Genetic relationships between Spanish Assaf (Assaf.E) and Spanish native dairy sheep breeds

At present, the Assaf is the main dairy sheep in Spain. The Spanish Assaf (Assaf.E) was formed by male-mediated absorption of native Spanish sheep. Here we assess the genetic relationships among the Assaf.E and major native Spanish dairy breeds using microsatellites to contribute to the knowledge of the formation and within-population genetic variability of the breed. Blood samples from 44 unrelated Assaf.E individuals from 23 different Assaf.E flocks spread throughout 6 different Spanish provinces were obtained and genotyped using 14 microsatellites. Up to 312 additional samples belonging to the Awassi and Milchschaf sheep breeds and to six native Spanish dairy sheep breeds (Castellana, Churra, Latxa, Manchega, and Rubia de El Molar) as well as samples from Merino individuals to be used as the outgroup were also analysed observed (H_o) and expected (H_e) heterozygosity, rarefacted number of alleles per locus and distances based on molecular coancestry information were computed. Probabilities of assignment of the Assaf.E individuals to native Spanish dairy sheep breeds and cryptic genetic structure in the whole dataset were also assessed. It can be concluded that the Assaf.E breed has low genetic variability and high genetic distance with respect native Spanish dairy sheep breeds. From our results, the formation of the Assaf.E breed basically occurred via the absorption of individuals belonging to the Entrefino type, particularly to the Castellana and Manchega populations. Furthermore, Churra individuals may have participated in the formation of the Assaf.E breed at an early moment of the introduction of the breed into Spain.     

Differentiation among Spanish sheep breeds using microsatellites

Genetic variability at 18 microsatellites was analysed on the basis of individual genotypes in five Spanish breeds of sheep – Churra, Latxa, Castellana, Rasa-Aragonesa and Merino -, with Awassi also being studied as a reference breed. The degree of population subdivision calculated between Spanish breeds from F_ST diversity indices was around 7% of total variability. A high degree of reliability was obtained for individual-breed assignment from the 18 loci by using different approaches among which the Bayesian method provided to be the most efficient, with an accuracy for nine microsatellites of over 99%. Analysis of the Bayesian assignment criterion illustrated the divergence between any one breed and the others, which was highest for Awassi sheep, while no great differences were evident among the Spanish breeds. Relationships between individuals were analysed from the proportion of shared alleles. The resulting dendrogram showed a remarkable breed structure, with the highest level of clustering among members of the Spanish breeds in Latxa and the lowest in Merino sheep, the latter breed exhibiting a peculiar pattern of clustering, with animals grouped into several closely set nodes. Analysis of individual genotypes provided valuable information for understanding intra- and inter-population genetic differences and allowed for a discussion with previously reported results using populations as taxonomic units.     

Genetic relationships among Spanish sheep using microsatellites

Five indigenous Spanish breeds of sheep, Churra, Latxa, Manchega, Rasa–Aragonesa and Merino, with Awassi sheep as a reference breed were genotyped for 19 DNA microsatellites. Allele frequencies and mean heterozygosities revealed the greatest genetic variation in Merino sheep and the lowest in Awassis. Differences in variability were not great in the other breeds studied. The dendrograms obtained based on genetic distances showed a large differentiation between Awassi sheep and the Spanish breeds, as was to be expected from their distinct genetic origin. Merinos appeared separated from the other four breeds, of which, according to a classification based on the fleece characteristics, Churra and Latxa belong to the churro type and Manchega and Rasa–Aragonesa to the so called entrefino type, though no clear separation was evident between the two types. These results suggest that morphological data alone are insufficient for determining relationships between breeds and that studies involving genetic markers may be of great assistance.     

Genetic diversity in the Churra tensina and Churra lebrijana endangered Spanish sheep breeds and relationship with other Churra group breeds and Spanish mouflon

The aim of the present study was to estimate the genetic intra-breed variability of Churra tensina and Churra lebrijana endangered breeds and to establish genetic relationships with Churra, Latxa and Merino breeds, as well as Spanish mouflon, by using 28 microsatellite markers, to provide useful information for their conservation. Allele frequencies and heterozygosity revealed high genetic variation in the two endangered breeds despite their small population size. Estimates of inbreeding coefficient (F_IS) were significant for all breeds studied, except for Churra lebrijana breed. The highest inbreeding coefficient (F_IS = 0.143) was found in the Spanish mouflon. Genetic differentiation tests (F_ST = 0.121) and assignment of individuals to populations indicated the existence of defined breed populations, and low genetic flow between these breeds. The highest pairwise Reynolds distance (D_R) values were observed between Mouflon and the domestic sheep breeds. Considering only domestic sheep breeds, the Churra lebrijana breed showed the highest pairwise D_R values_. The lowest values were found between Latxa and the other domestic sheep, except for Churra lebrijana. Results of pairwise D_R values, as well as phylogenetic tree and bottleneck analysis showed an important genetic isolation of the Churra lebrijana breed from the other Churra types, and genetic signatures of a demographic bottleneck. Finally, structure analysis of populations detected a population subdivision in the Latxa sheep breed. In conclusion, this study presents valuable insight into the existing genetic variability of two Spanish endangered breeds, as well as the first study in Spanish mouflon based on microsatellite analysis. The high degree of variability demonstrated in Churra tensina and Churra lebrijana implies that these populations are rich reservoirs of genetic diversity.     

Genetic variations in blood proteins within and between Spanish dairy sheep breeds

Eleven flocks of three Spanish sheep breeds have been studied: four Churra, four Lacha and three Manchega. Genetic variations between flocks of the same and different breeds have been analysed, using eight genetic blood systems and a wide range of statistical methods. The results show that the Churra, Lacha and Manchega breeds present significant differences between each other. Significant intraracial variations have been observed in Churras and Lachas, although they were almost non-existent in Manchegas. In some cases the genetic distances between flocks of the same breed were greater than the distances between flocks of different breeds. The data indicate that the closest breeds genetically are Lacha and Manchega.     

Carcass and meat quality characteristics of Churra and Assaf suckling lambs

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.     

Hemotypes in Spanish sheep breeds

The structure and the genetic diversity of the Churra, Lacha and Manchega sheep breeds have been analysed using hemotypes observed in eight loci. The three breeds are different in their hemotypes in terms both of quantity and quality. The proportions of unique hemotypes in Churra (60%), Lacha (61%) and Manchega (67%) revealed a high level of individual diversity within each breed. Racial genetic diversity follows the descending order of: Manchega-Lacha-Churra. The value of N:H (number of animals: total hemotypes) in the multi-racial population was 3.05.     

Influence of breed and age on morphometry and depth of inseminating catheter penetration in the ewe cervix: a postmortem study

A detailed examination of the cervical canal in the ewe was carried out. This analysis could be used to design new catheters for artificial insemination (AI) to achieve deeper cervical penetration and therefore better fertility results. Three hundred and sixty-five cervices from four sheep breeds (Churra, Assaf, Merino, Castellana) obtained postmortem were used. Cervix morphometry and depth of cervical penetration using two types of catheters were determined. A conventional straight catheter for ovine artificial insemination (IMV), and a bent catheter, ending in a stainless steel needle, 9 cm in length and with an 8 mm tip bent 45 degrees , were used. The results showed that the morphometry of the cervix depends on breed and age of the ewe. The cervices of Churra breed were shorter and narrower, and had a higher number of folds than those of other breeds. Postmortem cervical penetration was deeper when the cervices were longer and wider, and with fewer folds (Merino and Castellana breeds). In ageing ewes, the cervix tended to become longer and wider, with loose folds. This decreased structural complexity and significantly improved cervical penetration. The bent catheter allowed significantly greater cervical penetration than the straight IMV one.     

Assessing genetic diversity and differentiation in Portuguese coarse-wool sheep breeds with microsatellite markers

Population structure and genetic diversity in the Portuguese native breeds of sheep Algarvia (AL), Badana (BA), Galega Bragançana (GB), Galega Mirandesa (GM), Mondegueira (MO) and Churra da Terra Quente (TQ), as well as the exotic Assaf (AS), were analyzed by typing 25 microsatellite markers in 210 individuals. The markers used exhibited high levels of polymorphism, with means for total and effective number of alleles per locus of 13.0 and 4.2, respectively, and an expected heterozygosity of 0.72 across loci. The mean number of alleles per locus and expected heterozygosity were highest in GM and GB, and lowest in AS. Exclusive alleles were found in 10 of the 25 markers analysed, mostly in the AS breed. The proportion of loci which were not in Hardy–Weinberg equilibrium in each breed ranged between 0.12 (GB) and 0.40 (AL and GM), mostly due to a lower than expected number of heterozygotes in those loci. All breeds showed a significant deficit in heterozygosity, which was more pronounced in GM (F_IS = 0.113) and BA (F_IS = 0.103), suggesting that inbreeding might be a major concern in these breeds. The analysis of relationships among breeds, assessed by different methods, indicates that AS and AL are the more distanced breeds relative to the others, while the closest relationships were observed between TQ with MO and GM with GB. The estimated F_ST indicates that only 0.049 of the total genetic variability can be attributed to differences among breeds, and this ratio dropped to 0.029 when only the native breeds were considered. The analysis of individual distances based on allele-sharing indicates that only AS and AL had a tendency for animals of the same breed to cluster together, while for the other breeds there was overlapping among breeds. The results of this study confirm that native breeds of sheep represent an important reservoir of genetic diversity, even though the level of differentiation among closely located breeds tends to be rather small. For several of the breeds analyzed, the levels of inbreeding currently observed cause some apprehension, and recommend the establishment of appropriate conservation strategies, aimed at minimizing inbreeding to avoid further losses of genetic diversity.     

Genetic parameters affecting 180-days standardised milk yield, test-day milk yield and lactation length in Spanish Assaf (Assaf.E) dairy sheep

A total of 42,197 records of test-day milk yield (TDY) and 7654 lactations from 3854 individuals belonging to the Spanish Assaf (Assaf.E) breed were analysed using univariate and multivariate models to estimate genetic parameters affecting TDY, total milk yield standardised to 180 days (MY₁₈₀) and lactation length (LL) in order to asses their possibilities of use in the selection scheme of the Assaf.E population. Estimates of h² were, in general, on the lower limit of those usually reported for the three analysed traits. Estimates of heritability for MY₁₈₀ varied from 0.131 to 0.177. Estimates of h² for LL and TDY were consistent regardless the estimation model being of, roughly, 5% and 10%, respectively. The estimates for the permanent environmental effect (c²) of TDY were consistently the same (0.27–0.28) regardless the fitted model whilst they showed large differences for LL and MY₁₈₀. Genetic correlations were always positive and high ranging from 0.792 for the pair LL-TDY to 0.999 for the pair MY₁₈₀-TDY. Correlations between permanent environmental effects were even higher ranging from 0.932 for the pair LL-MY₁₈₀ and 0.999 for the pair MY₁₈₀-TDY. The advantages of using TDY as selection criterion in the Assaf.E improvement scheme are discussed.     

Microsatellite Analysis Characterizes Burkina Faso as a Genetic Contact Zone Between Sahelian and Djallonké Sheep

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

Genomic diversity and population structure of Mexican and Spanish bovine Lidia breed

The Lidia bovine breed is distinguished for its low genetic exchangeability given its selection on aggressive behavior, its management uniqueness and its subdivided structure. In this study, we present a comprehensive genome‐wide analysis of genetic diversity, population structure and admixture of 468 animals from Mexican and Spanish Lidia breed populations and 64 samples belonging to 10 Spanish native and American‐creole breeds using 37 148 single nucleotide polymorphisms. We found similar average inbreeding values in the Lidia breed, with different distributions within groups; variability of inbreeding values among Spanish lineages was significant and no differences were found among the Mexican sub‐populations. Together, the high F_IS of the lineages and the behavior of the runs of homozygosity are consequences of the lineage's small effective population sizes, contributing to their inbreeding increase. Population admixture analysis discarded any influence on the genetic structure of the Lidia populations from the Spanish native and American‐creole breeds. In addition, both Lidia populations depicted different genetic origins, with the exception of some Mexican individuals whose origins traced back to recent Spanish importations.     

Genetic variation at microsatellite loci in Spanish sheep

Genetic variation at 18 microsatellite loci was analysed in six indigenous Spanish sheep: Churra; Latxa; Manchega; Rasa-Aragonesa; Castellana and Merino. Merinos had frequently the highest number of alleles per locus, whereas Latxas showed the lowest one at many loci. Markers ordered decreasingly according to the number of variants differentiated in the whole population were: MAF70; TGLA13; CSSM66; BM143, BM6444; MAF36; MAF64; CSSM6; TGLA53; OarFCB11; MAF33; BM4621; MAF48; MAF65; BM1258; ILSTS002; ADCYC and OarCP34. Parameters of variability such as effective number of alleles and gene diversities corroborated the high level of variation frequently displayed by microsatellite markers. Comparison of allele distributions among populations and loci did not reveal consistent shapes. Distributions were centralised in some cases, whereas in others some kind of skewness was evident. Breed-specific alleles were detected at most loci, being frequent in Merinos and rare in Churras.     

Genetic diversity of three Chinese native sheep breeds

The sheep (Ovis aries L.) has been an important farm animal species since its domestication. A wide array of indigenous sheep breeds with abundant phenotypic diversity exists for domestication and selection. Therefore, assessing the genetic diversity of a local sheep resource using a multi-molecular system is helpful for maintaining and conserving those breeds. This study aimed to investigate the genetic diversity of three native Chinese sheep breeds (Tibetan sheep, Sishui Fur sheep, and Small-tailed Han sheep) using 15 microsatellite markers and the second exon of the DRA gene. In regards to the microsatellites, on average, 19 alleles per loci were observed among all individuals. Across loci, the HO within the population was 0.652 ± 0.022 in Tibetan sheep, 0.603 ± 0.023 in Small-tailed Han sheep and 0.635 ± 0.022 in SFS, and for most populations, the H _E and H _O were inconsistent. In addition, affluent private alleles within the breed indicated that the breeds have different domestication histories or sites. In regards to the 2 exon of the DRA gene, three haplotypes were constructed by seven single-nucleotide polymorphisms (SNPs), which were identified in the second DRA exon and inferred the potential for phenotypic variety in these Chinese native sheep. In summary, the current study reveals the importance of implementing effective conservation strategies for these three native Chinese sheep.     

Estimations of linkage disequilibrium,effective population size and ROH‐based inbreeding coefficients in Spanish Churra sheep using imputed high‐density SNP genotypes

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.     

The genetic structure of Spanish Celtic horse breeds inferred from microsatellite data

Partition of the genetic variability, genetic structure and relationships among seven Spanish Celtic horse breeds were studied using PCR amplification of 13 microsatellites on 481 random individuals. In addition, 60 thoroughbred horses were included. The average observed heterozygosity and the mean number of alleles were higher for the Atlantic horse breeds than for the Balearic Islands breeds. Only eight percentage of the total genetic variability could be attributed to differences among breeds (mean FST approximately 0.08; P < 0.01). Atlantic breeds clearly form a separate cluster from the Balearic Islands breeds and among the former only two form a clear clustering, while the rest of Atlantic breeds (Jaca Navarra, Caballo Gallego and Pottoka) are not consistently differentiated. Multivariate analysis showed that Asturcon populations, Losina and Balearic Islands breeds are clearly separated from each other and from the rest of the breeds. In addition to this, the use of the microsatellites proved to be useful for breed assignment.     

Genetic characterization and founder effect analysis of recently introduced Salers cattle breed population

Salers are a native French breed used for beef and dairy production that has expanded to all the continents. The Salers breed was introduced to the north of Spain in 1985 with only 15 individuals from France and has successfully increased to over 20 000 animals. Although over time new animals have been imported from France for breeding, it is possible that the limiting number of founder animals could have resulted in a reduction of the genetic diversity found in Spanish Salers. Thus, the purpose of the present study has been to characterize the genetic diversity of Salers breed in Spain and evaluate a possible founder effect due to reduced number of the first reproducers. A total of 403 individuals from 12 Salers herds were analyzed using 12 microsatellite markers and compared with phylogenetically and geographically close related Blonde d’Aquitaine, Limousin and Charolais French breeds but also other 16 European breeds. Microsatellites in Salers were polymorphic, with a mean allelic richness of 5.129 and an expected heterozygosity of 0.621 across loci (0.576 to 0.736 among all breeds). Average observed heterozygosity was 0.618. All the loci fit the Hardy–Weinberg (HW) equilibrium except TGLA227 locus due to a significant deficit of heterozygotes in only one of the herds, probably attributable to a sampling effect. When all loci were combined, Salers inbreeding coefficient did not differ statistically from 0 (F_IS=0.005), indicating not significant excess or deficit of heterozygotes (P=0.309). Based in allelic distribution, Salers revealed a frequency of 0.488 in BM2113-131 and 0.064 in BM2113-143 diagnostic alleles, which are specific to the African zebu. These zebu alleles are also found in some French breeds, supported by STR data previously postulated hypothesis of a migration route through Mediterranean route by which North African cattle may have left a genetic signature in southern Europe. Phylogenetic tree and population structure analyses could unambiguously differentiate Salers cattle from the other populations and 10% of the total genetic variability could be attributed to differences among breeds (mean R_ST=0.105; P<0.01). Mutation-drift equilibrium tests (sign test and Wilcoxon’s sign rank test) were in correspondence to the absence of founder effect when Bonferroni was applied. Gene diversity previously reported in French Salers was comparable with the observed in our population. Thus, high genetic diversity in Spanish Salers highlights the resources of this population, which looks toward future breeding and selection programs.     

Factors influencing the success of vaginal and laparoscopic artificial insemination in churra ewes: a field assay

Pregnancy rate following artificial insemination (AI) in sheep is variable depending on several factors. The Churra breed (milk breed of the North-West of Spain) yields lower fertility results compared with other local and European breeds (Manchega, Latxa, Merino, Lacaune, Sarde, etc.). In this work we studied the influence of many factors on the fertility of the Churra breed (insemination technique, year, farm, age, male, number of inseminations per ewe, lambing-insemination interval and technician), analyzing lambing data obtained after 44448 inseminations (39.67% cervical AI via vagina, AIV, and 60.33% intrauterine AI using laparoscopy, AIL) in a categorical model. The most important factors influencing fertility after AI were farm, year, season, AI technique, and technician. AIL showed significantly higher fertility results than AIV (44.89% versus 31.25%). Season significantly affected fertility in both cases, but differences were more evident in AIV. Fertility dropped 1.74% (AIV) and 2.07% (AIL) per year as the ewes aged. Finally, AI fertility decreased when the lambing-insemination interval was lower than 10 weeks.     

The role of bovine causal genes underlying dairy traits in Spanish Churra sheep

In dairy cattle, quantitative trait nucleotides (QTNs) underlying quantitative trait loci (QTL) for milk production traits have been identified in bovine DGAT1, GHR and ABCG2 genes. The SPP1 gene has also been proposed to be a regulator of lactation. In sheep, QTL underlying milk production traits have been reported only recently, and no proven QTN has been identified. Taking into account the close phylogenetic relationship between sheep and cattle, this study examined the possible effects of the aforementioned genes on sheep milk production traits. We first studied the genetic variability of the DGAT1, GHR, ABCG2 and SPP1 genes in 15 rams of the Spanish Churra dairy sheep breed. Second, we performed an association analysis between SNPs identified in these genes and three milk production traits recorded in a commercial population of Churra sheep. This analysis revealed only three significant associations at the nominal level (P-value <0.05) involving allelic variants of the ABCG2 gene, whereas no significant association was found for the DGAT1, GHR and SPP1 genes. When the Bonferroni correction was applied to take into account the multiple tests performed, none of the associations identified at the nominal level remained significant. Nevertheless, taking into account the high level of false-negative findings that can arise when applying the stringent Bonferroni correction, we think that our results provide a valuable primary assessment of strong candidate genes for milk traits in sheep.     

Genetic diversity in Spanish donkey breeds using microsatellite DNA markers

Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H_E) over all the populations varied between 0.637 and 0.684 in this study. The low G_ST value showed that only 3.6% of the diversity was between breeds (P < 0.01). Significant deviations from Hardy-Weinberg equilibrium were shown for a number of locus-population combinations, except HMS5 that showed agreement in all analysed populations. The cumulative exclusion probability (PE) was 0.999 in each breed, suggesting that the loci would be suitable for donkey parentage testing. The constructed dendrogram from the D_A distance matrix showed little differentiation between Spanish breeds, but great differentiation between them and the Moroccan ass and also with the horse, used as an outgroup. These results confirm the potential use of equine microsatellite loci as a tool for genetic studies in domestic donkey populations, which could also be useful for conservation plans.