Impact of oestrus synchronization devices on ewes vaginal microbiota and artificial insemination outcome.

Introduction: The low pregnancy rate by artificial insemination in sheep represents a fundamental challenge for breeding programs. In this species, oestrus synchronization is carried out by manipulating hormonal regimens through the insertion of progestogen intravaginal devices. This reproductive strategy may alter the vaginal microbiota affecting the artificial insemination outcome. Methods: In this study, we analyzed the vaginal microbiome of 94 vaginal swabs collected from 47 ewes with alternative treatments applied to the progesterone-releasing intravaginal devices (probiotic, maltodextrin, antibiotic and control), in two sample periods (before placing and after removing the devices). To our knowledge, this is the first study using nanopore-based metagenome sequencing for vaginal microbiome characterization in livestock. Results: Our results revealed a significant lower abundance of the genera Oenococcus (Firmicutes) and Neisseria (Proteobacteria) in pregnant compared to non-pregnant ewes. We also detected a significant lower abundance of Campylobacter in the group of samples treated with the probiotic. Discussion: Although the use of probiotics represents a promising practice to improve insemination results, the election of the suitable species and concentration requires further investigation. In addition, the use of progestogen in the synchronization devices seemed to increase the alpha-diversity and decrease the abundance of harmful microorganisms belonging to Gammaproteobacteria and Fusobacteriia classes, suggesting a beneficial effect of their use.

Exploring the ovine sperm transcriptome by RNAseq techniques. I Effect of seasonal conditions on transcripts abundance.

Understanding the cell molecular changes occurring as a results of climatic circumstances is crucial in the current days in which climate change and global warming are one of the most serious challenges that living organisms have to face. Sperm are one of the mammals' cells most sensitive to heat, therefore evaluating the impact of seasonal changes in terms of its transcriptional activity can contribute to elucidate how these cells cope with heat stress events. We sequenced the total sperm RNA from 64 ejaculates, 28 collected in summer and 36 collected in autumn, from 40 Manchega rams. A highly rich transcriptome (11,896 different transcripts) with 90 protein coding genes that exceed an average number of 5000 counts were found. Comparing transcriptome in the summer and autumn ejaculates, 236 significant differential abundance genes were assessed, most of them (228) downregulated. The main functions that these genes are related to sexual reproduction and negative regulation of protein metabolic processes and kinase activity. Sperm response to heat stress supposes a drastic decrease of the transcriptional activity, and the upregulation of only a few genes related with the basic functions to maintain the organisms' homeostasis and surviving. Rams' spermatozoids carry remnant mRNAs which are retrospectively indicators of events occurring along the spermatogenesis process, including abiotic factors such as environmental temperature.

Monitoring of Rabbit Hemorrhagic Disease Virus in European Wild Rabbit (Oryctolagus cuniculus) Populations by PCR Analysis of Rabbit Fecal Pellets.

European rabbits (Oryctolagus cuniculus) are affected by rabbit hemorrhagic disease (RHD), which is caused by a lagovirus responsible for significant mortality in European wild rabbit populations. Our study aimed to evaluate the potential for detecting viral RNA by duplex real-time PCR in rabbit fecal pellets collected in the field, as a noninvasive method to monitor RHD virus circulation in wild populations. To do this, monthly discoveries of rabbits that died from RHD and detection of viral RNA in fecal pellets were recorded in two enclosed populations of wild rabbits throughout a year. The results suggested a low performance of this procedure to monitor viral infection incidence and a weak concordance with monthly discoveries of rabbits that died from RHD. This poor association was probably due to the low amount of viral RNA in feces, the prolonged time of excretion after infection, and that the number of rabbits found dead from RHD does not necessarily correlate with RHD incidence. Nevertheless, this procedure may be a complementary noninvasive method to assist in determining the presence of RHD viruses in populations. Additional research is needed to determine the suitability of this methodology to perform epidemiologic surveys on wild populations of European rabbits and, especially, other European or North American lagomorph species affected by lagoviruses.

Changes in European wild rabbit population dynamics and the epidemiology of rabbit haemorrhagic disease in response to artificially increased viral transmission.

European wild rabbit (Oryctolagus cuniculus) populations are severely affected by rabbit haemorrhagic disease (RHD), currently aggravated by the spread of the new lagovirus serotype RHDV2 that replaced the classical RHDV strains (RHDV/RHDVa). This virus causes high mortality in both adult and young rabbits and to date, there is no management tool to effectively reduce its impact on wild rabbit populations. This hinders the success of common strategies, such as habitat management or restocking, in areas where rabbits are native. However, the present study, conducted on enclosed wild rabbit populations, showed that spreading RHDV2 on baits during breeding periods induced infection of young rabbits, reducing mortality rates, presumably due to maternal antibody protection. This reduced the young rabbit mortality hazard by a third, and more juvenile rabbits immune to RHDV2 were recruited into the adult breeding population. Young rabbits from populations in which the force of infection of RHDV2 was increased, however, exhibited considerably higher susceptibility to infection by RHDV than those from non-treated control populations. Since co-circulation of classical RHDVs was ruled out, differences in the type and degree of immunization, the level of cross-protection and/or other unknown factors, such as the circulation of undetected non-pathogenic lagoviruses, arose as possible explanations. This meant that although the present study demonstrated the possibility of successfully modulating the impact of RHD in wild populations, the epidemiological complexity of the situation where several lagoviruses circulate requires additional research to determine final applicability of the proposed method.

Infectivity of rabbit haemorrhagic disease virus excreted in rabbit faecal pellets.

Rabbit haemorrhagic disease (RHD) is caused by a lagovirus affecting European rabbits (Oryctolagus cuniculus). Viral RNA is detected in tissues or faeces of convalescent rabbits, suggesting persistent infections; however, this RNA has not been shown to be related to infective viruses to date. In the present work, seven laboratory rabbits were challenged with the RHDV2/b virus variant. Viral RNA was individually detected by duplex qPCR in faeces collected for four weeks after infection, and the infective capacity of viral RNA excreted in the faeces of surviving rabbits was tested by challenging new rabbits with faecal inocula. As results, viral RNA was detected in faeces until the end of the assay. Viral RNA detected in the fourth week was infective only in the case of one rabbit that did not exhibit clear seroconversion, suggesting persistent infection as a result of an impaired immune response. Since the surviving rabbits were apparently healthy individuals, the importance of detecting carriers and the correct management of faeces to control RHD outbreaks in rabbitries are highlighted.

Influence of the Ovine Genital Tract Microbiota on the Species Artificial Insemination Outcome. A Pilot Study in Commercial Sheep Farms.

To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3-V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes' vagina could be due to prior transmission by natural mating with rams reared in the herd.

Gene Expression and Fatty Acid Profiling in Longissimus thoracis Muscle, Subcutaneous Fat, and Liver of Light Lambs in Response to Concentrate or Alfalfa Grazing.

A better understanding of gene expression and metabolic pathways in response to a feeding system is critical for identifying key physiological processes and genes associated with polyunsaturated fatty acid (PUFA) content in lamb meat. The main objective of this study was to investigate transcriptional changes in L. thoracis (LT) muscle, liver, and subcutaneous fat (SF) of lambs that grazed alfalfa (ALF) and concentrate-fed (CON) slaughtered at 23 kg and using the Affymetrix Ovine Gene 1.1 ST whole-genome array. The study also evaluated the relationship between meat traits in LT muscle, including color, pigments and lipid oxidation during 7 days of display, α-tocopherol content, intramuscular fat (IMF) content and the fatty acid (FA) profile. Lambs that grazed on alfalfa had a greater α-tocopherol concentration in plasma than CON lambs (P < 0.05). The treatment did not affect the IMF content, meat color or pigments (P > 0.05). Grazing increased the α-tocopherol content (P < 0.001) and decreased lipid oxidation on day 7 of display (P < 0.05) in LT muscle. The ALF group contained a greater amount of conjugated linoleic acid (CLA), C18:3 n-3, C20:5 n-3, C22:5 n-3, and C22:6 n-3 than did the CON group (P < 0.05). We identified 41, 96 and four genes differentially expressed in LT muscle, liver, and subcutaneous fat, respectively. The most enriched biological processes in LT muscle were skeletal muscle tissue development, being the genes related to catabolic and lipid processes downregulated, except for CPT1B, which was upregulated in the ALF lambs. Animals grazing alfalfa had lower expression of desaturase enzymes in the liver (FADS1 and FADS2), which regulate unsaturation of fatty acids and are directly involved in the metabolism of n-3 PUFA series. The results found in the current study showed that ingesting diets richer in n-3 PUFA might have negative effects on the de novo synthesis of n-3 PUFA by downregulating the FADS1 and FADS2 expression. However, feeding diets poorer in n-3 PUFA can promote fatty acid desaturation, which makes these two genes attractive candidates for altering the content of PUFAs in meat.

Detection of Rabbit Hemorrhagic Disease Virus GI.2/RHDV2/b in the Mediterranean Pine Vole ( Microtus duodecimcostatus) and White-Toothed Shrew ( Crocidura russula).

The European wild rabbit ( Oryctolagus cuniculus) is a key prey species on the Iberian Peninsula, and several predator species that are at risk of extinction are dependent on them as prey. A new rabbit hemorrhagic disease (RHD) virus genotype (GI.2/RHDV2/b) emerged in 2010 and posed a threat to wild rabbit populations. During a survey aimed at investigating RHD epidemiology in wild rabbits, GI.2/RHDV2/b was detected by duplex real-time PCR in carcasses of one Mediterranean pine vole ( Microtus duodecimcostatus) and two white-toothed shrews ( Crocidura russula). Laboratory New Zealand white rabbits that were challenged with inocula obtained from the liver of the small mammals died showing RHD lesions, confirming the infectiousness of the isolates. Phylogenetic analysis of the VP60 gene nucleotide sequences showed complete homology between the isolates from the two small mammal species and a high degree of similarity, but not complete homology, to GI.2/RHDV2/b sequences from wild rabbits. The GI.2/RHDV2/b genotype has not been reported in species outside the order Lagomorpha.

Rabbit haemorrhagic disease: Cross-protection and comparative pathogenicity of GI.2/RHDV2/b and GI.1b/RHDV lagoviruses in a challenge trial.

European rabbits (Oryctolagus cuniculus) are severely affected by rabbit haemorrhagic disease (RHD). Caused by a lagovirus, the disease leads to losses in the rabbit industry and has implications for wildlife conservation. Past RHD outbreaks have been caused by GI.1/RHDV genotype viruses. A new virus belonging to the GI.2/RHDV2/b genotype emerged in 2010, quickly spreading and replacing the former in several countries; however, limited data are available on its pathogenicity and epidemiological factors. The present work extends these issues and evaluates cross-protection between both genotypes. Ninety-four and 88 domestic rabbits were challenged with GI.2/RHDV2/b and GI.1b/RHDV variant isolates, respectively. Cross-protection was determined by a second challenge on survivors with the corresponding strain. Mortality by GI.2/RHDV2/b was highly variable due to unknown individual factors, whereas mortality by GI.1b/RHDV was associated with age. Mortality in rabbits < 4 weeks old was 84%, higher than previously reported. Cross-protection was not identical between the two viruses because the ratio of mortality rate ratios for the first and second challenges was 3.80 ±â€¯2.68 times higher for GI.2/RHDV2/b than it was for GI.1b/RHDV. Rabbit susceptibility to GI.2/RHDV2/b varied greatly and appeared to be modulated by the innate functionality of the immune response and/or its prompt activation by other pathogens. GI.1b/RHDV pathogenicity appeared to be associated with undetermined age-related factors. These results suggest that GI.2/RHDV2/b may interact with other pathogens at the population level but does not satisfactorily explain the GI.1b/RHDV virus's quick replacement.

Genome-wide expression profiling in muscle and subcutaneous fat of lambs in response to the intake of concentrate supplemented with vitamin E.

BACKGROUND: The objective of this study was to acquire a broader, more comprehensive picture of the transcriptional changes in the L. Thoracis muscle (LT) and subcutaneous fat (SF) of lambs supplemented with vitamin E. Furthermore, we aimed to identify novel genes involved in the metabolism of vitamin E that might also be involved in meat quality. In the first treatment, seven lambs were fed a basal concentrate from weaning to slaughter (CON). In the second treatment, seven lambs received basal concentrate from weaning to 4.71 ± 2.62 days and thereafter concentrate supplemented with 500 mg dl-α-tocopheryl acetate/kg (VE) during the last 33.28 ± 1.07 days before slaughter. RESULTS: The addition of vitamin E to the diet increased the α-tocopherol muscle content and drastically diminished the lipid oxidation of meat. Gene expression profiles for treatments VE and CON were clearly separated from each other in the LT and SF. Vitamin E supplementation had a dramatic effect on subcutaneous fat gene expression, showing general up-regulation of significant genes, compared to CON treatment. In LT, vitamin E supplementation caused down-regulation of genes related to intracellular signaling cascade. Functional analysis of SF showed that vitamin E supplementation caused up-regulation of the lipid biosynthesis process, cholesterol, and sterol and steroid biosynthesis, and it down-regulated genes related to the stress response. CONCLUSIONS: Different gene expression patterns were found between the SF and LT, suggesting tissue specific responses to vitamin E supplementation. Our study enabled us to identify novel genes and metabolic pathways related to vitamin E metabolism that might be implicated in meat quality. Further exploration of these genes and vitamin E could lead to a better understanding of how vitamin E affects the oxidative process that occurs in manufactured meat products.

In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene.

Gene promoters are essential regions of DNA where the transcriptional molecular machinery to produce RNA molecules is recruited. In this process, DNA epigenetic modifications can acquire a fundamental role in the regulation of gene expression. Recently, in a previous work of our group, functional features and DNA methylation involved in the ovine HSP90AA1 gene expression regulation have been observed. In this work, we report a combination of methylation analysis by bisulfite sequencing in several tissues and at different developmental stages together with in silico bioinformatic analysis of putative regulating factors in order to identify regulative mechanisms both at the promoter and gene body. Our results show a "hybrid structure" (TATA box + CpG island) of the ovine HSP90AA1 gene promoter both in somatic and non-differentiated germ tissues, revealing the ability of the HSP90AA1 gene to be regulated both in an inducible and constitutive fashion. In addition, in silico analysis showed that several putative alternative spliced regulatory motifs, exonic splicing enhancers (ESEs), and G-quadruplex secondary structures were somehow related to the DNA methylation pattern found. The results obtained here could help explain the differences in cell-type transcripts, tissue expression rate, and transcription silencing mechanisms found in this gene.

Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extracted from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance.

Looking for adaptive footprints in the HSP90AA1 ovine gene.

BACKGROUND: Climatic factors play an important role in determining species distributions and phenotypic variation of populations over geographic space. Since domestic sheep is managed under low intensive systems animals could have retained some genome adaptive footprints. The gene encoding the Hsp90α has been extensively studied in sheep and some polymorphisms located at its promoter have been associates with differences in the transcription rate of the gene depending on climatic conditions. In this work the relationships among the distribution and frequencies of 11 polymorphisms of the ovine HSP90AA1 gene promoter in 31 sheep breeds and the climatic and geographic variables prevailing in their regions of origin have been studied. Also the promoter sequence has been characterized in 9 species of the Caprinae subfamily. RESULTS: Correlations among several climatic variables and allele frequencies of the polymorphisms of the HSP90AA1 gene promoter linked with differences in the transcription activity of the gene under heat stress conditions have been assessed. A group of breeds reared in semi dry climates have high frequencies of the insertion allele of the g.667-668insC associated with the heat stress response. Other group of breeds native to semi arid conditions showed very low frequencies of this same allele. However, in some cases, this previous correlation has not been achieved, revealing the high levels of gene flow among populations occurred following domestication. The Bayesian Test of Beaumont and Balding identified two outlier loci, the g.522A > G and g.703_704del(2)A candidates to balancing and directional selection, respectively. Polymorphisms detected in O. aries are also present in several species of the Caprinae subfamily being C. hircus, O. musimon and O. moschatus those sharing the highest number of them with O. aries. CONCLUSIONS: Despite domestication, sheep breeds showed some genetic footprints related to climatic variables. Adaptation of breeds to heat climates can suppose a selective advantage to cope with global warming caused by climatic change. Polymorphisms of the HSP90AA1 gene detected in the Ovis aries species are also present in wild species from the Caprinae subfamily, indicating a great antiquity of these mutations and its importance in the adaptation of species to past climatic conditions existing in its native environments.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): results from an international ring trial targeting four species of the subgenus Avaritia.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Influence of the temperature and the genotype of the HSP90AA1 gene over sperm chromatin stability in Manchega Rams.

The present study addresses the effect of heat stress on males' reproduction ability. For that, we have evaluated the sperm DNA fragmentation (DFI) by SCSA of ejaculates incubated at 37 °C during 0, 24 and 48 hours after its collection, as a way to mimic the temperature circumstances to which spermatozoa will be subject to in the ewe uterus. The effects of temperature and temperature-humidity index (THI) from day 60 prior collection to the date of semen collection on DFI were examined. To better understand the causes determining the sensitivity of spermatozoa to heat, this study was conducted in 60 males with alternative genotypes for the SNP G/C-660 of the HSP90AA1 promoter, which encode for the Hsp90α protein. The Hsp90α protein predominates in the brain and testis, and its role in spermatogenesis has been described in several species. Ridge regression analyses showed that days 29 to 35 and 7 to 14 before sperm collection (bsc) were the most critical regarding the effect of heat stress over DFI values. Mixed model analyses revealed that DFI increases over a threshold of 30 °C for maximum temperature and 22 for THI at days 29 to 35 and 7 to 14 bsc only in animals carrying the GG-660 genotype. The period 29-35 bsc coincide with the meiosis I process for which the effect of the Hsp90α has been described in mice. The period 7-14 bsc may correspond with later stages of the meiosis II and early stages of epididymal maturation in which the replacement of histones by protamines occurs. Because of GG-660 genotype has been associated to lower levels of HSP90AA1 expression, suboptimal amounts of HSP90AA1 mRNA in GG-660 animals under heat stress conditions make spermatozoa DNA more susceptible to be fragmented. Thus, selecting against the GG-660 genotype could decrease the DNA fragmentation and spermatozoa thermal susceptibility in the heat season, and its putative subsequent fertility gains.

Ovine HSP90AA1 expression rate is affected by several SNPs at the promoter under both basal and heat stress conditions.

The aim of this work was to investigate the association between polymorphisms located at the HSP90AA1 ovine gene promoter and gene expression rate under different environmental conditions, using a mixed model approach. Blood samples from 120 unrelated rams of the Manchega sheep breed were collected at three time points differing in environmental conditions. Rams were selected on the basis of their genotype for the transversion G/C located 660 base pairs upstream the gene transcription initiation site. Animals were also genotyped for another set of 6 SNPs located at the gene promoter. Two SNPs, G/C-660 and A/G-444, were associated with gene overexpression resulting from heat stress. The composed genotype CC-660-AG-444 was the genotype having the highest expression rates with fold changes ranging from 2.2 to 3.0. The genotype AG-522 showed the highest expression levels under control conditions with a fold change of 1.4. Under these conditions, the composed genotype CC-601-TT-524-AG-522-TT-468 is expected to be correlated with higher basal expression of the gene according to genotype frequencies and linkage disequilibrium values. Some putative transcription factors were predicted for binding sites where the SNPs considered are located. Since the expression rate of the gene under alternative environmental conditions seems to depend on the composed genotype of several SNPs located at its promoter, a cooperative regulation of the transcription of the HSP90AA1 gene could be hypothesized. Nevertheless epigenetic regulation mechanisms cannot be discarded.

Forage preservation (grazing vs. hay) fed to ewes affects the fatty acid profile of milk and CPT1B gene expression in the sheep mammary gland.

BACKGROUND: Alterations in lipid metabolism occur when animals are exposed to different feeding systems. In the last few decades, the characterisation of genes involved in fat metabolism and technological advances have enabled the study of the effect of diet on the milk fatty acid (FA) profile in the mammary gland and aided in the elucidation of the mechanisms of the response to diet. The aim of this study was to evaluate the effect of different forage diets (grazing vs. hay) near the time of ewe parturition on the relationship between the fatty acid profile and gene expression in the mammary gland of the Churra Tensina sheep breed. RESULTS: In this study, the forage type affected the C18:2 cis-9 trans-11 (CLA) and long-chain saturated fatty acid (LCFA) content, with higher percentages during grazing than during hay feeding. This may suggest that these FAs act as regulatory factors for the transcriptional control of the carnitine palmitoyltransferase 1B (CPT1B) gene, which was more highly expressed in the grazing group (GRE). The most highly expressed gene in the mammary gland at the fifth week of lactation is CAAT/ enhancer- binding protein beta (CEBPB), possibly due to its role in milk fat synthesis in the mammary gland. More stable housekeeping genes in the ovine mammary gland that would be appropriate for use in gene expression studies were ribosomal protein L19 (RPL19) and glyceraldehyde- 3- phosphate dehydrogenase (GAPDH). CONCLUSIONS: Small changes in diet, such as the forage preservation (grazing vs. hay), can affect the milk fatty acid profile and the expression of the CPT1B gene, which is associated with the oxidation of fatty acids. When compared to hay fed indoors, grazing fresh low mountain pastures stimulates the milk content of CLA and LCFA via mammary uptake. In this sense, LCFA in milk may be acting as a regulatory factor for transcriptional control of the CPT1B gene, which was more highly expressed in the grazing group.

Use of Maximum Likelihood-Mixed Models to select stable reference genes: a case of heat stress response in sheep.

BACKGROUND: Reference genes with stable expression are required to normalize expression differences of target genes in qPCR experiments. Several procedures and companion software have been proposed to find the most stable genes. Model based procedures are attractive because they provide a solid statistical framework. NormFinder, a widely used software, uses a model based method. The pairwise comparison procedure implemented in GeNorm is a simpler procedure but one of the most extensively used. In the present work a statistical approach based in Maximum Likelihood estimation under mixed models was tested and compared with NormFinder and geNorm softwares. Sixteen candidate genes were tested in whole blood samples from control and heat stressed sheep. RESULTS: A model including gene and treatment as fixed effects, sample (animal), gene by treatment, gene by sample and treatment by sample interactions as random effects with heteroskedastic residual variance in gene by treatment levels was selected using goodness of fit and predictive ability criteria among a variety of models. Mean Square Error obtained under the selected model was used as indicator of gene expression stability. Genes top and bottom ranked by the three approaches were similar; however, notable differences for the best pair of genes selected for each method and the remaining genes of the rankings were shown. Differences among the expression values of normalized targets for each statistical approach were also found. CONCLUSIONS: Optimal statistical properties of Maximum Likelihood estimation joined to mixed model flexibility allow for more accurate estimation of expression stability of genes under many different situations. Accurate selection of reference genes has a direct impact over the normalized expression values of a given target gene. This may be critical when the aim of the study is to compare expression rate differences among samples under different environmental conditions, tissues, cell types or genotypes. To select reference genes not only statistical but also functional and biological criteria should be considered. Under the method here proposed SDHA/MDH1 have arisen as the best set of reference genes to be used in qPCR assays to study heat shock in ovine blood samples.

Changes in HSP gene and protein expression in natural scrapie with brain damage.

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.