis). Furthermore, the observed contribution of the three main bovine ancestries (indicine, European and African taurine) in these different populations suggested that adaptation to local climate conditions may have either relied, on standing genomic variation of taurine origin or adaptive introgression from indicine origin, depending on the local breed origins. Taken together, our results highlight the genetic uniqueness of local Mediterranean cattle breeds and strongly support conservation of these populations

Mesenchymal stem cells (MSCs) are being investigated for the treatment of equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of equine bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for equine joint diseases.

Background This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. Results Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p

A 1-month-old Purebred Spanish Horse (PSH) foal presented with progressive hepatic failure culminating in death. Hepatic lesions were consistent with congenital hepatic fibrosis (CHF). Genetic studies in the PKHD1 gene in the affected foal revealed that it was heterozygous for the 2 previously described single-nucleotide polymorphisms (SNPs) linked to CHF in Swiss Franches-Montagnes (SFM) horses. In addition, 2 novel mutations were detected, the foal being homozygous for one of them and heterozygous for the other. Genetic studies in a healthy PSH population ( n = 35) showed a 3-fold higher genotypic frequency for PKHD1 SNP g.49,630,834G>A and a 5-fold higher genotypic frequency for PKHD1 SNP g.49,597,760A>T compared with those reported for SFM horses. SNPs in the PKHD1 gene in CHF-affected SFM horses might not fully explain the CHF observed in the PSH. Other mutations in the PKHD1 gene could play a more important role in the PSH.

Since the clinical use of mesenchymal stem cells (MSCs) for treating musculoskeletal injuries is gaining popularity, practitioners should be aware of the factors that may affect MSCs from tissue harvesting for MSC isolation to cell delivery into the injury site. This review provides equine practitioners with up-to-date, practical knowledge for the treatment of equine patients using MSCs. A brief overview of laboratory procedures affecting MSCs is provided, but the main focus is on shipping conditions, routes of administration, injection methods, and which commonly used products can be combined with MSCs and which products should be avoided as they have deleterious effects on cells. There are still several knowledge gaps regarding MSC-based therapies in horses. Therefore, it is important to properly manage the factors which are currently known to affect MSCs, to further strengthen the evidence basis of this treatment.

The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-β1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-β1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.

We have analyzed the 5′cis-regulatory regions of four genes coding for key proteins involved in lipid metabolism and energy homeostasis in Rasa Aragonesa, Assaf and Roja Mallorquina sheep breeds. We identified 10 novel polymorphisms in the 5′regulatory regions in fatty acid synthase (FASN), glycerol-3-phosphate acyltransferase mitochondrial (GPAM), melanocortin-4 receptor (MC4R) and perilipin (PLIN1) genes. Due to the involvement of these genes in fat quantitative traits and the effect of all polymorphic positions on transcription factors binding sites, we tested all of them in two relevant meat reared breeds which were subjected to different feeding systems. Although no relationship was detected between the mRNA expression level of the candidate genes and the genotypes, additional studies must be conducted in older individuals, since these polymorphisms have been detected by in silico studies to be putatively involved in transcriptional or posttranscriptional regulatory mechanisms. The expression level of GPAM, MC4R and PLIN1 genes was analyzed and compared between feeding groups detecting over expression of adipogenic genes in the intensive groups. These results suggest that nutritional stimulation affects the expression of candidates genes involved in lipid metabolic processes, and therefore the fat quality in meat ruminant-derived food products.

The aim of this Regional Research Communication was to validate a panel of 30 microsatellite markers recommended by FAO/ISAG for studies of biodiversity in cattle to improve the characterisation of Cuban buffalo populations. The water buffalo (Bubalus bubalis) is an economically important livestock species. Therefore, research focused on the study of the genetic relationships among water buffalo populations is useful to support conservation decisions and to design breeding schemes. Twenty-eight of the 30 tested regions were amplified, one of which (ETH10) turned out to be monomorphic. A total of 143 alleles were observed in the Cuban water buffalo population. The average number of alleles per locus was 5·04. The number of alleles per polymorphic locus ranged from two (INRA 63 and MM12) to nine (ETH185). The observed and expected heterozygosity ranged from 0·108 (HAUT24) to 0·851 (CSSM66) and 0·104 (MM12) to 0·829(INRA32), respectively. The polymorphic information content (PIC) ranged from 0·097 (MM12) to 0·806 (INRA32), and the overall value for these markers was 0·482. Within the population, inbreeding estimates (FIS) was positive in 14 of the 30 loci analysed. This study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in Cuban water buffalo breeds. Furthermore, the results can be utilised for future breeding strategies and conservation.

Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.

The standard treatment of peripherical nerve injuries with substance gap is to introduce the nerve free extremes in a biodegradable tube which, as a biocamera, allows the continuity of the nerve, promote the neuroconduction and save the lesion from the surrounding fibrosis. However, this procedure has not any direct effect on the neuroregeneration nor to resolve high severe lesions. The mesenchymal stem cells (MSC) can derivate "in vitro" in different lineages, including Schwann cells. Different studies have shown MSC can promote the nerve regeneration in rodents, dogs and primates. Moving to the human clinical application requires the procedure standardization, including the optimal cell dose which we have to use. In the sheep model animal we performed a study of 1 cm. nerve section-ressection and repair with a Neurolac™ biocamera, in whose gap we applied between 30 to 50×10(6) MSC from cancellous bone, all of them selected and cultured with GMP procedures. The results were compared with controls (saline serum ± platelet-rich plasma). We used radial nerve (sensitive) and tibial nerve (motor) from 7 sheep. In the first step we performed the surgical lesion and bone marrow aspiration, and in 3 weeks we performed the surgical repair. 3 sheep were sacrificed in 3 months, and 4 sheep in 6 months. In all surgeries we performed a neurophysiological register. When we obtained the tissue samples, we performed an histological, immunohistiquimical and morphometrical study. The recovery percentage was defined comparing the axonal density from the proximal and distal lesion margins. The 3 months samples results were wrong. In 6 months samples results we observed a significative myelined nervous fibers and conduction increasing, in front of controls, both radial and tibial nerves. These results suggest the MSC application in biodegradable scaffold in nerve injuries promotes good results in terms of regeneration and functional recovery.

Knowledge about the 5′ regulatory region variation of candidate genes may help to improve breeding and preserve genetic diversity in ruminant species and reveal insights about their evolutionary history. Cis-regulatory regions of candidate genes involved in lipid metabolism and related to energy homeostasis were sequenced in 3 Bos taurus breeds and 1 Bos indicus breed to identify potentially functional polymorphism in non-coding regions. Searching for promoter variability in Fatty Acid Synthase (FASN), Stearoyl-CoADesaturase (SCD), Perilipin (PLIN), Glycerol-3 phosphate acyltransferase mitochondrial (GPAM) and Melanocortin-4 receptor (MC4R), 42 polymorphisms were found. Forty one of them were reported for the first time in B. taurus and B. indicus species. Through in silico analysis we noticed that many of them fall within putative binding sites for transcription factors closely involved in the regulation of the expression of adipogenic genes. A higher nucleotide diversity was found in B. indicus for FASN, SCD, PLIN and MC4R, whereas GPAM was the only more polymorphic promoter in B. taurus. The lower diversity in the B. taurus breeds may reflect their population history with a stronger selection pressure for some economic and productive traits. Alternatively, the ancestors of the indicine breed may have had a high genetic diversity. This research underlines the significance of local breeds for preserving genetic diversity for a sustainable agriculture. The domestication history of ruminant species and the critical role of the analyzed genes in lipid metabolic processes, make these putatively regulatory polymorphisms important targets in breeding and in genetic diversity studies.

The Casta Navarra lineage was one of the populations used to establish the fighting bull (FB) breed, and it has also been reproductively isolated from the others FBs. A total of 1284 individuals from two generations of 16 Casta Navarra herds were sampled to analyse their diversity, their genetic structure and the ability of 28 microsatellite markers to assign individuals to closely related populations. These animals were compared with closely related phylogenetic (FB) or geographical (Pirenaica and Monchina) populations. Hardy-Weinberg equilibrium analysis showed that 82% of the loci had a significant heterozygote deficit as a consequence of the Wahlund effect. The average proportion of genetic variation explained by farm differences was 9% by Wright's FST index. A phylogenetic tree constructed with a neighbour-joining method based on Reynolds genetic distances and a Bayesian Markov chain Monte Carlo clustering approach revealed clear differences between farm groups that generally corresponded to historical information and could unambiguously differentiate Casta Navarra cattle from the other populations. The percentage of animals correctly assigned to the Casta Navarra population was 91.78% for a q threshold of >0.9. Admixture was only detected in 4.45% (q

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O2, a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O2) and hypoxia (5% O2). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.

Locomotor system disorders in equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and pheno-typing. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes. (C) 2016 Elsevier Inc. All rights reserved.

espanolFue evaluado el polimorfismo de los loci de seis proteinas lacteas en cinco razas bovinas cubanas mediante el analisis de ADN por las tecnicas combinadas de PCR y de creacion de sitio de restriccion (ACRS), metodologia de oligonucleotidos alelo-especificos (ASO) y polimorfismo de longitud de los fragmentos de restriccion (RFLP). Se estimaron las frecuencias alelicas para CASA1, CASAB, CASA2, CASK, LAA y LGB en las cinco razas (N = 324), siendo la cantidad de individuos por poblacion: Siboney de Cuba (SC = 85), Criollo Cubano (CC = 60), Cebu Cubano (ZC = 61), Mambi de Cuba (MC = 60) y Taino de Cuba (TC = 58). Se evaluo el estado de equilibrio Hardy�Weinberg para cada locus en cada poblacion, observandose que todas las poblaciones tienen al menos un locus desviado de esta condicion de equilibrio y siempre por exceso de heterocigotos. Se identificaron los alelos CASA1C y LAAAen el CC y TC, lo que evidencia la presencia de genes Bos indicus en estas poblaciones. Se aprecia un incremento de la heterocigosidad en estas poblaciones y las frecuencias genotipicas en cada poblacion permiten diferenciar estas de aquellas que las originaron. EnglishThe polymorphism of six milk protein loci in five Cuban breeds was investigated by means of DNA analysis by combined techniques of PCR and amplification created restriction sites (ACRS), the method of allele-specific oligonucleotides (ASO), and restriction fragment length polymorphism (RFLP). Allele frequencies of loci CASA1, CASAB, CASA2, CASK, LAA and LGB were estimated in the five breeds (N = 324), the number of individuals per population being: Siboney de Cuba (SC = 85), Cuban Creole (CC = 60) Cuban Zebu (ZC = 61), Cuba Mambi (MC = 60) and Taino of Cuba (TC = 58). Hardy�Weinberg equilibrium was estimated for each locus in each population, showing that all the populations have at least one locus deviated from this condition of equilibrium and always through heterozygote excess. Alleles CASA1C and LAAA were identified in the CC and CT breeds, showing the presence of Bos indicus genes in these populations. An increase of heterozygosis is observed in these populations, and genotype frequencies in each population allow differentiation of these from those which originated them. francaisLe polymorphisme de loci dans six proteines de lait de cinq races cubaines a ete evalue a travers de l'analyse de l'ADN par les techniques combinees de la PCR et la creation d'un site de restriction (ACRS), l'hybridation allele-specifique (ASO) et le polymorphisme de longueur des fragments de restriction (RFLP). Les frequences des alleles ont ete estimees pour CASA1, CASAB, CASA2, CASK, LAA et LGB au cours des cinq races (N = 324), le montant d'individus par population: Siboney de Cuba (SC = 85), Cubaine Creole (CC = 60) Cubaine Zebu (ZC = 61), Mambi de Cuba (MC = 60) et Taino de Cuba (TC = 58). Le statut d'equilibre Hardy�Weinberg a ete evalue pour chaque locus, dans chaque population, montrant que toutes les populations ont au moins un locus devie de cette condition d'equilibre et toujours a la suite d'un exces d'heterozygotes. Les alleles CASA1C et LAAA ont ete identifies dans le CC et CT, ce qui montre la presence de genes Bos indicus dans ces populations. Une augmentation de l'heterozygotie dans ces populations a ete observee, et les frequences genotypiques dans chaque population permettent de differencier ces dernieres de celles qui leur ont donne lieu.

Background: Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neurodegenerative disease characterized by ascending muscle weakness, atrophy and paralysis. Early muscle abnormalities that precede motor neuron loss in ALS may destabilize neuromuscular junctions, and we have previously demonstrated alterations in myogenic regulatory factor (MRF) expression in vivo and in the activation of myofiber-associated skeletal muscle satellite cells (SMSCs) in the mouse model of ALS (SOD1-G93A). Methods: To elucidate niche dependence versus cell-autonomous mutant SOD1 (mSOD1) toxicity in this model, we measured in vitro proliferation potential and MRF and cyclin gene expression in SMSC cultures derived from fast-twitch extensor digitorum longus and slow-twitch soleus muscles of SOD1-G93A mice. Results: SMSCs from early presymptomatic (p40) to terminal, semi-paralytic (p120) SOD1-G93A mice demonstrated generally lower proliferation potential compared with age-matched controls. However, induced proliferation was observed in surgically denervated wild-type animals and SOD1-G93A animals at p90, when critical denervation arises. SMSCs from fast and slow muscles were similarly affected by mSOD1 expression. Lowered proliferation rate was generally corroborated with decreased relative MRF expression levels, although this was most prominent in early age and was modulated by muscle type origin. Cyclins controlling cell proliferation did not show modifications in their mRNA levels; however, the expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a), which is known to promote myoblast differentiation, was decreased in SOD1-G93A cultures. Conclusions: Our data suggest that the function of SMSCs is impaired in SOD1-G93A satellite cells from the earliest stages of the disease when no critical motor neuron loss has been described.

Controlling meat traceability using SNPs is an effective method of ensuring food safety. We have analyzed several SNPs to create a panel for bovine genetic identification and traceability studies. One of these was the transversion g.329CT (Genbank accession no. AJ496781) on the cytochrome P450 17A1 gene, which has been included in previously published panels. Using minisequencing reactions, we have tested 701 samples belonging to eight Spanish cattle breeds. Surprisingly, an excess of heterozygotes was detected, implying an extreme departure from Hardy-Weinberg equilibrium (P0.001). By alignment analysis and sequencing, we detected that the g.329CT SNP is a false positive polymorphism, which allows us to explain the inflated heterozygotic value. We recommend that this ambiguous SNP, as well as other polymorphisms located in this region, should not be used in identification, traceability or disease association studies. Annotation of these false SNPs should improve association studies and avoid misinterpretations.

Summary Conservation and improvement strategies in farm animals should be based on a combination of genetic and phenotypic characteristics. Genotype data from 30 microsatellites were used to assess the genetic diversity and relationships among five Cuban cattle breeds (Siboney de Cuba, Criollo Cubano, Cebu Cubano, Mambi de Cuba and Taino de Cuba). All microsatellite markers were highly polymorphic in all the breeds. The expected heterozygosity ranged from 0.67 ± 0.02 in the Taino de Cuba breed to 0.75 ± 0.02 in the Mambi de Cuba breed, and the observed heterozygosity ranged from 0.66 ± 0.03 in the Cebu Cubano breed to 0.73 ± 0.02 in the Siboney de Cuba breed. The genetic differentiation between the breeds was significant (p

Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by reverse transcriptase real-time quantitative PCR along with the expression of 5 other MSC markers. Both populations of cells expressed CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD106, CD146 and CD166 transcripts. Significant differences in gene expression levels between BM- and AT-MSCs were observed for CD44, CD90, CD29 and CD34. Both cell types were negative for CD45 and CD31. The surface antigens tested revealed a similar phenotypic profile between horse and human MSCs, although specific differences in some surface antigens were noticed.

Summary Reasons for performing study: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objectives: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. Results: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. Conclusions: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. Potential relevance: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

Summary Genetic diversity in and relationships among 26 Creole cattle breeds from 10 American countries were assessed using 19 microsatellites. Heterozygosities, F-statistics estimates, genetic distances, multivariate analyses and assignment tests were performed. The levels of within-breed diversity detected in Creole cattle were considerable and higher than those previously reported for European breeds, but similar to those found in other Latin American breeds. Differences among breeds accounted for 8.4% of the total genetic variability. Most breeds clustered separately when the number of pre-defined populations was 21 (the most probable K value), with the exception of some closely related breeds that shared the same cluster and others that were admixed. Despite the high genetic diversity detected, significant inbreeding was also observed within some breeds, and heterozygote excess was detected in others. These results indicate that Creoles represent important reservoirs of cattle genetic diversity and that appropriate conservation measures should be implemented for these native breeds in order to minimize inbreeding and uncontrolled crossbreeding.

In Iberia there are 51 officially recognized cattle breeds of which 15 are found in Portugal and 38 in Spain. We present here a comprehensive analysis of the genetic diversity and structure of Iberian cattle. Forty of these breeds were genotyped with 19 highly polymorphic microsatellite markers. Asturiana de los Valles displayed the greatest allelic diversity and Mallorquina the least. Unbiased heterozygosity values ranged from 0.596 to 0.787. The network based on Reynolds distances was star-shaped with few pairs of interrelated breeds and a clear cluster of 4 breeds (Alistana/Arouquesa/Marinhoa/Mirandesa). The analysis of the genetic structure of Iberian cattle indicated that the most probable number of population clusters included in the study would be 36. Distance results were supported by the STRUCTURE software indicating a relatively recent origin or possible crossbreeding or both between pairs or small groups of breeds. Five clusters included 2 different breeds (Betizu/Pirenaica, Morucha/Avilena, Parda de Montana/Bruna de los Pirineos, Barrosa/Cachena, and Toro de Lidia/Brava de Lide), 3 breeds (Berrenda en Negro, Negra Andaluza, and Mertolenga) were divided in 2 independent clusters each, and 2 breeds were considered admixed (Asturiana de los Valles and Berrenda en Colorado). Individual assignation to breeds was not possible in the 2 admixed breeds and the pair Parda de Montana/Bruna de los Pirineos. The relationship between Iberian cattle reflects their geographical origin rather than their morphotypes. Exceptions to this geographic clustering are most probably a consequence of crossbreeding with foreign breeds. The relative genetic isolation within their geographical origin, the consequent genetic drift, the adaptation to specific environment and production systems, and the influence of African and European cattle have contributed to the current genetic status of Iberian cattle, which are grouped according to their geographical origin. The greater degree of admixture observed in some breeds should be taken into account before using molecular markers for genetic assignment of individuals to breeds.

We utilised DNA analysis to detect the presence of the digenean Phyllodistomum folium in three cyprinid species, Scardinius erythrophthalmus, Cyprinus carpio and Rutilus rutilus. DNA sequencing of the region containing the genes ITS1-5.8S-ITS2 revealed 100% sequence identity between DNA from the sporocysts found in zebra mussels and DNA from adults located in the urinary system of 29 cyprinid fish. A second genetically different (variation=1.6%) sequence was observed in two samples from R. rutilus. In our opinion, the existence of a complex of species reported as P. folium is supported by recent genetic studies, including our own results. The overall prevalence of P. folium in mussels from the Ebro River was 4.67% in 2006, although during the summer months the rates frequently exceeded 10%.

This study is aimed at establishing priorities for the optimal conservation of genetic diversity among a comprehensive group of 40 cattle breeds from the Iberian Peninsula. Different sets of breed contributions to diversity were obtained with several methods that differ in the relative weight attributed to the within- and between-breed components of the genetic variation. The contributions to the Weitzman diversity and the expected heterozygosity (He) account for between- and within-breed variation only, respectively. Contributions to the core set obtained for several kinship matrices, incorporate both sources of variation, as well as the combined contributions of Ollivier and Foulley and those of Caballero and Toro. In general, breeds that ranked high in the different core set applications also ranked high in the contribution to the global He, for example, Sayaguesa, Retinta, Monchina, Berrenda en Colorado or Marismeña. As expected, the Weitzman method prioritised breeds with low contributions to the He, like Mallorquina, Menorquina, Berrenda en Negro, Mostrenca, Vaca Palmera or Mirandesa, all showing highly negative contributions to He - that is, their removal would significantly increase the average He. Weighing the within- and between-breed components with the FST produced a balanced set of contributions in which all the breeds ranking high in both approaches show up. Unlike the other methods, the contributions to the diversity proposed by Caballero and Toro prioritised a good number of Portuguese breeds (Arouquesa, Barrosã, Mertolenga and Preta ranking highest), but this might be caused by a sample size effect. Only Sayaguesa ranked high in all the methods tested. Considerations with regard to the conservation scheme should be made before adopting any of these approaches: in situ v. cryoconservation, selection and adaptation within the breeds v. crossbreeding or the creation of synthetic breeds. There is no general consensus with regard to balancing within- and between-breed diversity and the decision of which source to favour will depend on the particular scenario. In addition to the genetic information, other factors, such as geographical, historical, economic, cultural, etc., also need to be considered in the formulation of a conservation plan. All these aspects will ultimately influence the distribution of resources by the decision-makers.

On the basis of QTL studies for milk-fat yield trait on BTA3, annexin 9 protein (ANXA9), fatty acid transport protein type 3 (SLC27A3) and diacylglycerol O-acyltransferase 1 (DGAT1) were selected as candidate genes. Three different single nucleotide polymorphisms (SNPs) of bovine ANXA9, SLC27A3 and DGAT1 genes have been tested in a selective genotyping design for milk-fat yield. Significant allele frequency differences were found for ANXA9 (p=0.02), in Holstein-Friesian animals with high and low breeding values for milk-fat yield. Regression analysis also showed a significant effect (p=0.0207) between estimated breeding values (EBVs) for fat milk content and ANXA9 polymorphism. So ANXA9 gene falls into a significant quantitative trait loci interval for milk-fat yield that was previously reported on bovine chromosome 3 in other dairy populations. Our results suggest that the ANXA9 gene polymorphism or a linked segregating QTL contributes to variation in milk-fat yield. © 2009 Elsevier Ltd.

The bovine transferrin gene (TF) is located at 125 cM on bovine chromosome 1 (BTA1); it codes for transferrin, a glycoprotein that is highly conserved in many species and that is responsible for iron transport. The TF gene has been located in several QTL regions, and some transferrin classes have been associated with fat and milk yields. We analyzed by means of allele-specific oligonucleotide real-time PCR the c.1455A>G SNP in exon 12 of the TF cDNA sequence (accession number U02564), which induces an Asp/Gly substitution at position 469 of the peptide. The c.1455A>G SNP was assayed in eight Spanish cattle breeds, as well as in two groups of Holstein-Friesian animals that had the highest and lowest estimated breeding values for milk fat yield. Analysis of the cSNP showed balanced frequencies in all breeds, with a mean of 0.44. Evaluation of a potential association between the cSNP and the groups of Holstein-Friesian animals selected for milk fat yield showed a significant association (P < 0.0006); the G allele was associated with high fat production. Significant differences in genotypic frequencies between the groups were also detected (P < 0.0028). These results lead us to suggest that the TF gene has an effect on milk fat yield.

Summary. Serum samples from 412 Spanish wild rabbits were analysed by starch and polyacrylamide gel electrophoresis. Three different transferrin (Tf) phenotypes (A, AB and B) were observed by both methods. The occurrence of two codominant alleles (TfA and TfB with frequencies of 0.89 and 0.11 respectively) at an autosomal locus (Tf) was supported by the population data on genetic equilibrium. Electrophoretic mobility differences between the Tf variants A and B could not be explained by differences in sialic acid or iron contents. Each of the two Tf variants were shown to have two sialic acid residues by neuraminidase treatment. These variants had similar affinities for iron, and iron binding did not lead to the conversion of one variant into the other.

Summary. Five erythrocyte proteins (Adenosine deaminase, Ada; 6-Phosphogluco-nate dehydrogenase, Pgd; Esterase 1, Es-1; Esterase 3, Es-3; NADH-Diaphorase 2, Dia-2) and a serum β-globulin protein (Esterase 7, Est-7) were studied in rabbits using starch gel electrophoresis. Samples were obtained from 317 Spanish Common individuals (38 families, 317 individuals). For the analyses of linkage, Morton's sequential probability ratio test was applied. Clear evidence for linkage between Es-1 and Est-7 (θ≅ 0.2) was obtained, and no evidence of linkage was obtained for the remaining pairwise combinations of loci studied.

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5′ flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed asin vitrocell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie — a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such asCD29were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential. The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPCwas upregulated in MSCs. This characteristic could be useful for developingin vitromodels for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt–Jakob disease patients, protein misfolding cyclic amplification did not detect PrPScin BM- or PB-MSCs from scrapie-infected sheep, which limits their use forin vivodiagnosis for scrapie.

Fatty acid synthase (FASN) is a multifunctional protein that carries out the synthesis of fatty acids so it plays a central role in de novo lipogenesis in mammals. Previously, we defined the genetic structure and expression of the bovine FASN gene. Our mapping studies placed FASN on BTA19 (19q22) where several quantitative trait loci (QTL) affecting milk-fat content and related traits have been described. This study was conducted to identify polymorphisms in the bovine FASN gene and to study their association with milk-fat content. The bovine FASN gene was screened for polymorphisms in two cattle breeds. Sequence analysis revealed several single nucleotide polymorphisms (SNPs), and two of them were analysed: a G>C substitution in the untranslated exon 1 (g.763G>C), altering a potential Sp1 transcription factor-binding site, and an A>G substitution in exon 34 (g.16009A>G), which determines a non-conservative substitution of threonine by alanine. Allele-specific amplification of the SNPs in FASN revealed significant frequency differences for both polymorphisms in Holsteins with high and low breeding values for milk-fat content. The intragenic haplotypes comprising exon 1 (alleles G and C) and exon 34 (alleles A and G) polymorphisms were studied, and the existence of linkage disequilibrium between these SNPs was found (D(CG) = 0.048, P < 0.001). Our results suggest that the FASN gene polymorphisms contribute to variation in milk-fat content. We propose that the bovine FASN gene is a candidate gene for a milk-fat content QTL.