Search

Dairy farming practices significantly affect the nutritional and functional properties of milk. This study compares miRNAs in milk exosomes from extensive and intensive dairy systems and explores their potential implications for human consumers. Extensive systems are believed to produce milk of higher quality with better animal welfare compared to intensive systems. Milk samples from eight extensive and nine intensive dairy farms were analysed. Milk-derived extracellular vesicles were isolated through sequential ultracentrifugation and characterised through Dynamic Light Scattering and Nanosight to determine the size and the concentration of the extracellular vesicles, in addition to immunoblotting to ensure the presence of exosome-specific proteins in their membrane. miRNA levels were quantified using RT-qPCR, and metabolic pathways associated with miRNAs showing significant differences between farm groups were analysed. EVs from extensive farms had higher concentrations. Notably, bta-miR-451 levels were significantly higher in milk from extensive farms ( p = 0.021). Like human miRNA hsa-miR-451 , it is linked to pathways related to Parkinson's disease and cancer. Our research suggests that milk production in extensive systems not only provides socioeconomic and environmental benefits but may also have positive effects on human health. Further research is warranted to explore the bioactive potential of these miRNAs and their implications for human health.

Dairy production systems significantly impact environmental sustainability, animal welfare, and human health. Intensive farming maximizes output through high-input practices, raising concerns about environmental degradation, animal welfare, and health risks from antibiotic residues. Conversely, organic farming emphasizes sustainable practices, animal welfare, and minimal synthetic inputs, potentially enhancing biodiversity, soil health, and milk quality. MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are promising biomarkers due to their response to various conditions. In this study, miRNAs bta-miR-103 and bta-miR-155, which are abundant in milk from pasture-fed cows, were selected. Additionally, bta-miR-215, which is abundant in milk fat from intensive systems, was also studied, in order to differentiate dairy production systems. A novel, cost-effective gold nanoparticle (AuNP)-based sensor was developed for miRNA detection, leveraging the unique plasmonic properties of AuNPs for visual detection. The method involves functionalizing AuNPs with complementary RNA probes and detecting miRNA-induced aggregation through colorimetric changes. This rapid, results in 30 min, and sensitive, visual limit of detection of 200 nM, assay requires minimal instrumentation and can be easily interpreted, offering significant advantages for field implementation in characterizing dairy production systems. This study demonstrates the successful application of this sensor in detecting miRNAs in 350 nM miRNA spiked raw milk, highlighting its potential for in situ dairy industry applications.

Multi-host communities are perfect scenarios for the emergence and spread of pathogens, threatening the recovery of endangered, isolated, or inbred populations, such as the brown bear ( Ursus arctos ) in northwestern Spain. The population recovery in recent years has forced bears to occupy highly anthropized areas, increasing their interaction with human and domestic animals, with potential consequences for global health. During 2022-2023 a survey of parasites, bacteria and viruses shared between wildlife, domestic animals and humans was performed in this population using non-invasive surveillance, i.e., bear fecal samples ( n  = 73) and sponge-based sampling of trees ( n  = 42; 14 rubbed trees and 28 control trees). Pathogen detection rates were defined as the percentage of qPCR or culture-positive samples. Generalized linear models were fitted to assess their relationship with environmental variables including dispersion of the human population, and percentage of agricultural and periurban habitats in a 6 km-buffer around each sample. Canine Adenovirus type 1 (45.2%), Giardia spp. (15.1%), Salmonella spp. (12.3%), and extended-spectrum-beta-lactamases (ESBL) Escherichia coli (1.4%) were identified in fecal samples. In contrast, only five sponges from three rubbed and two control trees resulted positive to E. coli (14.3%). The results suggest that several pathogens are common in the Cantabrian brown bear population and that anthropization of the territory modulates their prevalence and richness. The effective design of management programs for bear conservation will require a one-health approach, in which genetic analysis of non-invasive samples can be key tools for the sanitary surveillance at the wildlife-livestock-human interface., Competing Interests: The authors declare that they have no competing interests., (© 2024 The Author(s).)

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH 24 ), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated ( p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress ( bta-miR-1246 , bta-miR-2332 , bta-miR-23b-5p , and bta-miR-2411-3p ) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions ( p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem , as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem . These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

Understanding mortality causes is important for the conservation of endangered species, especially in small and isolated populations inhabiting anthropized landscapes where both natural and human-caused mortality may hinder the conservation of these species. We investigated the mortality causes of 53 free-ranging brown bears (Ursus arctos) found dead between 1998 and 2023 in the Cantabrian Mountains (northwestern Spain), a highly human-modified region where bears are currently recovering after being critically threatened in the last century. We detected natural traumatic injuries in 52.63% and infectious diseases in 39.47% of the 38 bears for which the mortality causes were registered, with 21.05% of these cases presenting signs of both infectious diseases and traumas. More specifically, almost 30% of the bears died during or after intraspecific fights, including sexually selected infanticide (10.53%). In addition, primary infectious diseases such as infectious canine hepatitis, distemper, clostridiosis and colibacillosis caused the death of 15.79% of the bears. The number of direct human-caused deaths (i.e., shooting, poisoning, snare) decreased over the study period. This study also reveals three new mortality causes triggered by pathogens, two of which-Clostridium novyi and verotoxigenic Escherichia coli-not previously described in ursids, and the other one, canine distemper virus, never reported in brown bears as cause of death. New management strategies for the conservation of Cantabrian bears, which are urgently needed due to the rapid expansion of the population, should consider the mortality causes described in this study and must promote further research to elucidate how the high prevalence of infectious diseases may threaten the current recovery of the population., (© 2024. The Author(s).)

Sustainable dairy farms are characterised by the self-production of forage for animal feed. These farms are sometimes located near industrial areas, entailing a risk of food chain contamination with hazardous metals and polycyclic aromatic hydrocarbons (PAHs). Accordingly, evaluating the impact of pollution on forage and milk is of great interest. In this study, the effects of industrial factors on sustainable forage from 43 dairy farms and possible correlations between inorganic elements and PAHs were studied. Spearman's correlation and principal component analysis (PCA) were performed for the forage and milk. Most of the inorganic elements in the forage were below the maximum residual limits for cadmium (Cd) and lead (Pb), established in EU 2013/1275 and EU 2019/1869, respectively. However, arsenic (As) and mercury (Hg) levels were above their respective limits in the forage (EU 2019/1869). No milk samples exceeded the maximum residual limits for Pb (EU 488/2014) or Cd (EU 1881/2006) in dairy products. Heavy-weight PAHs (HW-PAHs, four or more aromatic rings) were detected in forage but not in milk. In the forage samples, HW-PAHs were positively correlated with Zn and Cd. In addition, some hazardous metals (chromium (Cr), iron (Fe), As, Hg, and Pb) also were positively correlated with Zn and Cd. Interestingly, no correlations were found between forage pollutants and milk, suggesting that these pollutants have a low transfer rate to milk. The PCA results highlighted the predominant contribution of PAHs to the global variance in forage samples collected at different distances from industrial areas. In milk, the contributions of hazardous metals and PAHs were more balanced than in forages. Finally, when distances to potential pollution sources were included in the PCA of forage samples, a negative correlation was observed between the former and the concentrations of HW-PAHs, Cd, and Zn, suggesting that thermal power plants and steel factory emissions were the main sources of polluting forage in this area., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ana Soldado reports financial support has been included in acknowledgements., (© 2023 The Authors. Published by Elsevier Ltd.)

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson's disease (PD) and Alzheimer's disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1 *04 subtypes best accounted for the association, strongest with HLA-DRB1 *04:04 and HLA-DRB1 *04:07, and intermediary with HLA-DRB1 *04:01 and HLA-DRB1 *04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1 *04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1 *04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues.

MicroRNAs (miRNAs) regulate gene expression and might resist adverse physicochemical conditions, which makes them potential biomarkers. They are being investigated as biomarkers of dairy production systems, based on the variations in their levels in raw milk depending on animal diet and management. Whether miRNA levels can serve as biomarkers for dairy products remains unclear, since technological or culinary treatments, such as fermentation, may alter their levels. Here, 10 cow dairy farms were sampled in Asturias (north-west Spain) and milk samples were subjected to microwave heating or used to produce yogurt or cheese. Total RNA was isolated from raw milk and three derived products, and levels of seven miRNAs, selected based on previous studies as possible milk production system biomarkers, were assessed by RT-qPCR. The treatments decreased levels of all miRNAs to some extent. These results also imply that cheesemaking increases the concentration of miRNAs in this product; raw milk and cheese supposedly may provide similar concentrations of miRNAs, higher than those of yogurt and microwaved milk. They also indicate that the content of certain miRNAs in raw milk cannot necessarily be extrapolated to other dairy products.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples., (© 2023. The Author(s).)

Potentially toxic elements (PTEs) and polycyclic aromatic hydrocarbons (PAHs) frequently coexist in soils near industrial areas and sometimes in environmental compartments directly linked to feed (forage) and food (milk) production. However, the distribution of these pollutants along the dairy farm production chain is unclear. Here, we analyzed soil, forage, and milk samples from 16 livestock farms in Spain: several PTEs and PAHs were quantified. Farms were compared in terms of whether they were close to (<5 km) or far away from (>5 km) industrial areas. The results showed that PTEs and PAHs were enriched in the soils and forages from farms close to industrial areas, but not in the milk. In the soil, the maximum concentrations of PTEs reached 141, 46.1, 3.67, 6.11, and 138 mg kg -1 for chromium, arsenic, cadmium, mercury, and lead, respectively, while fluoranthene (172.8 µg kg -1 ) and benzo(b)fluoranthene (177.4 µg kg -1 ) were the most abundant PAHs. Principal component analysis of the soil PTEs suggested common pollution sources for iron, arsenic, and lead. In the forage, the maximum contents of chromium, arsenic, cadmium, mercury, and lead were 32.8, 7.87, 1.31, 0.47, and 7.85 mg kg -1 , respectively. The PAH found in the highest concentration in the feed forage was pyrene (120 µg kg -1 ). In the milk, the maximum PTE levels were much lower than in the soil or the feed forages: 74.1, 16.1, 0.12, 0.28, and 2.7 µg kg -1 for chromium, arsenic, cadmium, mercury, and lead, respectively. Neither of the two milk samples exceeded the 20 µg kg -1 limit for lead set in EU 1881/2006. Pyrene was the most abundant PAH found in the milk (39.4 µg kg -1 ), while high molecular weight PAHs were not detected. For PTEs, the results showed that soil-forage transfer factors were higher than forage-milk ratios. Our results suggest that soils and forages around farms near industries, as well as the milk produced from those farms, have generally low levels of PTE and PAH contaminants.

The Cantabrian capercaillie ( Tetrao urogallus cantabricus ) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli , Clostridium perfringens , or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

In autumn 2011, a disease outbreak caused by Spanish goat encephalitis virus (SGEV) was reported in a herd of goats from Asturias (north-western Spain), expanding the known geographic distribution of tick-borne encephalitis in Europe. The virus was classified as a new subtype (subspecies) within the Louping-ill virus species of the mammalian tick-borne flavivirus group. The aims of the present study were to describe the pathology in goats naturally infected with SGEV, as well as discuss the pathogenesis of the disease in that outbreak. A total of 22/85 (25.88%) goats (20 adults and 2 kids) died between October 2011 and June 2012, showing neurological clinical signs. Over three years, the mortality rate in the herd reached 100%. Neuropathological lesions caused by SGEV were severe and widespread throughout the central nervous system but were more severe and numerous in the proximal cervical spinal cord, medulla oblongata, pons and cerebellar cortex. They consisted of neuron necrosis, neuronophagia, mononuclear inflammatory cell perivascular cuffs (lymphocytes, plasma cells and macrophages) and gliosis. The distribution of viral antigens was restricted to the cytoplasm of neurons in several brain areas but not associated with inflammatory foci nor inflammatory cells. SGEV should be considered a significant pathogen of goats that results in severe neurological clinical disease and high mortality.

Studying microRNA (miRNAs) in certain agri-food products is attractive because (1) they have potential as biomarkers that may allow traceability and authentication of such products; and (2) they may reveal insights into the products' functional potential. The present study evaluated differences in miRNAs levels in fat and cellular fractions of tank milk collected from commercial farms which employ extensive or intensive dairy production systems. We first sequenced miRNAs in three milk samples from each production system, and then validated miRNAs whose levels in the cellular and fat fraction differed significantly between the two production systems. To accomplish this, we used quantitative PCR with both fractions of tank milk samples from another 20 commercial farms. Differences in miRNAs were identified in fat fractions: overall levels of miRNAs, and, specifically, the levels of bta-mir-215, were higher in intensive systems than in extensive systems. Bovine mRNA targets for bta-miR-215 and their pathway analysis were performed. While the causes of these miRNAs differences remain to be elucidated, our results suggest that the type of production system could affect miRNAs levels and potential functionality of agri-food products of animal origin.

Pasture-based milk presents several advantages over milk from intensive industrial farming in terms of human health, the environment, animal welfare, and social aspects. This highlights the need for reliable methods to differentiate milk according to its origin on the market. Here, we explored whether miRNA profiles could serve as a marker of milk production systems. We compared levels of previously described miRNAs in milk from four production systems (altogether 112 milk samples): grazing, zero grazing, grass silage or corn silage. Total RNA was extracted from the fat phase, and miRNAs levels were quantified by real-time quantitative PCR. The levels of the miRNAs bta-miR-155 and bta-miR-103 were higher in the grazing system than in corn silage farms. The levels of bta-miR-532 , bta-miR-103 and bta-miR-7863 showed differences between different farm managements. The miRNAs bta-miR-155 and bta-miR-103 were predicted to participate in common functions related to fat metabolism and fatty acid elongation. All four differentially expressed miRNAs were predicted to participate in transport, cell differentiation, and metabolism. These results suggest that the dairy production system influences the levels of some miRNAs in milk fat, and that bta-miR-155 and bta-miR-103 may be potential biomarkers to identify milk from pasture-managed systems.

Multi-host pathogens emerging and re-emerging at the wildlife-domestic animal interface affect wildlife management and conservation. This is the case of canine distemper virus (CDV), a paramyxovirus closely related to human measles virus and rinderpest virus of cattle. With an area of 10,603 km 2 , Asturias region in Atlantic Spain is a hotspot of carnivore diversity, which includes the largest Eurasian brown bear (Ursus arctos arctos) population and one of the largest wolf (Canis lupus) populations in south-western Europe. In 2020-2021, we recorded mortality due to distemper in four carnivore species including three mustelids (Eurasian badger Meles meles, European marten Martes martes and European polecat Mustela putorius) and one canid (red fox, Vulpes vulpes). Clinical signs and pathology were similar across species and consistent with the emergence of a highly pathogenic viral strain, with CDV antigen mainly located in the central nervous system, lungs, spleen and lymph nodes. A molecular study in eight wild carnivore species, also including the Iberian wolf, Eurasian brown bear, American mink (Neovison vison) and stone marten (Martes foina), revealed 19.51% (16/82) of positivity. Phylogenetic analysis demonstrated that CDV belonged to the previously described European lineage. A retrospective serosurvey (2008-2020) showed a high seroprevalence of CDV antibodies (43.4%) in 684 analyzed badgers, indicating a long-term though not stable viral circulation in this multi-host community. The possible triggers of the 2020-2021 outbreak and the implications for carnivore management and conservation are discussed., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Canine adenovirus type 1 (CAdV-1) causes infectious canine hepatitis (ICH) and has recently been described as a cause of death among endangered populations of European brown bear (Ursus arctos arctos) in the Cantabrian mountain range in Asturias, Spain. Sympatric wild and domestic carnivores can act as reservoirs of the virus and likely spread it into the environment and subsequently transmit it to brown bears. The present work investigates the prevalence and geo-temporal distribution of CAdV-1 among free-ranging wolves (Canis lupus) in Asturias from 2009 to 2018, during which three fatal cases of ICH were reported among brown bears in the region. A total of 149 wolves were analysed in this study, of which 21 (14%) were found to have CAdV-1 DNA based on real-time polymerase chain reaction (RT-PCR) of spleen samples. Prevalence of the virus was similar between males and females. All but one of the 20 CAdV-1-positive animals of estimable age were younger than 2 years, and only one of the 46 adult animals (>2 years) tested positive. Prevalence was highest in the western area of Asturias and during 2010 and 2011. Our results confirm that CAdV-1 is circulating in Asturian free-ranging wolves, supporting their possible role as virus reservoirs and sentinels in the region of this emerging disease in brown bears., (© 2021 Wiley-VCH GmbH.)

The optimization of milk production includes a rational use of forages, respect for the environment and offers the best quality to consumers. Milk production based on grass and forages produces healthier milk and it is widely spread throughout the Atlantic arc to maximize milk yield per hectare. However, the mode of offering the grass can have a major influence on milk composition. The aim of this study was to evaluate the effect of grass supply mode (grazing, zero-grazing or ensiling) on dairy cows' performance, with particular reference to fatty acids and fat-soluble antioxidants concentration. A three by three Latin square experiment was performed with 18 dairy cows. Experimental treatments consisted of exclusive feeding with grass silage and zero-grazing, both offered ad libitum indoors, or grazing for 24 h. The results showed that grazing cows had a higher dry matter intake and greater milk yield than cows feeding on grass silage and zero-grazing, as well as higher concentrations of protein, lactose, nonfat-solids and urea in milk than housed cows. Milk fat from grazing cows had a higher proportion of unsaturated fatty acids than from cows feeding on grass silage and zero-grazing, with significant differences in the proportion of vaccenic and rumenic acids. The 18:1 trans-11 to 18:1 trans-10 ratio is proposed as biomarker to identify the milk produced from the management system of grazing cattle. Milk from grazing cows had a greater proportion of lutein than cows eating grass silage, with the zero-grazing system having intermediate values. In conclusion, the mode of grass supply affects fatty acid and antioxidant profiles of milk.

This work summarizes the mortality cases of twenty-five free-ranging Eurasian wild brown bears ( Ursus arctos arctos ) from the Cantabrian mountain range submitted for necropsy in Asturias and Castilla y León (northwestern Spain) from 1998 to 2018. Mortality cases were classified both caused by (i) "non-human intervention" or "human intervention" causes and based on (ii) "non-infectious" or "infectious" etiology. In four cases (16%) it was not possible to determine the cause of death due to the inadequate preservation of collected specimens or insufficient tissue availability. Based on "non-human intervention" or "human intervention" causes, fourteen of the 21 (66.7%) brown bears died as a consequence of "non-human intervention" due to traumatic lesions (fights, unknown traumas or infanticide), infectious canine hepatitis, neoplasia or mushroom poisoning. In contrast, seven (33.3%) brown bears died by "human intervention" due to illegal hunting (shooting or snare), handling (during transit in an attempt to reintroduce a bear back into the wild) or strychnine poisoning. Based on "non-infectious" or "infectious" etiology, twelve of the 21 (57.1%) brown bears died due to "non-infectious" causes, namely traumatic lesions such as shooting, snare, fighting or infanticide, handling, strychnine poisoning, mushroom poisoning or neoplasia. The remaining nine (42.9%) animals died due to "infectious" diseases which included gangrenous myositis, infectious canine hepatitis or septicemia. In six of those cases traumatic lesions caused by non-human or human activities were complicated with bacterial infection (clostridiosis and septicemia) which finally caused the death of those animals. Additionally, exertional myopathy was observed in the handled animal and in one bear found in a snare. In a free-ranging population of Eurasian brown bear from the Cantabrian mountain range, main causes of death are attributed to non-human related traumatic lesions and infectious diseases (primary developed such as infectious canine hepatitis or secondary developed such as clostridiosis or septicemia) which is in contrast to previously reported data for other bear populations. These data are valuable and may help in the conservation and management of this recovering population.

Gold nanoparticles of different sizes have been synthesized and surface-functionalized with selected RNA probes in order to develop a rapid, low-cost and sensitive method for detection of microRNA146a (miR146a). The strategy is based on the change of colour that can be observed visually after aggregation of the RNA modified-gold nanoparticles (AuNPs) in presence of miR146a. Experimental conditions have been carefully selected in order to obtain a good sensitivity that allows to perform visual detection of microRNA at the nM level, achieving a detection limit of 5 nM. Good repeatability and selectivity versus other sequences that only differ from miR146a in 3 bases was achieved. miR146a has been described as one of the main microRNA involved in the immune response of bovine mastitis, being expressed in tissue, blood and milk samples. The method was successfully applied to the detection of miR146a in raw cow milk samples. The present scheme constitutes a rapid and low-cost alternative to perform highly sensitive detection of microRNA without the need of instrumentation and amplification steps for the early detection of bovine mastitis in the agrofood industry. Graphical abstract Schematic representation of the assay based on aggregation of RNA-modified gold nanoparticles (blue) in presence of microRNA146a generating a dark blue spot onto a solid support, versus a pink spot observed in absence of miR146a due to dispersed gold nanoparticles (red).

Canine adenovirus type 1 (CAdV-1) is responsible for infectious canine hepatitis. The disease has been described in captive American black bear (Ursus americanus) and European brown bear (Ursus arctos arctos), with just one recently reported case in a cub of a free-ranging brown bear (Ursus arctos horribilis) from Alaska. The aim of this work is to summarize findings related to presence and associated mortality of CAdV-1 in 21 free-ranging Cantabrian brown bears (Ursus arctos arctos) submitted to necropsy in Asturias and Castilla y León (northwestern Spain) from 1998 to 2018. On the basis of the anatomopathological findings and laboratory results three free-ranging brown bears died due to infectious canine hepatitis, which is to our knowledge the first description of death due to this disease in free-ranging bears in Europe. Gross lesions consisted of petechial haemorrhages and congestion in different internal organs, haemorrhagic fluid in internal cavities, friable and yellowish liver and thickening of gall bladder. Microscopic lesions were observed mainly in liver, kidney and brain and consisted of multifocal necrosis of cells with presence of basophilic intranuclear inclusions. Immunohistochemical (IHC) and real-time polymerase chain reaction (qPCR) techniques were used to assess the presence of CAdV-1 in paraffin-embedded liver samples. Viral antigens were detected by IHC labelling within hepatocytes and Küppfer cells in the three animals. The presence of viral DNA was confirmed by qPCR in one of them. In order to evaluate the circulation of CAdV-1 in brown bears, a retrospective study was performed using both IHC and qPCR techniques in 11 and 12 additional brown bears, respectively. An extra brown bear was found positive by IHC. This study shows that CAdV-1 surveillance of brown bears and sympatric carnivores should be considered as major concern for the monitoring the population evolution throughout time in this endangered species., (© 2018 Blackwell Verlag GmbH.)

Louping ill-like virus (LI) has been recently detected in two different locations in the north of Spain and separated by only around 400 km. Using molecular approaches, the viruses causing both outbreaks have been shown to be different to LI virus, but also different to each other. They have been called SSEV (Spanish sheep encephalitis virus) and SGEV (Spanish goat encephalitis virus) taking into account the species from which they were isolated. The aim of this paper was to design a quantitative TaqMan real-time RT-PCR protocol, for the specific diagnostic and quantitation of SGEV. Linearity, efficiency and dynamic range as well as reproducibility and specificity of the method has been tested and established. The method has proved to be valid for the specific detection and viral load quantitation of SGEV genome in virus isolates and tissue samples from infected animals. This assay will be a useful analytical tool in early diagnosis and epidemiological surveys., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this work was to perform a thorough analysis of the diversity of Y-haplotypes in Spanish cattle. A total of 207 Bos taurus males were sampled across 25 European breeds, with a special focus on rare, local Spanish populations. Animals were genotyped with five Y-specific microsatellites (INRA189, UMN0103, UMN0307, BM861 and BYM1), two indels (ZFY10 and USP9Y) and one SNP (UTY19). A new haplogroup, distinct from those described by Götherström et al. (2005), was identified and named Y1.2. Samples representing the three B. taurus Y-haplogroups were genotyped for four additional Y chromosome SNPs (rs121919254, rs121919281, rs121919323 and rs137049553). Among these SNPs, only rs121919281 was informative in B. taurus and helped to confirm the new Y1.2 haplogroup. Analysis of a larger dataset of standardized haplotypes for 1507 individuals from 57 populations from Spain, other European countries and Africa showed the new Y1.2 haplogroup to be found exclusively in Spanish breeds. This finding reinforces the importance of local Spanish cattle as reservoirs of genetic diversity as well as the importance of the Iberian Peninsula in the history of cattle., (© 2017 Stichting International Foundation for Animal Genetics.)

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field., (© 2013 Blackwell Verlag GmbH.)

The main objective of this work was to determine whether the five founding castes defined in the Lidia cattle breed actually have an important contribution to the Spanish paternal genetic stock as well as to the paternal genetic origin support. A total of 1300 Bos taurus male individuals were genotyped for five microsatellites (INRA189, UMN0103, UMN0307, BM861 and BYM1) and one indel (ZFY10). Microsatellite and indel alleles were combined into haplotypes, identifying a total of 38 haplotypes, 11 of them belonging to haplogroup Y1 and 27 to haplogroup Y2. Ten different haplotypes were found in the Lidia cattle breed, with five being exclusive to this breed. Our results agree with different male genetic stocks in the Lidia breed: one hypothetically representing the ancient Iberian bovine genetic stock (Gallardo, Navarra and Cabrera castes and some encastes from Vistahermosa) and a second one that is the result of the more recent breeding strategy of choosing the most aggressive individuals from traditional herds (including some Vistahermosa encastes and the Vazqueña caste). In terms of conservation, it would be better to not consider this breed as a unit but to consider the caste, or even better the encaste, as the target of putative conservation efforts., (© 2015 Stichting International Foundation for Animal Genetics.)

Recent studies suggest that badgers may be a potential reservoir of Mycobacterium bovis infection for cattle in Northern Spain. The objective of this study was to investigate potential epidemiological links between cattle and badgers. Culture and molecular typing data were available for cattle culled during the national tuberculosis (TB) eradication campaigns between 2008 and 2012, as well as from 171 necropsied badgers and 60 live animals trapped and examined over the same time period. Mycobacterium tuberculosis complex strains were isolated from pooled tissues of 14 (8.2%) necropsied badgers, of which 11 were identified as M. bovis: six different spoligotypes of M. bovis were subsequently identified. In two geographical locations where these isolates were shared between cattle and badgers, infected cattle herds and badgers lived in close contact. Although it remains unclear if badgers are a maintenance or spill-over host of M. bovis in this setting, it would appear prudent to have precautionary measures in place to reduce contact between cattle and badgers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Variation in mitochondrial DNA (mtDNA) and Y-chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon (Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt-3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt-3 respectively. In contrast, Y-chromosome analyses revealed extraordinary paternal allelic richness: HY-6, 89.3%; HY-8, 5.0%; HY-18, 3.1%; HY-7, 1.3%; and HY-5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY-18 (A-oY1/135-SRYM18), identified here for the first time, provides a link between the haplotype HY-12 (A-oY1/139-SRYM18) found in a few rams in Turkey and haplotype HY-9 (A-oY1/131-SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt-55), and all were paternally monomorphic for haplotype HY-6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y-chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to 'upgrade' local populations using rams with different paternal origins., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Meat is a significant source of high-quality nutrients, which are very important in the diet. Among meat products, one of the most prized is bovine meat, of which male beef has been designated to be of a higher quality. However, because of its similarity with female beef, deliberate or unintentional substitutions can occur. To avoid this, methodology based on the fast real-time polymerase chain reaction has been developed to authenticate the species and gender origin of beef. This technique consists of two polymerase chain reactions: one bovine-specific reaction and another Y-chromosome-specific multiplex reaction. This methodology has been validated for all kinds of beef products, including those subjected to intensive processing treatments, and it has subsequently been applied to 10 commercial samples labelled as ox to determine whether they are properly labelled. This assay has been shown to have high specificity, sensitivity and rapidity, with the potential to be a powerful tool in enforcing food labelling regulations.

A total of 162 individuals, belonging to three Burkinabé and one Niger sheep populations, were analysed for prion protein (PrP) gene polymorphism at codons 136, 154 and 171. The ARQ allele was the most frequent in both the Burkinabé (86.7%) and the Niger (67.5%) sheep populations. The highly sensitive allele VRQ was not found in the sampled individuals. The highly resistant ARR allele was in very low frequency in the Burkina-Sahel (4.4%) and Mossi (3.2%) populations and was not present in the Djallonké and Touareg populations. Only 4 out of 15 possible PrP genotypes were identified in the sampled individuals. No favourable ARR/ARR genotypes were found in either of the breeds. Sequencing a subgroup of the samples allowed the identification of other five polymorphisms on the PrP gene sequence at codons 116, 138, 151, 237 and 240. The very low frequency of the ARR allele in the West African sheep should dissuade the implementation of a preventive selection programme aimed to increase resistance to scrapie, to avoid an extreme erosion of the genetic stock.

Although louping ill affects mainly sheep, a 2011 outbreak in northern Spain occurred among goats. Histopathologic lesions and molecular genetics identified a new strain of louping ill virus, 94% identical to the strain from Britain. Surveillance is needed to minimize risk to domestic and wildlife species and humans.

Empirical evidence of the usefulness of different molecular-based methods to estimate the effective population size (N(e)) for conservation purposes in endangered livestock populations is reported. The black-coated Asturcón pony pedigree (1,981 individuals) was available. Additionally, a total of 267 Asturcón individuals born in 1998, 2002, and 2008 were typed for 15 microsatellites. These yearly cohorts (cohort(1998, 2002, 2008)) included almost all individuals kept for reproduction at the end of the corresponding foaling season. The genealogical realized N(e) was estimated for each cohort by using the individual increase in inbreeding. Molecular N(e) was computed by using 1) linkage disequilibrium [N(e)(()(D)())], 2) a temporal method based on F-statistics [N(e)(()(T)())], 3) an unbiased temporal method [N(e)(()(JR)())], and 4) a Bayesian temporal method [N(e)(()(B)())]. Estimates of increased from cohort(1998) (18.8 ± 5.1) to cohort(2008) (24.9 ± 5.2), illustrating the history of the population and its breeding policy of avoiding matings between close relatives. The estimates of N(e)(()(D)()) were highly biased upward, with the maximum N(e)(()(D)()) value obtained for cohort(2002) (137.0). The estimates of N(e)(()(T)()), N(e)(()(JR)()), and N(e)(()(B)()) showed similar performance. However, N(e)(()(JR)()) estimates were very consistent across cohorts, varying from 14.9 to 15.5 after correcting for the effect of overlapping generations. When the drift signal was not strong (pair cohort(1998)-cohort(2002)), estimates of N(e)(()(T)()) and N(e)(()(B)()) were not realistic. Estimates of N(e)(()(B)()) tended to be biased downward (being 9.0 or below for the sampling pairs including cohort(2008)). Results of N(e)(()(D)()) are more likely to be estimates of the effective number of breeders producing the sample, rather than the effective size for a generation. The temporal methods were strongly affected by a weak drift signal, particularly when samplings were not spaced a sufficient number of generations or a sufficient time apart. The use of molecular-based estimates of N(e) is not straightforward, and their use in livestock conservation programs should be carried out with caution. Sampling strategies (including sampling sizes, sampling periods, and the age structure of the sampled individuals) must be carefully planned to ensure that robust estimates of N(e) are obtained.

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

The effect of selection for scrapie resistance on genetic variability in three endangered Spanish sheep breeds (Colmenareña, Mallorquina and Rubia de El Molar) was studied using two different criteria for quantifying contributions to genetic variability: (a) molecular coancestry or genetic identity; and (b) average number of alleles per locus or allelic richness. A total of 236 (81 Colmenareña, 76 Mallorquina and 79 Rubia de El Molar) individuals were genotyped for the PrP gene and for 22 microsatellite markers. The analyses assumed a selective policy aimed at the elimination of the VRQ allele and the reduction of the frequency of the ARQ/ARQ genotype. These goals are approached by rejecting for breeding those individuals with the highest susceptibility for scrapie (risk groups R4 and R5) in a genetic scenario with no previous selection programmes considering the PrP gene polymorphism carried out. When all the individuals classified into risk groups R4 and R5 were removed from the dataset, the total molecular coancestry slightly increased in the Colmenareña breed illustrating that the carriers of undesirable PrP genotypes are not essential to maintain its overall gene diversity. When the allelic richness was considered, the removal of the R4 and R5 individuals gave high losses in the Rubia de El Molar breed. The analyses carried out considering the sex of the individuals informed that most increases in genetic identity in the Colmenareña breed resulted from the removal of the R4 and R5 males while in the Mallorquina breed resulted from the removal of the undesirable females. Losses of diversity in the Rubia de El Molar breed were basically independent of the sex of the individuals due to the balanced contributions to diversity of both sexes. As a general recommendation, not all the individuals of undesirable risk groups should be rejected for reproduction at the same time to avoid irretrievable loses of genetic diversity but according to the sex of the individuals.

Here we tested the segregation and paternal compatibility of markers INRA124 and INRA126 on female DNA in 10 different cattle families, in order to clarify the usefulness of these microsatellites for the study of male-mediated population processes in cattle. Their performance was compared with that of four microsatellites located in the PAR-BTAY (UMN0108, UMN0803, UMN0929 and UMN0905) and another one male-specific microsatellite (INRA189). INRA124 and INRA126 amplified the same sized fragment in both sexes. Same size alleles were sequenced and the high homology found allowed us to rule out non-specific female amplification. INRA124 showed full parental compatibility, whilst the locus INRA126 showed 55% parental incompatibility. Based on these observations, it is recommended that markers INRA124 and INRA126 should not be used in studies to characterize male-mediated genetic events in cattle.

To date, no comprehensive study has been performed on mitochondrial genetic diversity of the West African goat. Here, we analysed a 481-bp fragment of the HVI region of 111 goats representing four native West African populations, namely the three main Burkina Faso breeds, zoo-farm kept Dwarf goats and endangered Spanish goat breeds used as the outgroup. Analyses gave 83 different haplotypes with 102 variable sites. Most haplotypes (65) were unique. Only three haplotypes were shared between populations. Haplotypes were assigned to cluster A except for H45 (belonging to the Spanish Bermeya goat) which was assigned to cluster C. amova analysis showed that divergence between groups (Phi(CT)) was not statistically significant regardless of whether the partition in two hierarchical levels that was fitted included Spanish samples or not. The West African goat scenario shown here is consistent with that previously reported for the species: haplogroup A is predominant and has a very high haplotype diversity regardless of the geographic area or sampled breed. The large phenotypic differences observable between the West African Dwarf and Sahelian long-legged goat populations are not detectable with mitochondrial markers. Moreover, a previously suggested introgression of Sahelian goat southwards because of desertification could not be assessed using mtDNA information.

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.

A total of 123 sheep belonging to the Djallonke, Mossi, and Burkina-Sahel breeds, along with 41 Spanish Xalda sheep were genotyped for 27 microsatellites. The pair Djallonke-Mossi had the highest between breeds molecular coancestry. Admixture analysis informed on the parental role of the Burkina-Sahel and Djallonke breeds. The Mossi breed was a hybrid population nearer to the Djallonke breed. Only half of the Mossi individuals were correctly assigned to their breed. The Burkina-Sahel and Djallonke breeds can be considered ancestrally different genetic entities. Differentiation between the Djallonke and Mossi breeds may be due to introgression of Sahelian sheep.

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100&#95;104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software&#95;i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

Background: Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue., Results: Our findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated., Conclusion: We propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 microM, 1 microM and 10 microM), ATRA 0.7 microM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 microM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 microM, while a dose-dependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r=0.93; p<0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation.

Here we study 109 Iberian roe deer individuals corresponding to 9 Spanish populations. Individuals were sampled at locations that are expected to have acted as refugia for the species during the 20th century. Samples were analyzed for a 436 bp mtDNA fragment from the HVI region of mitochondrial DNA and 10 microsatellites. The 109 sequences gave 31 different haplotypes that enabled identification of a new haplogroup (mainly present in Northwestern Iberian populations and representing roughly a third of our samples) that is not present in other European roe deer populations. Using microsatellites, correspondence analysis and molecular coancestry information revealed high molecular differentiation among Northwestern and Central-Southern Spain roe deer populations. Both sequence and microsatellite analysis reveal that the Spanish roe deer populations are genetically heterogeneous and have high genetic structure clearly separating the Pyrenean-reintroduced populations and two main areas for the species in Spain (Northwestern and Central-Southern) coinciding with the two main areas acting as refugia for the majority of the mammal species during glaciations. The implications of the obtained information as regards the phylogeography of the species are discussed together with suggestions as to appropriate strategies for the conservation and management of populations.