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Mammary gland infections constitute a significant challenge in dairy sheep, affecting productivity and welfare. Temporal RNA sequencing (RNA-Seq) provide a valuable approach to evaluate the evolution of the host defensive molecular mechanisms triggered by mastitis caused by external agents or events. This study aimed to characterize the transcriptomic response of sheep mammary glands to an intramammary inflammation induced with an Escherichia coli LPS inoculation based on RNA-Seq samples generated from milk somatic cells collected at 3 time points: pre-inoculation (0 h), and 6 h and 24 h post-LPS inoculation. The differential expression analyses between the analyzed time points were performed using 2 statistical approaches: one parametric (DESeq2) and one nonparametric (Wilcoxon rank-sum test). The differentially expressed genes (DEG) commonly identified by both approaches encompass 5,872 for the 0 h versus 6 h comparison, 4,063 for the 0 h versus 24 h comparison, and 1,034 for the 6 h versus 24 h comparison. At both 6 and 24 h, transcriptomic data highlighted a significant decrease in the expression of genes linked to metabolic processes crucial for milk protein and lipid synthesis within the mammary gland. Concurrently, increased expression of genes related to the neutrophil attraction was observed for 6 and 24 h, with differences in gene expression between DEG with the highest expression at 6 h, related to T cell activation, type I interferon-mediated signaling pathway, and 24 h, related to cell-cell neutrophil adhesion extravasation or epithelial cell proliferation. In summary, this study reveals how the sheep mammary gland transcriptome responds dynamically to an LPS inoculation, providing a comprehensive understanding of how gene expression patterns evolve over time and shedding light on the molecular mechanisms driving the initial defensive response of the mammary gland against potential inflammatory challenges., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Underutilized sheep and goat breeds can adapt to challenging environments due to their genetics. Integrating publicly available genomic datasets with new data will facilitate genetic diversity analyses; however, this process is complicated by data discrepancies, such as outdated assembly versions or different data formats. Here, we present the SMARTER-database, a collection of tools and scripts to standardize genomic data and metadata, mainly from SNP chip arrays on global small ruminant populations, with a focus on reproducibility. SMARTER-database harmonizes genotypes for about 12,000 sheep and 6,000 goats to a uniform coding and assembly version. Users can access the genotype data via File Transfer Protocol and interact with the metadata through a web interface or using their custom scripts, enabling efficient filtering and selection of samples. These tools will empower researchers to focus on the crucial aspects of adaptation and contribute to livestock sustainability, leveraging the rich dataset provided by the SMARTER-database., Availability and Implementation: The code is available as open-source software under the MIT license at https://github.com/cnr-ibba/SMARTER-database., Competing Interests: The author(s) declare that they have no competing interests., (© The Author(s) 2024.)

In recent years, rising prices for high-quality protein-based feeds have significantly increased nutrition costs. Consequently, investigating strategies to reduce these expenses and improve feed efficiency (FE) have become increasingly important for the dairy sheep industry. This research investigates the impact of nutritional protein restriction (NPR) during prepuberty and FE on the milk transcriptome of dairy Assaf ewes (sampled during the first lactation). To this end, we first compared transcriptomic differences between NPR and control ewes. Subsequently, we evaluated gene expression differences between ewes with divergent FE, using feed conversion ratio (FCR), residual feed intake (RFI), and consensus classifications of high- and low-FE animals for both indices. Lastly, we assess milk gene expression as a predictor of FE phenotype using random forest. No effect was found for the prepubertal NPR on milk performance or FE. Moreover, at the milk transcriptome level, only one gene, HBB, was differentially expressed between the NPR (n = 14) and the control group (n = 14). Further, the transcriptomic analysis between divergent FE sheep revealed 114 differentially expressed genes (DEGs) for RFI index (high-FE RFI = 10 vs low-FE RFI = 10), 244 for FCR (high-FE FCR = 10 vs low-FE FCR = 10), and 1 016 DEGs between divergent consensus ewes for both indices (high-FE consensus  = 8 vs low-FE consensus  = 8). These results underscore the critical role of selected FE indices for RNA-Seq analyses, revealing that consensus divergent animals for both indices maximise differences in transcriptomic responses. Genes overexpressed in high-FE consensus ewes were associated with milk production and mammary gland development, while low-FE consensus genes were linked to higher metabolic expenditure for tissue organisation and repair. The best prediction accuracy for FE phenotype using random forest was obtained for a set of 44 genes consistently differentially expressed across lactations, with Spearman correlations of 0.37 and 0.22 for FCR and RFI, respectively. These findings provide insights into potential sustainability strategies for dairy sheep, highlighting the utility of transcriptomic markers as FE proxies., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Estimating feed efficiency (FE) in dairy sheep is challenging due to the high cost of systems that measure individual feed intake. Identifying proxies that can serve as effective predictors of FE could make it possible to introduce FE into breeding programs. Here, 39 Assaf ewes in first lactation were evaluated regarding their FE by 2 metrics, residual feed intake (RFI) and feed conversion ratio (FCR). The ewes were classified into high, medium and low groups for each metric. Milk samples of the 39 ewes were subjected to untargeted metabolomics analysis. The complete milk metabolomic signature was used to discriminate the FE groups using partial least squares discriminant analysis. A total of 41 and 26 features were selected as the most relevant features for the discrimination of RFI and FCR groups, respectively. The predictive ability when utilizing the complete milk metabolomic signature and the reduced data sets were investigated using 4 machine learning (ML) algorithms and a multivariate regression method. The orthogonal partial least squares algorithm outperformed other ML algorithms for FCR prediction in the scenarios using the complete milk metabolite signature (R 2 = 0.62 ± 0.06) and the 26 selected features (R 2 = 0.62 ± 0.15). Regarding RFI predictions, the scenarios using the 41 selected features outperformed the scenario with the complete milk metabolite signature, where the multilayer feedforward artificial neural network (R 2 = 0.18 ± 0.14) and extreme gradient boosting (R 2 = 0.17 ± 0.15) outperformed other algorithms. The functionality of the selected metabolites implied that the metabolism of glucose, galactose, fructose, sphingolipids, amino acids, insulin, and thyroid hormones was at play. Compared with the use of traditional methods, practical applications of these biomarkers might simplify and reduce costs in selecting feed-efficient ewes., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Long non-coding RNAs (lncRNAs) are being studied in farm animals due to their association with traits of economic interest, such as fat deposition. Based on the analysis of perirenal fat transcriptomes, this research explored the relevance of these regulatory elements to fat deposition in suckling lambs. To that end, meta-analysis techniques have been implemented to efficiently characterize and detect differentially expressed transcripts from two different RNA-seq datasets, one including samples of two sheep breeds that differ in fat deposition features, Churra and Assaf ( n  = 14), and one generated from Assaf suckling lambs with different fat deposition levels ( n  = 8). The joint analysis of the 22 perirenal fat RNA-seq samples with the FEELnc software allowed the detection of 3953 novel lncRNAs. After the meta-analysis, 251 differentially expressed genes were identified, 21 of which were novel lncRNAs. Additionally, a co-expression analysis revealed that, in suckling lambs, lncRNAs may play a role in controlling angiogenesis and thermogenesis, processes highlighted in relation to high and low fat deposition levels, respectively. Overall, while providing information that could be applied for the improvement of suckling lamb carcass traits, this study offers insights into the biology of perirenal fat deposition regulation in mammals.

Background: Livestock populations are under constant selective pressure for higher productivity levels for different selective purposes. This pressure results in the selection of animals with unique adaptive and production traits. The study of genomic regions associated with these unique characteristics has the potential to improve biological knowledge regarding the adaptive process and how it is connected to production levels and resilience, which is the ability of an animal to adapt to stress or an imbalance in homeostasis. Sheep is a species that has been subjected to several natural and artificial selective pressures during its history, resulting in a highly specialized species for production and adaptation to challenging environments. Here, the data from multiple studies that aim at mapping selective sweeps across the sheep genome associated with production and adaptation traits were integrated to identify confirmed selective sweeps (CSS)., Results: In total, 37 studies were used to identify 518 CSS across the sheep genome, which were classified as production (147 prodCSS) and adaptation (219 adapCSS) CSS based on the frequency of each type of associated study. The genes within the CSS were associated with relevant biological processes for adaptation and production. For example, for adapCSS, the associated genes were related to the control of seasonality, circadian rhythm, and thermoregulation. On the other hand, genes associated with prodCSS were related to the control of feeding behaviour, reproduction, and cellular differentiation. In addition, genes harbouring both prodCSS and adapCSS showed an interesting association with lipid metabolism, suggesting a potential role of this process in the regulation of pleiotropic effects between these classes of traits., Conclusions: The findings of this study contribute to a deeper understanding of the genetic link between productivity and adaptability in sheep breeds. This information may provide insights into the genetic mechanisms that underlie undesirable genetic correlations between these two groups of traits and pave the way for a better understanding of resilience as a positive ability to respond to environmental stressors, where the negative effects on production level are minimized., (© 2024. The Author(s).)

Background: As the prepubertal stage is a crucial point for the proper development of the mammary gland and milk production, this study aims to evaluate how protein restriction at this stage can affect methylation marks in milk somatic cells. Here, 28 Assaf ewes were subjected to 42.3% nutritional protein restriction (14 animals, NPR) or fed standard diets (14 animals, C) during the prepubertal stage. During the second lactation, the milk somatic cells of these ewes were sampled, and the extracted DNA was subjected to whole-genome bisulfite sequencing., Results: A total of 1154 differentially methylated regions (DMRs) were identified between the NPR and C groups. Indeed, the results of functional enrichment analyses of the genes harboring these DMRs suggested their relevant effects on the development of the mammary gland and lipid metabolism in sheep. The additional analysis of the correlations of the mean methylation levels within these DMRs with fat, protein, and dry extract percentages in the milk and milk somatic cell counts suggested associations between several DMRs and milk production traits. However, there were no phenotypic differences in these traits between the NPR and C groups., Conclusion: In light of the above, the results obtained in the current study might suggest potential candidate genes for the regulation of milk production traits in the sheep mammary gland. Further studies focusing on elucidating the genetic mechanisms affected by the identified DMRs may help to better understand the biological mechanisms modified in the mammary gland of dairy sheep as a response to nutritional challenges and their potential effects on milk production., (© 2023. BioMed Central Ltd., part of Springer Nature.)

This study evaluated the influence of a temporary nutritional protein restriction (NPR) performed, under commercial conditions, in prepubertal female lambs on first lactation milk production traits and the inflammatory response triggered by an inflammatory challenge of the. From 40 Assaf female lambs, we defined a control group (Cn = 20), which received a standard diet for replacement lambs and the NPR group (n = 20), which received the same diet but without soybean meal between 3 and 5 months of age. About 150 days after lambing, 24 of these ewes (13 NPR, 11C) were subjected to an intramammary infusion of E. coli lipopolysaccharide (LPS). Our dynamic study identified indicator traits of local (SCC) and systemic (rectal T a , IL-6, CXCL8, IL-10, IL-36RA, VEGF-A) response to the LPS challenge. The NPR did not show significant effects on milk production traits and did not affect the SCC and rectal T a after the LPS challenge. However, the NPR had a significant influence on 8 of the 14 plasma biomarkers analysed, in all the cases with higher relative values in the C group. The effects observed on VEGF-A (involved in vasculogenesis during mammary gland development and vascular permeability) and IL-10 (a regulatory cytokine classically known by its anti-inflammatory action) are the most remarkable to explain the differences found between groups. Whereas further studies should be undertaken to confirm these results, our findings are of interest considering the current concern about the future world's demand for protein and the need for animal production systems to evolve toward sustainability., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Suckling lamb meat is highly appreciated in European Mediterranean countries because of its mild flavor and soft texture. In suckling lamb carcasses, perirenal and pelvic fat depots account for a large fraction of carcass fat accumulation, and their proportions are used as an indicator of carcass quality., Material and Methods: This study aimed to characterize the genetic mechanisms that regulate fat deposition in suckling lambs by evaluating the transcriptomic differences between Spanish Assaf lambs with significantly different proportions of kidney knob and channel fat (KKCF) depots in their carcasses (4 High-KKCF lambs vs. 4 Low-KKCF lambs)., Results: The analyzed fat tissue showed overall dominant expression of white adipose tissue gene markers, although due to the young age of the animals (17-36 days), the expression of some brown adipose tissue gene markers (e.g., UCP1 , CIDEA ) was still identified. The transcriptomic comparison between the High-KKCF and Low-KKCF groups revealed a total of 80 differentially expressed genes (DEGs). The enrichment analysis of the 49 DEGs with increased expression levels in the Low-KKCF lambs identified significant terms linked to the biosynthesis of lipids and thermogenesis, which may be related to the higher expression of the UCP1 gene in this group. In contrast, the enrichment analysis of the 31 DEGs with increased expression in the High-KKCF lambs highlighted angiogenesis as a key biological process supported by the higher expression of some genes, such as VEGF-A and THBS1 , which encode a major angiogenic factor and a large adhesive extracellular matrix glycoprotein, respectively., Discussion: The increased expression of sestrins, which are negative regulators of the mTOR complex, suggests that the preadipocyte differentiation stage is being inhibited in the High-KKCF group in favor of adipose tissue expansion, in which vasculogenesis is an essential process. All of these results suggest that the fat depots of the High-KKCF animals are in a later stage of development than those of the Low-KKCF lambs. Further genomic studies based on larger sample sizes and complementary analyses, such as the identification of polymorphisms in the DEGs, should be designed to confirm these results and achieve a deeper understanding of the genetic mechanisms underlying fat deposition in suckling lambs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alonso-García, Suárez-Vega, Fonseca, Marina, Pelayo, Mateo, Arranz and Gutiérrez-Gil.)

Introduction: As higher feed efficiency in dairy ruminants means a higher capability to transform feed nutrients into milk and milk components, differences in feed efficiency are expected to be partly linked to changes in the physiology of the mammary glands. Therefore, this study aimed to determine the biological functions and key regulatory genes associated with feed efficiency in dairy sheep using the milk somatic cell transcriptome., Material and Methods: RNA-Seq data from high (H-FE, n = 8) and low (L-FE, n = 8) feed efficiency ewes were compared through differential expression analysis (DEA) and sparse Partial Least Square-Discriminant analysis (sPLS-DA)., Results: In the DEA, 79 genes were identified as differentially expressed between both conditions, while the sPLS-DA identified 261 predictive genes [variable importance in projection (VIP) > 2] that discriminated H-FE and L-FE sheep., Discussion: The DEA between sheep with divergent feed efficiency allowed the identification of genes associated with the immune system and stress in L-FE animals. In addition, the sPLS-DA approach revealed the importance of genes involved in cell division (e.g., KIF4A and PRC1 ) and cellular lipid metabolic process (e.g., LPL, SCD, GPAM , and ACOX3 ) for the H-FE sheep in the lactating mammary gland transcriptome. A set of discriminant genes, commonly identified by the two statistical approaches, was also detected, including some involved in cell proliferation (e.g., SESN2, KIF20A , or TOP2A ) or encoding heat-shock proteins ( HSPB1 ). These results provide novel insights into the biological basis of feed efficiency in dairy sheep, highlighting the informative potential of the mammary gland transcriptome as a target tissue and revealing the usefulness of combining univariate and multivariate analysis approaches to elucidate the molecular mechanisms controlling complex traits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Suárez-Vega, Frutos, Gutiérrez-Gil, Esteban-Blanco, Toral, Arranz and Hervás.)

In sheep, nutrition during the prepubertal stage is essential for growth performance and mammary gland development. However, the potential effects of nutrient restriction in a prepuberal stage over the progeny still need to be better understood. Here, the intergenerational effect of maternal protein restriction at prepubertal age (2 months of age) on methylation patterns was evaluated in the perirenal fat of Assaf suckling lambs. In total, 17 lambs from ewes subjected to dietary protein restriction (NPR group, 44% less protein) and 17 lambs from control ewes (C group) were analyzed. These lambs were ranked based on their carcass proportion of perirenal and cavitary fat and classified into HighPCF and LowPCF groups. The perirenal tissue from 4 NPR-LowPCF, 4 NPR-HighPCF, 4 C-LowPCF, and 4 C-HighPCF lambs was subjected to whole-genome bisulfite sequencing and differentially methylated regions (DMRs) were identified. Among other relevant processes, these DMRs were mapped in genes responsible for regulating the transition of brown to white adipose tissue and nonshivering thermoregulation, which might be associated with better adaptation/survival of lambs in the perinatal stage. The current study provides important biological insights about the intergenerational effect on the methylation pattern of an NPR in replacement ewes., (© 2023. The Author(s).)

Wallachian and Sumava sheep are autochthonous breeds that have undergone a significant bottleneck effect and subsequent restoration efforts. The first objective of this study was to evaluate the degree of genetic variability of both breeds and, therefore, the current management of the breeding. The second was to determine whether these two breeds still retain their genetic uniqueness in relation to each other and other breeds, despite regenerative interventions. Our data consisted of 48 individuals of Sumava and 37 individuals of Wallachian sheep. The comparison data contained 25 other breeds (primarily European) from the HapMap dataset generated by the International Sheep Genomics Consortium. When comparing all 27 breeds, the Czech breeds clustered with 15 other breeds and formed a single branch with them according to Nei's distances. At the same time, however, the clusters of both breeds were integral and easily distinguishable from the others when displayed with principal component analysis (PCA). Population substructure analysis did not show any common genetic ancestry of the Czech national breeds and breeds used for regeneration or, eventually, breeds whose ancestral population was used for regeneration. The average values of F ST were higher in Wallachian sheep (F ST  = 0.14) than in Sumava sheep (F ST  = 0.08). The linkage disequilibrium (LD) extension per autosome was higher in Wallachian than in Sumava sheep. Consequently, the N e estimates five generations ago were 68 for Sumava versus 34 for Wallachian sheep. Both native Czech breeds exhibit a wide range of inbreeding based on the excess of homozygosity (F HOM ) among individuals, from -0.04 to 0.16 in Sumava and from -0.13 to 0.12 in Wallachian. Average inbreeding based on runs of homozygosity was 0.21 in Sumava and 0.27 in Wallachian. Most detected runs of homozygosity (ROH) were less than 5 Mb long for both breeds. ROH segments longer than 15 Mb were absent in Wallachian sheep. Concerning putative selection signatures, a total of 471 candidate genes in Wallachian sheep within 11 hotspots and 653 genes within 13 hotspots in Sumava sheep were identified. Czech breeds appear to be well differentiated from each other and other European breeds. Their genetic diversity is low, especially in the case of the Wallachian breed. Sumava is not so threatened by low diversity but has a larger share of the non-native gene pool., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dietary supplementation with marine lipids modulates ruminant milk composition toward a healthier fatty acid profile for consumers, but it also causes milk fat depression (MFD). Because the dairy goat industry is mainly oriented toward cheese manufacturing, MFD can elicit economic losses. There is large individual variation in animal susceptibility with goats more (RESPO+) or less (RESPO-) responsive to diet-induced MFD. Thus, we used RNA-Seq to examine gene expression profiles in mammary cells to elucidate mechanisms underlying MFD in goats and individual variation in the extent of diet-induced MFD. Differentially expression analyses (DEA) and weighted gene co-expression network analysis (WGCNA) of RNA-Seq data were used to study milk somatic cell transcriptome changes in goats consuming a diet supplemented with marine lipids. There were 45 differentially expressed genes (DEGs) between control (no-MFD, before diet-induced MFD) and MFD, and 18 between RESPO+ and RESPO-. Biological processes and pathways such as "RNA transcription" and "Chromatin modifying enzymes" were downregulated in MFD compared with controls. Regarding susceptibility to diet-induced MFD, we identified the "Triglyceride Biosynthesis" pathway upregulated in RESPO- goats. The WGCNA approach identified 9 significant functional modules related to milk fat production and one module to the fat yield decrease in diet-induced MFD. The onset of MFD in dairy goats is influenced by the downregulation of SREBF1 , other transcription factors and chromatin-modifying enzymes. A list of DEGs between RESPO+ and RESPO- goats (e.g., DBI and GPD1 ), and a co-related gene network linked to the decrease in milk fat ( ABCD3, FABP3 , and PLIN2 ) was uncovered. Results suggest that alterations in fatty acid transport may play an important role in determining individual variation. These candidate genes should be further investigated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Suárez-Vega, Gutiérrez-Gil, Toral, Frutos, Loor, Arranz and Hervás.)

In sheep, differences were observed regarding fat accumulation and fatty acid (FA) composition between males and females, which may impact the quality and organoleptic characteristics of the meat. The integration of different omics technologies is a relevant approach for investigating biological and genetic mechanisms associated with complex traits. Here, the perirenal tissue of six male and six female Assaf suckling lambs was evaluated using RNA sequencing and whole-genome bisulfite sequencing (WGBS). A multiomic discriminant analysis using multiblock (s)PLS-DA allowed the identification of 314 genes and 627 differentially methylated regions (within these genes), which perfectly discriminate between males and females. These candidate genes overlapped with previously reported QTLs for carcass fat volume and percentage of different FAs in milk and meat from sheep. Additionally, differentially coexpressed (DcoExp) modules of genes between males (nine) and females (three) were identified that harbour 22 of these selected genes. Interestingly, these DcoExp were significantly correlated with fat percentage in different deposits (renal, pelvic, subcutaneous and intramuscular) and were associated with relevant biological processes for adipogenesis, adipocyte differentiation, fat volume and FA composition. Consequently, these genes may potentially impact adiposity and meat quality traits in a sex-specific manner, such as juiciness, tenderness and flavour., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fonseca, Alonso-García, Pelayo, Marina, Esteban-Blanco, Mateo, Gutiérrez-Gil, Arranz and Suárez-Vega.)

Gastrointestinal nematodes (GIN) are a major threat to health and welfare in small ruminants worldwide. Teladorsagia circumcincta is a nematode that inhabits the abomasum of sheep, especially in temperate regions, causing important economic losses. Given that T. circumcincta and microbiome share the same niche, interactions between them and the host are expected. Although it is known that within a sheep breed there are animals that are more resistant than others to infection by GIN, it is not known if the microbiome influences the phenotype of these animals. Under this condition, 12 sheep were classified according to their cumulative faecal egg count (cFEC) at the end of a first experimental infection, 6 as resistant group (RG) and 6 as susceptible group (SG) to T. circumcincta infection. Then, all sheep were experimentally infected with 70,000 L3 of T. circumcincta and at day 7 days post-infection were euthanized. At necropsy, gastric mucosa and gastric content from abomasum were collected to extract bacterial DNA and sequence V3-V4 region from 16S rRNA gene using Ilumina technology. After bioanalysis performed, results showed that α-diversity and β-diversity remained similar in both groups. However, resistant phenotype sheep showed a higher number of bacteria butyrate-fermenting species as Clostridium sensu stricto 1 (abundance in RG: 1.29% and in SG: 0.069%; p = 0.05), and Turicibacter (abundance in RG: 0.31% and in SG: 0.027%; p = 0.07) in gastric content but also Serratia spp in gastric mucosa (abundance in RG: 0.12% and in SG: 0.041%; p = 0.07). A trend towards a significant negative correlation between cFEC and Clostridium sensu stricto 1 abundance in gastric content was detected (r = - 0.537; p = 0.08). These data suggest that microbiome composition could be another factor associated with the development of the resistant phenotype modifying the interaction with the host and the in last instance affecting the individual risk of infection., (© 2022. The Author(s).)

The present study aimed to ascertain how different strategies for leveraging genomic information enhance the accuracy of estimated breeding values for milk and cheese-making traits and to evaluate the implementation of a low-density (LowD) SNP chip designed explicitly for that aim. Thus, milk samples from a total of 2,020 dairy ewes from 2 breeds (1,039 Spanish Assaf and 981 Churra) were collected and analyzed to determine 3 milk production and composition traits and 2 traits related to milk coagulation properties and cheese yield. The 2 studied populations were genotyped with a customized 50K Affymetrix SNP chip (Affymetrix Inc.) containing 55,627 SNP markers. The prediction accuracies were obtained using different multitrait methodologies, such as the BLUP model based on pedigree information, the genomic BLUP (GBLUP), and the BLUP at the SNP level (SNP-BLUP), which are based on genotypic data, and the single-step GBLUP (ssGBLUP), which combines both sources of information. All of these methods were analyzed by cross-validation, comparing predictions of the whole population with the test population sets. Additionally, we describe the design of a LowD SNP chip (3K) and its prediction accuracies through the different methods mentioned previously. Furthermore, the results obtained using the LowD SNP chip were compared with those based on the 50K SNP chip data sets. Finally, we conclude that implementing genomic selection through the ssGBLUP model in the current breeding programs would increase the accuracy of the estimated breeding values compared with the BLUP methodology in the Assaf (from 0.19 to 0.39) and Churra (from 0.27 to 0.44) dairy sheep populations. The LowD SNP chip is cost-effective and has proven to be an accurate tool for estimating genomic breeding values for milk and cheese-making traits, microsatellite imputation, and parentage verification. The results presented here suggest that the routine use of this LowD SNP chip could potentially increase the genetic gains of the breeding selection programs of the 2 Spanish dairy sheep breeds considered here., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Single-step genomic best linear unbiased prediction (ssGBLUP) allows the inclusion of information from genotyped and ungenotyped individuals in a single analysis. This avoids the need to genotype all candidates with the potential benefit of reducing overall costs. The aim of this study was to assess the effect of genotyping strategies, the proportion of genotyped candidates and the genotyping criterion to rank candidates to be genotyped, when using ssGBLUP evaluation. A simulation study was carried out assuming selection over several discrete generations where a proportion of the candidates were genotyped and evaluation was done using ssGBLUP. The scenarios compared were: (i) three genotyping strategies defined by their protocol for choosing candidates to be genotyped (RANDOM: candidates were chosen at random; TOP: candidates with the best genotyping criterion were genotyped; and EXTREME: candidates with the best and worse criterion were genotyped); (ii) eight proportions of genotyped candidates (p); and (iii) two genotyping criteria to rank candidates to be genotyped (candidates' own phenotype or estimated breeding values). The criteria of the comparison were the cumulated gain and reliability of the genomic estimated breeding values (GEBV)., Results: The genotyping strategy with the greatest cumulated gain was TOP followed by RANDOM, with EXTREME behaving as RANDOM at low p and as TOP with high p. However, the reliability of GEBV was higher with RANDOM than with TOP. This disparity between the trend of the gain and the reliability is due to the TOP scheme genotyping the candidates with the greater chances of being selected. The extra gain obtained with TOP increases when the accuracy of the selection criterion to rank candidates to be genotyped increases., Conclusions: The best strategy to maximise genetic gain when only a proportion of the candidates are to be genotyped is TOP, since it prioritises the genotyping of candidates which are more likely to be selected. However, the strategy with the greatest GEBV reliability does not achieve the largest gain, thus reliability cannot be considered as an absolute and sufficient criterion for determining the scheme which maximises genetic gain., (© 2022. The Author(s).)

Different SNP genotyping technologies are commonly used in multiple studies to perform QTL detection, genotype imputation, and genomic predictions. Therefore, genotyping errors cannot be ignored, as they can reduce the accuracy of different procedures applied in genomic selection, such as genomic imputation, genomic predictions, and false-positive results in genome-wide association studies. Currently, whole-genome resequencing (WGR) also offers the potential for variant calling analysis and high-throughput genotyping. WGR might overshadow array-based genotyping technologies due to the larger amount and precision of the genomic information provided; however, its comparatively higher price per individual still limits its use in larger populations. Thus, the objective of this work was to evaluate the accuracy of the two most popular SNP-chip technologies, namely, Affymetrix and Illumina, for high-throughput genotyping in sheep considering high-coverage WGR datasets as references. Analyses were performed using two reference sheep genome assemblies, the popular Oar_v3.1 reference genome and the latest available version Oar_rambouillet_v1.0. Our results demonstrate that the genotypes from both platforms are suggested to have high concordance rates with the genotypes determined from reference WGR datasets (96.59% and 99.51% for Affymetrix and Illumina technologies, respectively). The concordance results provided in the current study can pinpoint low reproducible markers across multiple platforms used for sheep genotyping data. Comparing results using two reference genome assemblies also informs how genome assembly quality can influence genotype concordance rates among different genotyping platforms. Moreover, we describe an efficient pipeline to test the reliability of markers included in sheep SNP-chip panels against WGR datasets available on public databases. This pipeline may be helpful for discarding low-reliability markers before exploiting genomic information for gene mapping analyses or genomic prediction., (© 2021 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

This study aimed to perform a GWAS to identify genomic regions associated with milk and cheese-making traits in Assaf and Churra dairy sheep breeds; second, it aimed to identify possible positional and functional candidate genes and their interactions through post-GWAS studies. For 2,020 dairy ewes from 2 breeds (1,039 Spanish Assaf and 981 Churra), milk samples were collected and analyzed to determine 6 milk production and composition traits and 6 traits related to milk coagulation properties and cheese yield. The genetic profiles of the ewes were obtained using a genotyping chip array that included 50,934 SNP markers. For both milk and cheese-making traits, separate single-breed GWAS were performed using GCTA software. The set of positional candidate genes identified via GWAS was subjected to guilt-by-association-based prioritization analysis with ToppGene software. Totals of 84 and 139 chromosome-wise significant associations for the 6 milk traits and the 6 cheese-making traits were identified in this study. No significant SNPs were found in common between the 2 studied breeds, possibly due to their genetic heterogeneity of the phenotypes under study. Additionally, 63 and 176 positional candidate genes were located in the genomic intervals defined as confidence regions in relation to the significant SNPs identified for the analyzed traits for Assaf and Churra breeds. After the functional prioritization analysis, 71 genes were identified as promising positional and functional candidate genes and proposed as targets of future research to identify putative causative variants in relation to the traits under examination. In addition, this multitrait study allowed us to identify variants that have a pleiotropic effect on both milk production and cheese-related traits. The incorporation of variants among the proposed functional and positional candidate genes into genomic selection strategies represent an interesting approach for achieving rapid genetic gains, specifically for those traits difficult to measure, such as cheese-making traits., (© 2021, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Several recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in regulating the defense mechanism against parasite infections, but no studies are available that investigated their relevance for immune response to nematode infection in sheep. Thus, the aim of the current study was to (i) detect putative lncRNAs that are expressed in the abomasal lymph node of adult sheep after an experimental infection with the gastrointestinal nematode (GIN) Teladorsagia circumcincta and (ii) to elucidate their potential functional role associated with the differential host immune response. We hypothesized that putative lncRNAs differentially expressed (DE) between samples from animals that differ in resistance to infection may play a significant regulatory role in response to nematode infection in adult sheep. To obtain further support for our hypothesis, we performed co-expression and functional gene enrichment analyses with the differentially expressed lncRNAs (DE lncRNAs). In a conservative approach, we included for this predictive analysis only those lncRNAs that are confirmed and supported by documentation of expression in gastrointestinal tissues in the current sheep gene atlas. We identified 9,105 putative lncRNA transcripts corresponding to 7,124 gene loci. Of these, 457 were differentially expressed lncRNA loci (DELs) with 683 lncRNA transcripts. Based on a gene co-expression analysis via weighted gene co-expression network analysis, 12 gene network modules (GNMs) were found significantly correlated with at least one of 10 selected target DE lncRNAs. Based on the principle of "guilt-by-association," the DE genes from each of the three most significantly correlated GNMs were subjected to a gene enrichment analysis. The significant pathways associated with DE lncRNAs included ERK5 Signaling, SAPK/JNK Signaling, RhoGDI Signaling, EIF2 Signaling, Regulation of eIF4 and p70S6K Signaling and Oxidative Phosphorylation pathways. They belong to signaling pathway categories like Cellular Growth, Proliferation and Development, Cellular Stress and Injury, Intracellular and Second Messenger Signaling and Apoptosis. Overall, this lncRNA study conducted in adult sheep after GIN infection provided first insights into the potential functional role of lncRNAs in the differential host response to nematode infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chitneedi, Weikard, Arranz, Martínez-Valladares, Kuehn and Gutiérrez-Gil.)

This study describes early immunological mechanisms that underlie resistance to Teladorsagia circumcincta infection in adult Churra sheep. After a first experimental infection, 6 animals were classified as resistant (RG) and 6 as susceptible (SG) to T. circumcincta infection based on their cumulative faecal egg count (cFEC) at the end of the infection. RG showed higher IgA levels against somatic antigen of T. circumcincta fourth-larvae stage (L4) in serum at day 3 post-infection (pi) (p < 0.05) and close to significance at day 21 pi (p = 0.06). Moreover, a strong negative correlation between cFEC and specific IgA was only significant in RG at day 3 pi (r = - 0.870; p < 0.05), but absent in SG. At the end of this infection, sheep were treated with moxidectin and infected again 3 weeks later to be slaughtered at day 7 pi. At necropsy, the specific IgA levels in gastric mucosa were similar between groups; the absence differences at day 7 pi could be due to a previous increase in the IgA response, probably around day 3 pi, as described during the first infection. L4 burden, 68% lower in RG than in SG, was influenced by the specific IgA in gastric mucus and the number of γδ T cells. RG group showed a positive correlation between γδ T cells and eosinophils (r = 0.900; p = 0.037); however, this correlation was not found in SG. These results show that these two phenotypes show different early immune response pattern to T. circumcincta infection in Churra sheep.

The global production of sheep milk is growing, and the main industrial use of sheep milk is cheese making. The Spanish Churra sheep breed is one of the most important native dairy breeds in Spain. The present study aimed to estimate genetic parameters for a wide range of traits influencing the cheese-making ability of Churra sheep milk. Using a total of 1,049 Churra ewes, we studied the following cheese-making traits: 4 traits related to milk coagulation properties (rennet coagulation time, curd-firming time, and curd firmness at 30 and 60 min after addition of rennet), 2 traits related to cheese yield (individual laboratory cheese yield and individual laboratory dried curd yield), and 3 traits measuring curd firmness over time (maximum curd firmness, time to attain maximum curd firmness, and syneresis). In addition, a list of milk traits, including the native pH of the milk and several milk production and composition traits (milk yield; the fat, protein, and dried extract percentages; and the somatic cell count), were also analyzed for the studied animals. After discarding the noncoagulating samples (only 3.7%), data of 1,010 ewes were analyzed with multiple-trait animal models by using the restricted maximum likelihood method to estimate (co)variance components, heritabilities, and genetic correlations. In general, the heritability estimates were low to moderate, ranging from 0.08 (for the individual laboratory dried curd yield trait) to 0.42 (for the fat percentage trait). High genetic correlations were found within pairs of related traits (i.e., 0.93 between fat and dried extract percentages, -0.93 between the log of the curd-firming time and curd firmness at 30 min, 0.70 between individual laboratory cheese yield and individual laboratory dried curd yield, and -0.94 between time to attain maximum curd firmness and syneresis). Considering all the information provided here, we suggest that in addition to the current consideration of the protein percentage trait for improving cheese yield traits, the inclusion of the pH of milk as a measured trait in the Churra dairy breeding program would represent an efficient strategy for improving the cheese-making ability of milk from this breed., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Sperm quality traits routinely collected by artificial insemination (AI) center for rams progeny test are related with the capacity to produce sperm doses for AI and, in more or less grade, with males' fertility. Low-quality ejaculates are unuseful to perform AI sperm doses, which suppose high economic loses for the AI center. Moreover, sperm quality traits have low heritability values which make traditional genetic selection little efficient to its improvement. In this work, a genome-wide association study (GWAS) was conducted by using sperm quality traits data and 50 K Affymetrix custom chip genotypes of 429 rams of Assaf breed from OVIGEN AI centre. Furthermore, 47 of these rams were also genotyped with the Illumina HD Ovine BeadChip, and therefore HD genotypes were imputed for all rams with phenotype data. Previous to the GWAS, a linear regression model was fitted including sperm traits as dependent variables; the flock of origin, date of sperm collection, and jump number as fixed effects; rams age at collection in months as covariate; and ram permanent effect as random. Pseudo-phenotypes obtained from this model were used as input for GWAS. Associations at the chromosome-wise level (FDR 10%) of 76 single-nucleotide polymorphisms (SNPs) in 4 chromosomes for ejaculate concentration (CON), 20 SNPs in 3 chromosomes for ejaculate volume (VOL), 32 SNPs in 1 chromosome for ejaculate number of spermatozoa (SPZ), and 23 SNPs for spermatozoa mass motility (MOT) in 17 chromosomes were found. Only SNPs associated with MOT overcame the genome-wide significance level. Some candidate genes for sperm traits variability were SLC9C1 (OAR1), TSN (OAR2), and FUT10 (OAR26) for MOT;. DOCK2, CPLANE1, SPEF2, and RAI14 (OAR16) for CON; SCAPER and PSMA4 (OAR18) for VOL; and PARM1 and LOC101110593 (OAR6) for SPZ. SNPs associated with sperm traits were not found to be correlated with milk production genetic variation; however, the high frequencies of some SNPs with negative effect over sperm traits found in animals at the top milk yield estimated breeding values (EBVs) ranking would allow to exert some selective presure to improve rams sperm performances. Effects and frequencies of some of the SNPs detected over sperm quality traits make these variants good candidates to be used in marker-assisted selection to improve sperm characteristics of Assaf rams and AI center efficiency to produce sperm doses., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Transitioning from traditional to new genotyping technologies requires the development of bridging methodologies to avoid extra genotyping costs. This study aims to identify the optimum number of single nucleotide polymorphisms (SNPs) necessary to accurately impute microsatellite markers to develop a low-density SNP chip for parentage verification in the Assaf sheep breed. The accuracy of microsatellite marker imputation was assessed with three metrics: genotype concordance (C), genotype dosage (length r 2 ), and allelic dosage (allelic r 2 ), for all imputation scenarios tested (0.5-10 Mb microsatellite flanking SNP windows). The imputation accuracy for the three metrics analyzed for all haplotype lengths tested was higher than 0.90 (C), 0.80 (length r 2 ), and 0.75 (allelic r 2 ), indicating strong genotype concordance. The window with 2 Mb length provides the best accuracy for the imputation procedure and the design of an affordable low-density SNP chip for parentage testing. We additionally evaluated imputation performance under two null models, naive (imputing the most common allele) and random (imputing by randomly selecting the allele), which in comparison showed weak genotype concordances (0.41 and 0.15, respectively). Therefore, we describe a precise methodology in the present article to impute multiallelic microsatellite genotypes from a low-density SNP chip in sheep and solve the problem of parentage verification when different genotyping platforms have been used across generations.

Sheep milk is mainly intended to manufacture a wide variety of high-quality cheeses. The ovine cheese industry would benefit from an improvement, through genetic selection, of traits related to the milk coagulation properties (MCPs) and cheese yield-related traits, broadly denoted as "cheese-making traits." Considering that routine measurements of these traits needed for genetic selection are expensive and time-consuming, this study aimed to evaluate the accuracy of a cheese-making phenotype imputation method based on the information from official milk control records combined with the pH of the milk. For this study, we analyzed records of milk production traits, milk composition traits, and measurements of cheese-making traits available from a total of 1,145 dairy ewes of the Spanish Assaf sheep breed. Cheese-making traits included five related to the MCPs and two cheese yield-related traits. The milk and cheese-making phenotypes were adjusted for significant effects based on a general linear model. The adjusted phenotypes were used to define a multiple-phenotype imputation procedure for the cheese-making traits based on multivariate normality and Markov chain Monte Carlo sampling. Five of the seven cheese-making traits considered in this study achieved a prediction accuracy of 0.60 computed as the correlation between the adjusted phenotypes and the imputed phenotypes. Particularly the logarithm of curd-firming time since rennet addition (logK20) (0.68), which has been previously suggested as a potential candidate trait to improve the cheese ability in this breed, and the logarithm of the ratio between the rennet clotting time and the curd firmness at 60 min (logRCT/A60) (0.65), which has been defined by other studies as an indicator trait of milk coagulation efficiency. This study represents a first step toward the possible use of the phenotype imputation of cheese-making traits to develop a practical methodology for the dairy sheep industry to impute cheese-making traits only based on the analysis of a milk sample without the need of pedigree information. This information could be also used in future planning of specific breeding programs considering the importance of the cheese-making efficiency in dairy sheep and highlights the potential of phenotype imputation to leverage sample size on expensive, hard-to-measure phenotypes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Different studies have shown that polymorphisms in the sequence of genes coding for the milk proteins and milk fatty acids are associated with milk composition traits as well as with cheese-making traits. However, the lack of coincident results across sheep populations has prevented the use of this information in sheep breeding programs. The main objective of this study was to exploit the information derived from a total of 175 whole genome resequencing (WGR) datasets from 43 domestic sheep breeds and three wild sheep to evaluate the genetic diversity of 24 candidate genes for milk composition and identify genetic variants with a potential phenotypic effect. The functional annotation of the identified variants highlighted five single nucleotide polymorphisms (SNPs) predicted to have a high impact on the protein function and 42 missense SNPs with a putative deleterious effect. When comparing the allelic frequencies at these 47 polymorphisms with relevant functional effects between the genomes of Assaf and Churra sheep breeds, two missense deleterious variants were identified as potential markers associated to the milk composition differences found between the Churra and Assaf: XDH:92215727C>T and LALBA:137390760T>C . Future research is required to confirm the effect of the potential functionally relevant variants identified in the present study on milk composition and cheese-making traits.

Milk from healthy animals has classically been considered a sterile fluid. With the development of massively parallel sequencing and its application to the study of the microbiome of different body fluids, milk microbiota has been documented in several animal species. In this study, the main objective of this work was to access bacterial profiles of healthy milk samples using the next-generation sequencing of amplicons from the 16S rRNA gene to characterise the milk microbiome of the Churra breed. A total of 212 samples were collected from two Churra dairy farms with a different management system. The core milk microbiota in Churra ewes includes lesser genera (only two taxa: Staphylococcus and Escherichia/Shigella) than studies reported in other dairy species or even in a previous study in Assaf sheep milk. We found that diversity values in the two flocks of Churra breed were lower than the diversity of the milk microbiota in Assaf. The non-metric multidimensional scaling (NMDS) ordination using Bray-Curtis distance separates samples based on their microbiota composition. The information reported here might be used to understand the complex issue of milk microbiota composition.

Most of the milk produced by sheep is used for the production of high-quality cheese. Consequently, traits related to milk coagulation properties and cheese yield are economically important to the Spanish dairy industry. The present study aims to identify candidate genes and their regulators related to 14 milk and cheese-making traits and to develop a low-density panel of markers that could be used to predict an individual's genetic potential for cheese-making efficiency. In this study, we performed a combination of the classical genome-wide association study (GWAS) with a stepwise regression method and a pleiotropy analysis to determine the best combination of the variants located within the confidence intervals of the potential candidate genes that may explain the greatest genetic variance for milk and cheese-making traits. Two gene networks related to milk and cheese-making traits were created using the genomic relationship matrices built through a stepwise multiple regression approach. Several co-associated genes in these networks are involved in biological processes previously found to be associated with milk synthesis and cheese-making efficiency. The methodology applied in this study enabled the selection of a co-association network comprised of 374 variants located in the surrounding of genes showing a potential influence on milk synthesis and cheese-making efficiency.

In dairy sheep flocks from Mediterranean countries, replacement and adult ewes are the animals most affected by gastrointestinal nematode (GIN) infections. In this study, we have exploited the information derived from an RNA-Seq experiment with the aim of identifying potential causal mutations related to GIN resistance in sheep. Considering the RNA-Seq samples from 12 ewes previously classified as six resistant and six susceptible animals to experimental infection by Teladorsagia circumcincta, we performed a variant calling analysis pipeline using two different types of software, gatk version 3.7 and Samtools version 1.4. The variants commonly identified by the two packages (high-quality variants) within two types of target regions - (i) QTL regions previously reported in sheep for parasite resistance based on SNP-chip or sequencing technology studies and (ii) functional candidate genes selected from gene expression studies related to GIN resistance in sheep - were further characterised to identify mutations with a potential functional impact. Among the genes harbouring these potential functional variants (930 and 553 respectively for the two types of regions), we identified 111 immune-related genes in the QTL regions and 132 immune-related genes from the initially selected candidate genes. For these immune-related genes harbouring potential functional variants, the enrichment analyses performed highlighted significant GO terms related to apoptosis, adhesion and inflammatory response, in relation to the QTL related variants, and significant disease-related terms such as inflammation, adhesion and necrosis, in relation to the initial candidate gene list. Overall, the study provides a valuable list of potential causal mutations that could be considered as candidate causal mutations in relation to GIN resistance in sheep. Future studies should assess the role of these suggested mutations with the aim of identifying genetic markers that could be directly implemented in sheep breeding programmes considering not only production traits, but also functional traits such as resistance to GIN infections., (© 2020 Stichting International Foundation for Animal Genetics.)

This work aimed to use 16S ribosomal RNA sequencing with the Illumina MiSeq platform to describe the milk microbiota from 50 healthy Assaf ewes. The global observed microbial community for clinically healthy milk samples analysed was complex and showed a vast diversity. The core microbiota of the sheep milk includes five genera: Staphylococcus, Lactobacillus, Corynebacterium, Streptococcus and Escherichia/Shigella. Although there are some differences, some of these genera are common with the microbiota core pattern of milk from other species, especially with dairy cows. The microbial composition of the studied samples, based on the definition of amplicon sequence variants, was analysed through a correlation network. A preliminary analysis by grouping the milk samples based on their somatic cell count (SCC), which is considered an indicator of subclinical mastitis (SM), showed certain differences for the core of the samples identified as SM. The differences in the microbiota diversity pattern among samples might also suggest that subclinical mastitis would be associated with the significant increase in some genera that are inhabitants of the mammary gland and a remarkable concomitant reduction in the microbial diversity. Additionally, we have also presented here a preliminary analysis to assess the impact of the sheep milk microbiome on SCC, as an indicator of subclinical mastitis. The results here reported provide a first characterization of the sheep milk microbiota and settle the basis for future studies in this field., (© 2019 Blackwell Verlag GmbH.)

This work aimed to estimate genetic parameters for traits related to semen production and quality in Spanish dairy sheep breeds. For that, ejaculates of rams from Assaf, Churra, Latxa Cara Negra, Latxa Cara Rubia, and Manchega breeds were analyzed to measure volume, semen concentration, and motility. Estimates of variance components were obtained with multiple-trait animal models using the average information REML method in the BLUPF90 family of programs. Repeatability estimates for all the traits were also calculated, with values ranging from 0.077 to 0.304 for the motility and the semen concentration traits, respectively. Heritability estimates were of low to moderate magnitude, ranging from 0.014 (motility in Latxa Cara Rubia) to 0.198 (volume in Churra), although the estimates differed among the breeds. The estimated genetic correlations among the three semen traits showed adequate precision only in the MAN breed. The heritability estimates for the semen traits reported in the present paper suggest an adequate response to selection. The practical extension of these results to the other breeds studied here will be secondary to the estimation of more reliable genetic correlations in these breeds.

The population of Spanish sheep has decreased from 24 to 15 million heads in the last 75 years due to multiple social and economic factors. Such a demographic reduction might have caused an increase in homozygosity and inbreeding, thus limiting the viability of local breeds with excellent adaptations to harsh ecosystems. The main goal of our study was to investigate the homozygosity patterns of 11 Spanish ovine breeds and to elucidate the relationship of these Spanish breeds with reference populations from Europe, Africa and the Near East. By using Ovine SNP50 BeadChip data retrieved from previous publications, we have found that the majority of studied Spanish ovine breeds have close genetic relatedness with other European populations; the one exception is the Canaria de Pelo breed, which is similar to North African breeds. Our analysis has also demonstrated that, with few exceptions, the genomes of Spanish sheep harbor fewer than 50 runs of homozygosity (ROH) with a total length of less than 350 Mb. Moreover, the frequencies of very long ROH (>30 Mb) are very low, and the inbreeding coefficients (F ROH ) are generally small (F ROH  < 0.10), ranging from 0.008 (Rasa Aragonesa) to 0.086 (Canaria de Pelo). The low levels of homozygosity observed in the 11 Spanish sheep under analysis might be due to their extensive management and the high number of small to medium farms., (© 2019 Stichting International Foundation for Animal Genetics.)

Milk fat depression (MFD) is characterized by a reduction in the content of milk fat, presumably caused by the anti-lipogenic effects of rumen biohydrogenation intermediates, such as trans-10 cis-12 conjugated linoleic acid (CLA). In this study, RNA-Seq technology was used to help elucidate the mammary responses involved in CLA-induced MFD in lactating ewes. To this end, we compared the milk somatic cell transcriptome of ewes suffering from CLA-induced MFD with control ewes (i.e., those without MFD), as well as with ewes fed a diet supplemented with fish oil (FO-MFD) that we previously reported affects the mammary transcriptome. In the differential expression analysis between CLA-MFD and controls, we identified 1,524 differentially expressed genes (DEGs), whereas 653 were detected between CLA- and FO-MFD groups. Although this article focuses on lipid metabolism, CLA affected the expression of many genes related to other biological processes, especially immunity. Among the 55 genes shared by both MFD conditions, some genes linked to fatty acid synthesis, such as ACACA, AACS, ACSS2, or ACSS3, were downregulated. In addition, this study provides a list of candidate genes that are not usually considered in the nutrigenomics of MFD but that may act as key regulators of this syndrome in dairy ewes.

Adipose deposits influence the quality of ruminant carcasses, and in suckling lambs, internal types of adipose deposits represent a notable proportion of total fat. The aim of this study was to perform a comparative analysis of the perirenal fat transcriptomes of suckling lambs from two breeds with different growth and carcass characteristics. The perirenal fat tissue from 14 suckling lambs (Assaf, n = 8; Churra, n = 6) was used for the RNA-seq analysis. The functional enrichment analysis of the 670 highly expressed genes (>150 fragments per kilobase of exon per million fragments mapped) in the perirenal fat transcriptome of both breeds revealed that the majority of these genes were involved in energy processes. The expression of the UCP1 gene, a classical biomarker of brown fat, and the presence of multilocular adipocytes in the two breeds supported the presence of brown fat at the transition stage towards white fat tissue. The differential expression analysis performed identified 373 differentially expressed genes (DEGs) between the two compared breeds. Brown/white fat gene biomarkers were not included in the list of DEGs. In Assaf lambs, DEGs were enriched in Gene Ontology (GO) biological processes related to fatty-acid oxidation, whereas in Churra lambs, the majority of the significantly enriched GO terms were related to cholesterol synthesis, which suggests that upregulated DEGs in Assaf lambs are implicated in fat burning, whereas the Churra upregulated DEGs are linked to fat accumulation. These results can help to increase knowledge of the genes controlling early fat deposition in ruminants and shed light on fundamental aspects of adipose tissue growth., (© 2018 Stichting International Foundation for Animal Genetics.)

This study presents a scan of the ovine genome to identify quantitative trait loci (QTL) influencing the somatic cell score (SCS), a classical indicator of subclinical mastitis in sheep, and a subsequent high-resolution analysis of one of the identified QTL regions based on the analysis of whole-genome sequence data sets. A half-sib commercial population of Churra sheep genotyped with a 50K SNP chip was analyzed using linkage analysis (LA) and combined linkage and linkage disequilibrium analysis (LDLA). By LA, 2 5% chromosome-wide significant QTL on OAR5 and OAR25 and one 5% genome-wide significant QTL on ovine chromosome 20 (OAR20) were detected, whereas 22 significant associations were identified by LDLA. Two of the associations detected by LDLA replicated LA-detected effects (OAR20, OAR25). We compared the detected associations with previously reported QTL in sheep and cattle, and functional candidate genes were identified within the estimated confidence intervals. We then performed a high-resolution analysis of the OAR20 QTL region, the most significant QTL region identified by LA that replicated a QTL previously described in Churra sheep for SCS using microsatellite markers. For that, 2 segregating trios of 2 segregating families for the OAR20 QTL (each including the Qq sire and 2 daughters, QQ and qq) were selected for whole-genome sequencing. The bioinformatic analysis of the 6 sequenced samples performed across the genomic interval considered (14.2-41.7 Mb) identified a total of 227,030 variants commonly identified by 2 independent software packages. For the 3 different concordance tests considered, due to discrepancies regarding the QTL peak in the segregating families, the list of mutations concordant with the QTL segregating pattern was processed to identify the variants identified in immune-related genes that show a moderate/high impact on the encoded protein function. Among a list of 85 missense variants concordant with the QTL segregation pattern that were within candidate immune-related genes, 13 variants distributed across 7 genes [PKHD1, NOTCH4, AGER, ENSOARG00000009395 (HLA-C, Homo sapiens), ENSOARG00000015002 (HLA-B, H. sapiens), MOG, and ENSOARG00000018075 (BoLA, Bos taurus, orthologous to human HLA-A] were predicted to cause deleterious effects on protein function. Future studies should assess the possible associations of the candidate variants identified herein in commercial populations with indicator traits of udder inflammation (SCS, clinical mastitis)., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Suckling lamb meat is traditionally produced in Mediterranean Europe. Breed can affect the quality of the lamb carcass and meat. This study is aimed at comparing the carcass and meat quality between suckling lambs from a local and a non-native dairy breed, Churra and Assaf. Churra is included in the Spanish Protected Geographical Indication (PGI) 'Lechazo de Castilla y León', whereas Assaf is not. However, Assaf breeders have requested the inclusion of the breed in the PGI. Carcasses and meat from 16 male lambs (eight Churra and eight Assaf) were used in this study. The lambs were all raised under an intensive rearing system and fed on a milk substitute to minimise maternal influence. The carcasses were evaluated for conformation, fatness, joint and leg tissue proportions and the meat was analysed for composition (i.e. proximate composition, iron, haematin, fatty acids and volatiles) and technological quality traits (i.e. texture, water holding capacity, colour and lipid stability). Churra carcasses were larger than Assaf carcasses. However, the proportions of commercial joints and main tissues did not differ between breeds. Cavity and intermuscular leg fat, but not total leg fat, were higher in Churra carcasses. Churra meat showed a higher proportion of n-6 fatty acids, higher redness and better colour stability during aerobic storage. In contrast, Assaf lamb was more resistant to lipid oxidation after cooking. This is a preliminary study to measure the influence of breed on a wide range of quality characteristics in Churra and Assaf suckling lamb carcass and meat. It may be of relevance for breeders, consumers and food policy makers, setting the basis for future studies that include larger commercial populations.

The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep.

Background: With the aim of identifying selection signals in three Merino sheep lines that are highly specialized for fine wool production (Australian Industry Merino, Australian Merino and Australian Poll Merino) and considering that these lines have been subjected to selection not only for wool traits but also for growth and carcass traits and parasite resistance, we contrasted the OvineSNP50 BeadChip (50 K-chip) pooled genotypes of these Merino lines with the genotypes of a coarse-wool breed, phylogenetically related breed, Spanish Churra dairy sheep. Genome re-sequencing datasets of the two breeds were analyzed to further explore the genetic variation of the regions initially identified as putative selection signals., Results: Based on the 50 K-chip genotypes, we used the overlapping selection signals (SS) identified by four selection sweep mapping analyses (that detect genetic differentiation, reduced heterozygosity and patterns of haplotype diversity) to define 18 convergence candidate regions (CCR), five associated with positive selection in Australian Merino and the remainder indicating positive selection in Churra. Subsequent analysis of whole-genome sequences from 15 Churra and 13 Merino samples identified 142,400 genetic variants (139,745 bi-allelic SNPs and 2655 indels) within the 18 defined CCR. Annotation of 1291 variants that were significantly associated with breed identity between Churra and Merino samples identified 257 intragenic variants that caused 296 functional annotation variants, 275 of which were located across 31 coding genes. Among these, four synonymous and four missense variants (NPR2&#95;His847Arg, NCAPG&#95;Ser585Phe, LCORL&#95;Asp1214Glu and LCORL&#95;Ile1441Leu) were included., Conclusions: Here, we report the mapping and genetic variation of 18 selection signatures that were identified between Australian Merino and Spanish Churra sheep breeds, which were validated by an additional contrast between Spanish Merino and Churra genotypes. Analysis of whole-genome sequencing datasets allowed us to identify divergent variants that may be viewed as candidates involved in the phenotypic differences for wool, growth and meat production/quality traits between the breeds analyzed. The four missense variants located in the NPR2, NCAPG and LCORL genes may be related to selection sweep regions previously identified and various QTL reported in sheep in relation to growth traits and carcass composition.

In this study, the availability of the Ovine HD SNP BeadChip (HD-chip) and the development of an imputation strategy provided an opportunity to further investigate the extent of linkage disequilibrium (LD) at short distances in the genome of the Spanish Churra dairy sheep breed. A population of 1686 animals, including 16 rams and their half-sib daughters, previously genotyped for the 50K-chip, was imputed to the HD-chip density based on a reference population of 335 individuals. After assessing the imputation accuracy for beagle v4.0 (0.922) and fimpute v2.2 (0.921) using a cross-validation approach, the imputed HD-chip genotypes obtained with beagle were used to update the estimates of LD and effective population size for the studied population. The imputed genotypes were also used to assess the degree of homozygosity by calculating runs of homozygosity and to obtain genomic-based inbreeding coefficients. The updated LD estimations provided evidence that the extent of LD in Churra sheep is even shorter than that reported based on the 50K-chip and is one of the shortest extents compared with other sheep breeds. Through different comparisons we have also assessed the impact of imputation on LD and effective population size estimates. The inbreeding coefficient, considering the total length of the run of homozygosity, showed an average estimate (0.0404) lower than the critical level. Overall, the improved accuracy of the updated LD estimates suggests that the HD-chip, combined with an imputation strategy, offers a powerful tool that will increase the opportunities to identify genuine marker-phenotype associations and to successfully implement genomic selection in Churra sheep., (© 2017 Stichting International Foundation for Animal Genetics.)

In this study, RNA sequencing was used to obtain a comprehensive profile of the transcriptomic changes occurring in the mammary gland of lactating sheep suffering from fish oil-induced milk fat depression (FO-MFD). The milk somatic cell transcriptome analysis of four control and four FO-MFD ewes generated an average of 42 million paired-end reads per sample. In both conditions, less than 220 genes constitute approximately 89% of the total counts. These genes, which are considered as core genes, were mainly involved in cytoplasmic ribosomal proteins and electron transport chain pathways. In total, 117 genes were upregulated, and 96 genes were downregulated in FO-MFD samples. Functional analysis of the latter indicated a downregulation of genes involved in the SREBP signaling pathway (e.g., ACACA, ACSL, and ACSS) and Gene Ontology terms related to lipid metabolism and lipid biosynthetic processes. Integrated interpretation of upregulated genes indicated enrichment in genes encoding plasma membrane proteins and proteins regulating protein kinase activity. Overall, our results indicate that FO-MFD is associated with the downregulation of key genes involved in the mammary lipogenesis process. In addition, the results also suggest that this syndrome may be related to upregulation of other genes implicated in signal transduction and codification of transcription factors.

Background: The identification of genetic variation underlying desired phenotypes is one of the main challenges of current livestock genetic research. High-throughput transcriptome sequencing (RNA-Seq) offers new opportunities for the detection of transcriptome variants (SNPs and short indels) in different tissues and species. In this study, we used RNA-Seq on Milk Sheep Somatic Cells (MSCs) with the goal of characterizing the genetic variation within the coding regions of the milk transcriptome in Churra and Assaf sheep, two common dairy sheep breeds farmed in Spain., Results: A total of 216,637 variants were detected in the MSCs transcriptome of the eight ewes analyzed. Among them, a total of 57,795 variants were detected in the regions harboring Quantitative Trait Loci (QTL) for milk yield, protein percentage and fat percentage, of which 21.44% were novel variants. Among the total variants detected, 561 (2.52%) and 1,649 (7.42%) were predicted to produce high or moderate impact changes in the corresponding transcriptional unit, respectively. In the functional enrichment analysis of the genes positioned within selected QTL regions harboring novel relevant functional variants (high and moderate impact), the KEGG pathway with the highest enrichment was "protein processing in endoplasmic reticulum". Additionally, a total of 504 and 1,063 variants were identified in the genes encoding principal milk proteins and molecules involved in the lipid metabolism, respectively. Of these variants, 20 mutations were found to have putative relevant effects on the encoded proteins., Conclusions: We present herein the first transcriptomic approach aimed at identifying genetic variants of the genes expressed in the lactating mammary gland of sheep. Through the transcriptome analysis of variability within regions harboring QTL for milk yield, protein percentage and fat percentage, we have found several pathways and genes that harbor mutations that could affect dairy production traits. Moreover, remarkable variants were also found in candidate genes coding for major milk proteins and proteins related to milk fat metabolism. Several of the SNPs found in this study could be included as suitable markers in genotyping platforms or custom SNP arrays to perform association analyses in commercial populations and apply genomic selection protocols in the dairy production industry.

Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (-29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Reelin is a signaling protein that plays a crucial role in synaptic function, which expression is influenced by β-amyloid (Aβ). We show that Reelin and Aβ oligomers co-immunoprecipitated in human brain extracts and were present in the same size-exclusion chromatography fractions. Aβ treatment of cells led to increase expression of Reelin, but secreted Reelin results trapped together with Aβ aggregates. In frontal cortex extracts an increase in Reelin mRNA, and in soluble and insoluble (guanidine-extractable) Reelin protein, was associated with late Braak stages of Alzheimer's disease (AD), while expression of its receptor, ApoER2, did not change. However, Reelin-dependent induction of Dab1 phosphorylation appeared reduced in AD. In cells, Aβ reduced the capacity of Reelin to induce internalization of biotinylated ApoER2 and ApoER2 processing. Soluble proteolytic fragments of ApoER2 generated after Reelin binding can be detected in cerebrospinal fluid (CSF). Quantification of these soluble fragments in CSF could be a tool to evaluate the efficiency of Reelin signaling in the brain. These CSF-ApoER2 fragments correlated with Reelin levels only in control subjects, not in AD, where these fragments diminished. We conclude that while Reelin expression is enhanced in the Alzheimer's brain, the interaction of Reelin with Aβ hinders its biological activity.

Because ewe milk is principally used for cheese making, its quality is related to its content of total solids and the way in which milk constituents influence cheese yield and determine the technological and organoleptic characteristics of dairy products. Therefore, an in-depth knowledge of the expression levels of milk genes influencing cheese-related traits is essential. In the present study, the milk transcriptome data set of 2 dairy sheep breeds, Assaf and Spanish Churra, was used to evaluate the expression levels of 77 transcripts related to cheese yield and quality traits. For the comparison between both breeds, we selected the RNA sequencing (RNA-Seq) data at d 10 of lactation because this is the time point at which within and between breed differences due to lactation length are minimal. The evaluated genes encode major milk proteins (caseins and whey proteins), endogenous proteases, and enzymes related to fatty acid metabolism and citrate content. Through this analysis, we identified the genes predominantly expressed in each of the analyzed pathways that appear to be key genes for traits related to sheep milk cheese. Among the highly expressed genes in both breeds were the genes encoding caseins and whey proteins (CSN2, CSN3, CSN1S1, ENSOARG00000005099/PAEP, CSN1S2, LALBA), genes related to lipid metabolism (BTN1A1, XDH, FASN, ADFP, SCD, H-FABP, ACSS2), and one endogenous protease (CTSB). Moreover, a differential expression analysis between Churra and Assaf sheep allowed us to identify 7 genes that are significantly differentially expressed between the 2 breeds. These genes were mainly linked to endogenous protease activity (CTSL, CTSK, KLK10, KLK6, SERPINE2). Additionally, there were 2 differentially expressed genes coding for an intracellular fatty acid transporter (FABP4), an intermediate molecule of the citric acid cycle (SUCNR1), and 2 heat shock proteins (HSP70, HSPB8) that could be related to high protein production. The differential expression of these genes could have a direct influence on the different phenotypes observed between the 2 analyzed breeds., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism), to quantify expression and to identify differential regulation in a single experiment. Our aim in this study was to take advantage of using RNA-Seq high-throughput technology to provide a comprehensive transcriptome profiling of the sheep lactating mammary gland. Eight ewes of two dairy sheep breeds with differences in milk production traits were used in this experiment (four Churra and four Assaf ewes). Milk samples from these animals were collected on days 10, 50, 120 and 150 after lambing to cover the various physiological stages of the mammary gland across the complete lactation. RNA samples were extracted from milk somatic cells. The RNA-Seq dataset was generated using an Illumina HiSeq 2000 sequencer. The information reported here will be useful to understand the biology of lactation in sheep, providing also an opportunity to characterize their different patterns on milk production aptitude.

Background: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses., Results: In this dataset, 64% of animals had zero FEC while the ZINB model suggested that 38% of sheep had not been recently infected with GIN. Therefore 26% of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed., Conclusions: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasites.

The goals of the current work were to analyse the population structure of 11 Spanish ovine breeds and to detect genomic regions that may have been targeted by selection. A total of 141 individuals were genotyped with the Infinium 50 K Ovine SNP BeadChip (Illumina). We combined this dataset with Spanish ovine data previously reported by the International Sheep Genomics Consortium (N = 229). Multidimensional scaling and Admixture analyses revealed that Canaria de Pelo and, to a lesser extent, Roja Mallorquina, Latxa and Churra are clearly differentiated populations, while the remaining seven breeds (Ojalada, Castellana, Gallega, Xisqueta, Ripollesa, Rasa Aragonesa and Segureña) share a similar genetic background. Performance of a genome scan with BayeScan and hapFLK allowed us identifying three genomic regions that are consistently detected with both methods i.e. Oar3 (150-154 Mb), Oar6 (4-49 Mb) and Oar13 (68-74 Mb). Neighbor-joining trees based on polymorphisms mapping to these three selective sweeps did not show a clustering of breeds according to their predominant productive specialization (except the local tree based on Oar13 SNPs). Such cryptic signatures of selection have been also found in the bovine genome, posing a considerable challenge to understand the biological consequences of artificial selection.

Background: Persistence of gastrointestinal nematode (GIN) infection and the related control methods have major impacts on the sheep industry worldwide. Based on the information generated with the Illumina OvineSNP50 BeadChip (50 K chip), this study aims at confirming quantitative trait loci (QTL) that were previously identified by microsatellite-based genome scans and identifying new QTL and allelic variants that are associated with indicator traits of parasite resistance in adult sheep. We used a commercial half-sib population of 518 Spanish Churra ewes with available data for fecal egg counts (FEC) and serum levels of immunoglobulin A (IgA) to perform different genome scan QTL mapping analyses based on classical linkage analysis (LA), a combined linkage disequilibrium and linkage analysis (LDLA) and a genome-wide association study (GWAS)., Results: For the FEC and IgA traits, we detected a total of three 5 % chromosome-wise significant QTL by LA and 63 significant regions by LDLA, of which 13 reached the 5 % genome-wise significance level. The GWAS also revealed 10 significant SNPs associated with IgAt, although no significant associations were found for LFEC. Some of the significant QTL for LFEC that were detected by LA and LDLA on OAR6 overlapped with a highly significant QTL that was previously detected in a different half-sib population of Churra sheep. In addition, several new QTL and SNP associations were identified, some of which show correspondence with effects that were reported for different populations of young sheep. Other significant associations that did not coincide with previously reported associations could be related to the specific immune response of adult animals., Discussion: Our results replicate a FEC-related QTL located on OAR6 that was previously reported in Churra sheep and provide support for future research on the identification of the allelic variant that underlies this QTL. The small proportion of genetic variance explained by the detected QTL and the large number of functional candidate genes identified here are consistent with the hypothesis that GIN resistance/susceptibility is a complex trait that is not determined by individual genes acting alone but rather by complex multi-gene interactions. Future studies that combine genomic variation analysis and functional genomic information may help elucidate the biology of GIN disease resistance in sheep.