Key genes and metabolites that regulate wool fibre diameter identified by combined transcriptome and metabolome analysis.

BACKGROUND: Fibre diameter is an important economic trait of wool fibre. As the fibre diameter decreases, the economic value of wool increases. Therefore, understanding the mechanism of wool fibre diameter regulation is important in improving the value of wool. RESULTS: In this study, we used non-targeted metabolome and reference transcriptome data to detect differences in metabolites and genes in groups of Alpine Merino sheep with different wool fibre diameter gradients, and integrated metabolome and transcriptome data to identify key genes and metabolites that regulate wool fibre diameter. We found 464 differentially abundant metabolites (DAMs) and 901 differentially expressed genes (DEGs) in four comparisons of groups with different wool fibre diameters. Approximately 25% of the differentially abundant metabolites were lipid and lipid-like molecules. These molecules were predicted to be associated with skin development and keratin filament by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Key genes, including COL5A2, COL5A3, CREB3L4, COL1A1, and SFRP4, were identified by gene set enrichment analysis. CONCLUSIONS: Key genes regulating wool fibre diameter were identified, the effects of lipid molecules on wool performance were investigated, and potential synergies between genes and metabolites were postulated, providing a theoretical framework for fine wool sheep breeding.

Genome-wide association study identifies genetic variants underlying footrot in Portuguese Merino sheep.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D. nodosus) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses. RESULTS: Phenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction (p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes. CONCLUSION: This is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

Analysis of the Long Non-Coding and Messenger RNA Expression Profiles in the Skin Tissue of Super Merino and Small-Tailed Han Sheep.

Wool quality and yield are two important economic livestock traits. However, there are relatively few molecular studies on lncRNA for improving sheep wool, so these require further exploration. In this study, we examined skin tissue from the upper scapula of Super Merino (SM) and Small-Tailed Han (STH) sheep during the growing period. The apparent difference was verified via histological examination. High-throughput RNA sequencing identified differentially expressed (DE) long non-coding (lncRNAs) and messenger RNAs (mRNAs). The target gene of DE lncRNA and DE genes were enrichment analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was used to verify randomly selected DE lncRNAs and mRNAs. Finally, the DE, RAC2, WNT11, and FZD2 genes, which were enriched in the Wnt signaling pathway, were detected via immunohistochemistry. The results showed that a total of 20,888 lncRNAs and 31,579 mRNAs were identified in the skin tissues of the two sheep species. Among these, 56 lncRNAs and 616 mRNAs were differentially expressed. Through qRT-PCR, the trends in the randomly selected DE genes' expression were confirmed to be aligned with the RNA-seq results. GO and KEGG enrichment analysis showed that DE lncRNA target genes were enriched in GO terms as represented by epidermal and skin development and keratin filature and in KEGG terms as represented by PI3K-Akt, Ras, MAPK, and Wnt signaling pathways, which were related to hair follicle growth and development. Finally, immunohistochemistry staining results indicated that RAC2, WNT11, and FZD2 were expressed in dermal papilla (DP). The lncRNAs MSTRG.9225.1 and MSTRG.98769.1 may indirectly participate in the regulation of hair follicle growth, development, and fiber traits by regulating their respective target genes, LOC114113396(KRTAP15-1), FGF1, and IGF1. In addition, MSTRG.84658.1 may regulate the Wnt signaling pathway involved in the development of sheep hair follicles by targeting RAC2. This study provides a theoretical reference for improving sheep breeding in the future and lays a foundation for further research on the effects of MSTRG.84658.1 and the target gene RAC2 on dermal papilla cells (DPC).

Climate-influenced performance and offspring development of merino sheep in a dry temperate-cold valley.

This study aims to explore the effects of climate on the performance and offspring development of aged Merino sheep relocated from an arid, cold environment with harsh grazing conditions to a dry, temperate-cold valley with irrigated pasture production. We utilized time series data from merino sheep in a dry temperate-cold climate in southern Argentina to characterize their growth curves, assess the impact of climate on performance, and compare offspring growth with maternal growth. Our approach involved developing a dynamic model, a non-autonomous differential equation growth curve based on the widely used Brody model. The model considered variables such as local temperature, age, sex, origin, and pregnancy status to determine the optimal combination of parameters for sheep growth in our dataset. The results have shown that moving the old sheep from the steppe to the valley resulted in an increase of an average of 1 kg in weight, but their offspring had an asymptotic weight of 65 kg, 17 kg more than their mothers. The optimum temperature for the growth rate was 15.7+/-0.56 C and 8.7+/-6.3C for the asymptotic weight.

The impact of retrieval method and breed on the motility and kinematic parameters of fresh and post-thaw ram epididymal spermatozoa.

This study was conducted to develop ideal post-mortem gamete retrieval and conservation methods to establish a Hungarian ex-situ in vitro gene bank. Pairs of testes from German Mutton Merino (n = 7) and Hungarian Black Racka (n = 7) rams were collected at a slaughterhouse, transported to the laboratory and stored overnight (4-5 °C) before processing. Post mortem ram epididymal spermatozoa (REPS) were obtained from the cauda epididymidis by slice or incision methods. Fresh samples were extended to 200 × 106/mL cell concentration, filled into mini straws and equilibrated at 5 °C for 2 h. Freezing was performed manually in a Styrofoam box. The fresh and post-thaw total motility, progressive motility and kinematic parameters of REPS were assessed using the CASA technique. The collection method did not affect significantly the fresh and post-thaw motility and kinematic parameters. Merino had higher (P < 0.05) testicular weight. Racka had significantly better fresh and post-thaw linear movement but had statistically the same (P > 0.05) cryotolerance as Merino. In conclusion, both collection methods were found suitable for REPS retrieval. The REPS from Racka exhibited better linear movement values than those from the Merino breed. The cryotolerance of REPS of both breeds was comparable.

A SNP in the ovine cathepsin K (CTSK) gene is associated with yearling growth performance in a crossbred sheep population.

Cathepsin K (CTSK) is a lysosomal protease existent in the skeletal muscles which is involved in biochemical processes related to obesity. Several studies have reported the effects of CTSK gene on body weight and fat deposition in human, mice and pigs. However, information about its structure and functions in sheep is very limited. Thus, this study was performed to evaluate the association between CTSK gene variants and yearling growth performance in Afshari × Booroola-Merino crossbred sheep. A fragment of 500 bp in exon 6 and partial of intron 5 of CTSK gene was amplified with polymerase chain reaction (PCR). All animals were genotyped by single-stranded conformation polymorphism (SSCP) and further confirmed by sequencing. Association analysis using a fixed linear model indicated that g.106510225G > A SNP was significantly related to average daily weight gain (ADWG) per year, fat-tail weight to carcass weight ratio (FW/CW), muscle thickness (MT) and muscle cross-sectional area (MCSA) of animals (p < 0.05). Due to the low polymorphic information content (PIC <0.25) for targeted locus in studied population, more association studies are needed to confirm the CTSK gene effects on growth traits in sheep.

Genetic diversity and substructuring of the Hungarian merino sheep breed using microsatellite markers.

The Hungarian Merino sheep breed (Ovis aries) is the most significant animal resource of the Hungarian sheep sector which, unfortunately, has gone through a huge reduction in number during the last decades and became endangered in 2014. A modern molecular genetic survey is now becoming more than necessary in order to characterize the within-breed genetic diversity and structure. For that reason, six Hungarian Merino flocks were genotyped in 16 microsatellite markers. In total, 144 different alleles were found and the mean values of observed and expected heterozygosity were 0.714 and 0.705, respectively, suggesting a noticeable genetic variability of the breed. The genetic differentiation of the Hungarian flocks was generally low, as reflected by the estimated total FST value (0.036), the extended pattern of admixture in Structure analysis, as well as, by the noticeable level of genetic clustering in UPGMA and FCA analyses. However, two out of the six studied flocks tended to be genetically more distant. The outcome of our study could be a starting point for a planned breeding strategy of the Hungarian Merino breed, by keeping the within-flock genetic variability in priority, as well as, by preserving the potential genetic uniqueness with close monitoring of the inbreeding.

Proteome Analysis of Alpine Merino Sheep Skin Reveals New Insights into the Mechanisms Involved in Regulating Wool Fiber Diameter.

Wool fiber is a textile material that is highly valued based on its diameter, which is crucial in determining its economic value. To analyze the molecular mechanisms regulating wool fiber diameter, we used a Data-independent acquisition-based quantitative proteomics approach to analyze the skin proteome of Alpine Merino sheep with four fiber diameter ranges. From three contrasts of defined groups, we identified 275, 229, and 190 differentially expressed proteins (DEPs). Further analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that pathways associated with cyclic adenosine monophosphate and peroxisome proliferator-activated receptor signaling are relevant to wool fiber diameter. Using the K-means method, we investigated the DEP expression patterns across wool diameter ranges. Using weighted gene co-expression network analysis, we identified seven key proteins (CIDEA, CRYM, MLX, TPST2, GPD1, GOPC, and CAMK2G) that may be involved in regulating wool fiber diameter. Our findings provide a theoretical foundation for identifying DEPs and pathways associated with wool fiber diameter in Alpine Merino sheep to enable a better understanding of the molecular mechanisms underlying the genetic regulation of wool fiber quality.

Bacteria associated with ovine gut parasites Trichuris ovis and Haemonchus contortus.

An associated microbiome of any host helps it in different metabolic processes ranging from the decomposition of food to the maturation of gametes. Organisms with a parasitic mode of life, though present at nutritious sites inside their host, maintain their own microbiome. Nevertheless, the comprehensive characterization and functionality of microbiome in parasitic organisms remain understudied. We selected two nematode parasites of Kashmir Merino sheep viz;Haemonchus contortus and Trichuris ovis based on their higher prevalence, difference in mode of nutrition, habitation site and effect on host. The objective of the study was to explore the bacteria associated with these parasitic nematodes of sheep. We adopted a 16S rRNA metagenomic sequencing approach to estimate and compare the bacterial communities present in these two nematode species. Nematode parasites from Kashmir Merino sheep were identified morphologically and confirmed with DNA characterization. H. contortus was dominated by phylum Proteobacteria (57%), Firmicutes (25%), Bacteroidota (15%) and Actinobacteriota (3%). Conversely, T. ovis showed Proteobacteria (78%) followed by Firmicutes (8%), Bacteroidota (8%), Actinobacteriota (1%), Fusobacteriota (1%) and other phyla (4%). This study provides a comprehensive account of the microbiome composition of H. contortus and T. ovis, both of which are highly prevalent among Kashmir Merino sheep. Additionally, T. ovis exhibited a greater bacterial diversity compared to H. contortus. Notably, these nematodes were found to harbor certain pathogenic bacteria. This study can further be carried forward in gaining insights into the complex relationship between the microbiota of a parasite and its pathogenicity, reproductive potential and host microbiome modification.

Verification of pedigree information with paternity testing in Karacabey Merino sheep.

Complex tests of DNA sequences, genetic research, and the use of specific DNA polymorphisms as genetic markers have all advanced rapidly in recent years. It is widely used in DNA-based parental testing. Therefore, it has replaced the blood types routinely used in pedigree tests. The study aimed to evaluate the 14 microsatellite loci in the study in terms of paternity test parameters and to investigate the usability of the relevant loci in determining the parentage of the Karacabey Merino breed. In this study, paternity testing was performed on 5 rams of the Karacabey Merino breed and their probable 98 offspring using 14 microsatellite loci. A total of 290 alleles were observed in the study. The observed heterozygosity ratio (Ho) based on loci was between 0.82 and 0.98, and the expected heterozygosity ratio (He) was between 0.87 and 0.93. Individual exclusion probability (PE) values for the loci were calculated to be between 0.596 and 0.761, with combined exclusion probability (CPE) values of 0.99999995. According to the results of the study, paternity tests using 14 microsatellite markers in Karacabey Merino sheep can be used with high accuracy.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

The genomic structure of isolation across breed, country and strain for important South African and Australian sheep populations.

BACKGROUND: South Africa and Australia shares multiple important sheep breeds. For some of these breeds, genomic breeding values are provided to breeders in Australia, but not yet in South Africa. Combining genomic resources could facilitate development for across country selection, but the influence of population structures could be important to the compatability of genomic data from varying origins. The genetic structure within and across breeds, countries and strains was evaluated in this study by population genomic parameters derived from SNP-marker data. Populations were first analysed by breed and country of origin and then by subpopulations of South African and Australian Merinos. RESULTS: Mean estimated relatedness according to the genomic relationship matrix varied by breed (-0.11 to 0.16) and bloodline (-0.08 to 0.06) groups and depended on co-ancestry as well as recent genetic links. Measures of divergence across bloodlines (FST: 0.04-0.12) were sometimes more distant than across some breeds (FST: 0.05-0.24), but the divergence of common breeds from their across-country equivalents was weak (FST: 0.01-0.04). According to mean relatedness, FST, PCA and Admixture, the Australian Ultrafine line was better connected to the SA Cradock Fine Wool flock than with other AUS bloodlines. Levels of linkage disequilibrium (LD) between adjacent markers was generally low, but also varied across breeds (r2: 0.14-0.22) as well as bloodlines (r2: 0.15-0.19). Patterns of LD decay was also unique to breeds, but bloodlines differed only at the absolute level. Estimates of effective population size (Ne) showed genetic diversity to be high for the majority of breeds (Ne: 128-418) but also for bloodlines (Ne: 137-369). CONCLUSIONS: This study reinforced the genetic complexity and diversity of important sheep breeds, especially the Merino breed. The results also showed that implications of isolation can be highly variable and extended beyond breed structures. However, knowledge of useful links across these population substructures allows for a fine-tuned approach in the combination of genomic resources. Isolation across country rarely proved restricting compared to other structures considered. Consequently, research into the accuracy of across-country genomic prediction is recommended.

Integrated analysis of miRNAs and mRNA profiling reveals the potential roles of miRNAs in sheep hair follicle development.

BACKGROUND: Merino sheep exhibit high wool production and excellent wool quality. The fleece of Merino sheep is predominantly composed of wool fibers grown from hair follicles (HFs). The HF is a complex biological system involved in a dynamic process governed by gene regulation, and gene expression is regulated by microRNAs (miRNAs). miRNA inhibits posttranscriptional gene expression by specifically binding to target messenger RNA (mRNA) and plays an important role in regulating gene expression, the cell cycle and biological development sequences. The purpose of this study was to examine mRNA and miRNA binding to identify key miRNAs and target genes related to HF development. This will provide new and important insights into fundamental mechanisms that regulate cellular activity and cell fate decisions within and outside of the skin. RESULTS: We analyzed miRNA data in skin tissues collected from 18 Merino sheep on four embryonic days (E65, E85, E105 and E135) and two postnatal days (D7 and D30) and identified 87 differentially expressed miRNAs (DE-miRNAs). These six stages were further divided into two longer developmental stages based on heatmap cluster analysis, and the results showed that DE-mRNAs in Stage A were closely related to HF morphogenesis. A coanalysis of Stage A DE-mRNAs and DE-miRNAs revealed that 9 DE-miRNAs and 17 DE-mRNAs presented targeting relationships in Stage A. We found that miR-23b and miR-133 could target and regulate ACVR1B and WNT10A. In dermal fibroblasts, the overexpression of miR-133 significantly reduced the mRNA and protein expression levels of ACVR1B. The overexpression of miR-23b significantly reduced the mRNA and protein expression levels of WNT10A. CONCLUSION: This study provides a new reference for understanding the molecular basis of HF development and lays a foundation for further improving sheep HF breeding. miRNAs and target genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine-wool sheep.

Gene network analysis reveals candidate genes related with the hair follicle development in sheep.

BACKGROUND: Merino sheep are the most famous fine wool sheep in the world. They have high wool production and excellent wool quality and have attracted worldwide attention. The fleece of the Merino sheep is composed predominantly of wool fibers grown from secondary wool follicles. Therefore, it is necessary to study the development of hair follicles to understand the mechanism of wool production. The hair follicle is a complex biological system involved in a dynamic process governed by gene regulation. The hair follicle development process is very complex and poorly understood. The purpose of our research is to identify candidate genes related to hair follicle development, provide a theoretical molecular breeding basis for the cultivation of fine wool sheep, and provide a reference for the problems of hair loss and alopecia areata that affect human beings. RESULTS: We analyzed mRNAs data in skin tissues of 18 Merino sheep at four embryonic days (E65, E85, E105 and E135) and two postnatal days (P7 and P30). G1 to G6 represent hair follicles developmental at six stages (i.e. E65 to P30). We identified 7879 differentially expressed genes (DEGs) and 12623 novel DEGs, revealed different expression patterns of these DEGs at six stages of hair follicle development, and demonstrated their complex interactions. DEGs with stage-specific expression were significantly enriched in epidermal differentiation and development, hair follicle development and hair follicle morphogenesis and were enriched in many pathways related to hair follicle development. The key genes (LAMA5, WNT10A, KRT25, SOSTDC1, ZDHHC21, FZD1, BMP7, LRP4, TGFß2, TMEM79, SOX10, ITGB4, KRT14, ITGA6, and GLI2) affecting hair follicle morphogenesis were identified by network analysis. CONCLUSION: This study provides a new reference for the molecular basis of hair follicle development and lays a foundation for further improving sheep hair follicle breeding. Candidate genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine wool sheep. These results are a valuable resource for biological investigations of fleece evolution in animals.

Estimates of (co)variance components and phenotypic and genetic parameters of growth traits and wool traits in Alpine Merino sheep.

The (co)variance components and corresponding phenotypic and genetic parameters for growth traits and wool traits of economic importance were estimated in the Alpine Merino sheep population maintained at Gansu Provincial Sheep Breeding Technology Extension Station in northwestern China. Data from a maximum of 49,474 animals sired by 526 rams and born from 22,531 ewes over 20 years from 2000 to 2019 were used in this study. Birth type, age of dam, birth year, sex and/or management group, and age at measurement were initially fitted as fixed effects in an animal model with various random effects. Genetic groups were defined for all animals by the sire breed and breed genotype interacted with dam-strain flocks and were fitted as one of the random effects. Analyses were conducted using a residual maximum likelihood procedure (ASReml). Seven different animal models were fitted for all traits, and the most appropriate model with relevant random effects was selected through log-likelihood ratio testing. After identifying the appropriate model through single-trait analysis, bivariate analyses were used to obtain the phenotypic and genetic correlations among the traits. The estimates of additive direct heritability for birth weight (BWT), weaning weight (WWT), preweaning growth rate (prwADG), postweaning growth rate (powADG), yearling body weight (YWT), average fibre diameter (AFD), greasy fleece weight (GFW), clean fleece weight (CFW), yield (YLD), yearling wool staple length (YSL), coefficient of variation of average fibre diameter (FDcv) and wool visual fineness counts (VFC) were 0.30, 0.18, 0.18, 0.20, 0.29, 0.20, 0.19, 0.20, 0.35, 0.19, 0.16 and 0.13, respectively, with standard errors ranging from 0.02 to 0.05. The corresponding ratios of genetic group variance to additive genetic variance were significant and, respectively, 0.35, 0.80, 0.62, 0.26, 0.13, 1.06, 0.38, 0.64, 0.09, 0.12, 0.06 and 0.58. These results suggest for these traits that there is potential to exploit both the additive genetic variation and between genetic group variation although for most traits the between group variation was smaller than the variation within groups. Favourable genetic correlations were found among the growth traits, and between growth traits and fleece production traits, and among wool traits GFW, CFW, YSL and YLD. This study provides the required estimates of genetic parameters of both growth and wool traits of the new breed for the design of more effective breeding programmes.

Factors affecting wool characteristics of sheep reared in Kashmir.

This study was conducted on 82,908 records of purebred and upgraded Kashmir Merino sheep to evaluate the performance of breed over the years. The data pertaining to fiber diameter (FD), staple length (SL), clean wool yield percent (CWY %), number of crimps/cm (NCPC), and medullation percent (MP) spread over a period of 15 years (2013-2017) was collected from Fleece Testing Laboratory Nowshera, Srinagar. The highest CV (%) was observed for MP, whereas the lowest CV (%) was observed for FD (2.07%). The least-squares means were 20.96 ± 0.002 µm, 4.05 ± 0.01 cm, 66.68 ± 0.01%, 4.38 ± 0.02 No/cm and 0.79 ± 0.05% for FD, SL, CWY (%), NCPC and MP, respectively. The year of shearing had highly significant (p < 0.01) effect on all the traits under the study. The study concludes that crossbreeding with exotic fine wool breeds has resulted improved genetic potential of native germplasm with respect to wool quality traits with Merino sheep performing better in the agro-climatic conditions of the State. Environment was also found to play a significant role in expression of wool quality traits during the period of the study.

Association analysis of polymorphisms in six keratin genes with wool traits in sheep.

OBJECTIVE: The purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals. METHODS: To explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used. RESULTS: We report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in Ile→Thr, Glu→Asp, Gly→Ala, Ala→Ser, Se→His; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p< 0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05). CONCLUSION: Our results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

The uptake of transdermal fentanyl in a pregnant sheep model.

OBJECTIVE: To evaluate the maternal and foetal uptake of transdermal fentanyl patch applied to the groin of pregnant sheep following surgery. STUDY DESIGN: Prospective series. ANIMALS: A group of 16 singleton pregnant sheep underwent anaesthesia for laparotomy, hysterotomy and instrumentation of the foetus. Of these ewes 10 (101 ± 12 days of gestation) were used to evaluate the maternal uptake of transdermal fentanyl, and the efficacy of the drug in the postoperative period (n = 10). To determine the extent of transplacental transfer of fentanyl, six ewes from the group of 10, and six other ewes (92 ± 1 days' gestation) were studied. METHODS: A 75 µg hour-1 fentanyl patch was placed onto the woolless skin of the medial thigh close to the groin at the end of surgery. Maternal blood samples were collected from the cephalic or jugular vein, and pain and sedation scores were determined, prior to application of the patch (time 0) and at 3, 6, 12, 24, 36 and 48 hours after. A commercial Fentanyl ELISA kit was used to determine the concentration of fentanyl. Paired maternal and foetal blood samples were collected 48 hours after surgery. Animals were euthanized at the end of the study. Data were tested for normality and compared with Student t test or one-way anova and are expressed as mean ± standard deviation or median (range). RESULTS: Recovery from anaesthesia and surgery was uneventful in all ewes. The dose of fentanyl was 1.4 ± 0.2 µg kg-1 hour-1. The maximum maternal plasma concentration of fentanyl was 0.547 ng mL-1 (range, 0.349-0.738 ng mL-1) at 12 hours. After 48 hours, the concentration of fentanyl was 0.381 ng mL-1 (range, 0.211-0.487 ng mL-1; maternal) and 0.295 ng mL-1 (range, 0.185-0.377 ng mL-1; foetal; p = 0.175). The placental transfer rate of fentanyl was 77%. CONCLUSIONS AND CLINICAL RELEVANCE: The uptake of fentanyl varied between animals. The placental transfer rate of fentanyl was 77%.

Genetic diversity and population structure of South African smallholder farmer sheep breeds determined using the OvineSNP50 beadchip.

A population structure study was performed in South African ovine populations using the OvineSNP50 beadchip. Blood samples were obtained from 295 sheep of which 172 had been identified as smallholder Dorpers, 4 smallholder White Dorpers, 46 purebred Dorpers, 26 purebred South African Mutton Merinos and 47 purebred Namaqua Afrikaners. Blood from the latter three breeds were obtained from a resource flock maintained on the Nortier research farm. Genetic diversity was estimated using allelic richness (A r), observed heterozygosity (H o), expected heterozygosity (H e) and inbreeding coefficient (F). Population structure analysis was performed using fastSTRUCTURE to determine the breed composition of each genotyped individual. The Namaqua Afrikaner had the lowest H e of 0.280 ± 0.18 while the H e of smallholder Dorper, Dorper and South African Mutton Merino did not differ and were 0.364 ± 0.13, 0.332 ± 0.16 and 0.329 ± 0.17, respectively. The average inbreeding coefficient was highest for the pure breeds, Namaqua Afrikaner, Dorper and South African Mutton Merino compared to the average inbreeding coefficient for the smallholder Dorper population. The smallholder Dorper were introgressed with Namaqua Afrikaner, South African Mutton Merino and White Dorpers. Similarly, the smallholder Dorper population was more genetically diverse than the purebred Dorper, South African Mutton Merino and Namaqua Afrikaner from the research farm. The higher genetic diversity among the smallholder sheep may be advantageous for their fitness and can be used to facilitate selective breeding.

Associations Between Newly Discovered Polymorphisms of the CEBPD GENE LOCUS and Body Parameters in Sheep.

An understanding of what effects particular genes can have on body parameters in productive animals is particularly significant for the process of marker-assisted selection. The gene of transcriptional factor CCAAT/enhancer-binding protein delta (CEBPD gene) is involved in the process of growth in animals and is known to be a promising candidate for use as a genomic marker. The structure of the CEBPD gene locus was determined using NimbleGen sequencing technology (Roche, USA). The effect of polymorphisms, which were identified using the aforementioned technology, was investigated in 30 rams of the Manych Merino sheep breed. Twenty-two single nucleotide polymorphisms (SNP) were detected in the CEBPD gene locus. Significantly, two SNPs, namely, g.315T>G and g.327C>T, have been identified for the first time. It was demonstrated that the complex of linked SNPs, consisting of g.301A>T, g.426T>C, and g.1226T>C, had a negligible effect on body parameters in Manych Merino sheep. Animals with the heterozygous type of SNP g.1142C>T exhibited changes solely in the chest and croup width. The newly discovered SNP g.327C>T was proven to have a negative effect on live weight and body size (p < 0.05) in Manych Merino sheep. Sheep with the heterozygous type of g.562G>A and g.3112C>G SNP complex showed an increase in live weight and dimensions (p < 0.05) compared with those of wild homozygous type. Consequently, SNPs g.327C>T, g.562G>A, and g.3112C>G in the CEBPD gene locus can be successfully used as markers in sheep breeding. Future research will evaluate the influence of the aforementioned SNPs on slaughter indicators for sheep meat production.