Transcriptome analysis of perirenal fat from Spanish Assaf suckling lamb carcasses showing different levels of kidney knob and channel fat.

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.

Influence of a temporary restriction of dietary protein in prepubertal ewe lambs on first lactation milk traits and response to a mammary gland inflammatory challenge.

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 Ta, 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 Ta 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.

Intergenerational impact of dietary protein restriction in dairy ewes on epigenetic marks in the perirenal fat of their suckling lambs.

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.

Genetic diversity of two native sheep breeds by genome-wide analysis of single nucleotide polymorphisms.

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 FST were higher in Wallachian sheep (FST = 0.14) than in Sumava sheep (FST = 0.08). The linkage disequilibrium (LD) extension per autosome was higher in Wallachian than in Sumava sheep. Consequently, the Ne 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 (FHOM) 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.

Integrated analyses of the methylome and transcriptome to unravel sex differences in the perirenal fat from suckling lambs.

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.

Accuracy of Imputation of Microsatellite Markers from a 50K SNP Chip in Spanish Assaf Sheep.

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 r2), and allelic dosage (allelic r2), 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 r2), and 0.75 (allelic r2), 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.

A multiple-phenotype imputation procedure as a method for prediction of cheese-making efficiency in Spanish Assaf sheep.

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.

Analysis of Whole Genome Resequencing Datasets from a Worldwide Sample of Sheep Breeds to Identify Potential Causal Mutations Influencing Milk Composition Traits.

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.

The Milk Microbiota of the Spanish Churra Sheep Breed: New Insights into the Complexity of the Milk Microbiome of Dairy Species.

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.

Estimation of the Genetic Parameters for Semen Traits in Spanish Dairy Sheep.

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.