Genome-wide SNP analysis clearly distinguished the Belarusian Red cattle from other European cattle breeds.

Local breeds can serve as an important source of genetic variability in domestic animal species. This study aimed to assess the genetic diversity and population structure of Belarusian Red cattle and their differentiation from other European cattle populations based on genome-wide SNP genotypes. Twenty pedigree-recorded non-closely related cows of Belarusian Red cattle were genotyped using the Illumina BovineHD BeadChip. Genotypes of 22 other European cattle breeds were included in the study for comparison. A total of 28 562 SNPs passed through the quality control checks and were selected for analysis. The Belarusian Red cattle displayed a moderate level of genetic variability (U HE  = 0.341, HO  = 0.368), and the highest heterozygote excess (U FIS  = -0.066), among the studied breeds; this reflects the contribution of multiple breeds to their formation. The principal component analysis, FST -based Neighbor-Net tree and Admixture clustering, clearly distinguished the Belarusian Red cattle from the other European cattle breeds. Moreover, the presence of ancestral genomic components of Danish Red and Brown Swiss breeds were clearly visible, which agrees with the breed's history and its recent development. Our study highlights the importance of maintaining the specific genomic components, which makes a significant contribution to the global genetic diversity in the modern population of Belarusian Red cattle, allowing us to consider them a valuable national genetic resource. Our research results will be useful for the development of conservation programs for this local cattle breed.

Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle.

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.

Analysis of ROH patterns in the Noriker horse breed reveals signatures of selection for coat color and body size.

Overlapping runs of homozygosity (ROH islands) shared by the majority of a population are hypothesized to be the result of selection around a target locus. In this study we investigated the impact of selection for coat color within the Noriker horse on autozygosity and ROH patterns. We analyzed overlapping homozygous regions (ROH islands) for gene content in fragments shared by more than 50% of horses. Long-term assortative mating of chestnut horses and the small effective population size of leopard spotted and tobiano horses resulted in higher mean genome-wide ROH coverage (SROH ) within the range of 237.4-284.2 Mb, whereas for bay, black and roan horses, where rotation mating is commonly applied, lower autozygosity (SROH from 176.4-180.0 Mb) was determined. We identified seven common ROH islands considering all Noriker horses from our dataset. Specific islands were documented for chestnut, leopard spotted, roan and bay horses. The ROH islands contained, among others, genes associated with body size (ZFAT, LASP1 and LCORL/NCAPG), coat color (MC1R in chestnut and the factor PATN1 in leopard spotted horses) and morphogenesis (HOXB cluster in all color strains except leopard spotted horses). This study demonstrates that within a closed population sharing the same founders and ancestors, selection on a single phenotypic trait, in this case coat color, can result in genetic fragmentation affecting levels of autozygosity and distribution of ROH islands and enclosed gene content.

Novel insights into Sabino1 and splashed white coat color patterns in horses.

Within the framework of genome-wide analyses using the novel Axiom® genotyping array, we investigated the distribution of two previously described coat color patterns, namely sabino1 (SBI), associated with the KIT gene (KI16+1037A), and splashed white, associated with the PAX3 gene (ECA6:g.11429753C>T; PAX3C70Y ), including a total of 899 horses originating from eight different breeds (Achal Theke, Purebred Arabian, Partbred Arabian, Anglo-Arabian, Shagya Arabian, Haflinger, Lipizzan and Noriker). Based on the data we collected we were able to demonstrate that, besides Quarter horses, the PAX3C70Y allele is also present in Noriker (seven out of 189) and Lipizzan (three out of 329) horses. The SB1 allele was present in three breeds (Haflinger, 14 out of 98; Noriker, four out of 189; Lipizzan one out of 329). Furthermore, we examined the phenotypes of SB1- and PAX3C70Y -carrier horses for their characteristic white spotting patterns. None of the SB1/sb1-carrier horses met the criteria defining the Sabino1 pattern according to current applied protocols. From 10 heterozygous PAX3C70Y -carrier horses, two had nearly a splashed white phenotype. The results of this large-scale experiment on the genetic association of white spotting patterns in horses underline the influence of gene interactions and population differences on complex traits such as Sabino1 and splashed white.

Asian horses deepen the MSY phylogeny.

Humans have shaped the population history of the horse ever since domestication about 5500 years ago. Comparative analyses of the Y chromosome can illuminate the paternal origin of modern horse breeds. This may also reveal different breeding strategies that led to the formation of extant breeds. Recently, a horse Y-chromosomal phylogeny of modern horses based on 1.46 Mb of the male-specific Y (MSY) was generated. We extended this dataset with 52 samples from five European, two American and seven Asian breeds. As in the previous study, almost all modern European horses fall into a crown group, connected via a few autochthonous Northern European lineages to the outgroup, the Przewalski's Horse. In total, we now distinguish 42 MSY haplotypes determined by 158 variants within domestic horses. Asian horses show much higher diversity than previously found in European breeds. The Asian breeds also introduce a deep split to the phylogeny, preliminarily dated to 5527 ± 872 years. We conclude that the deep splitting Asian Y haplotypes are remnants of a far more diverse ancient horse population, whose haplotypes were lost in other lineages.

Association between population structure and allele frequencies of the glycogen synthase 1 mutation in the Austrian Noriker draft horse.

The aim of this study was to determine the allele frequency of the glycogen synthase 1 (GYS1) mutation associated with polysaccharide storage myopathy type 1 in the Austrian Noriker horse. Furthermore, we examined the influence of population substructures on the allele distribution. The study was based upon a comprehensive population sample (208 breeding stallions and 309 mares) and a complete cohort of unselected offspring from the year 2014 (1553 foals). The mean proportion of GYS1 carrier animals in the foal cohort was 33%, ranging from 15% to 50% according to population substructures based on coat colours. In 517 mature breeding horses the mutation carrier frequency reached 34%, ranging on a wider scale from 4% to 62% within genetic substructures. We could show that the occurrence of the mutated GYS1 allele is influenced by coat colour; genetic bottlenecks; and assortative, rotating and random mating strategies. Highest GYS1 carrier frequencies were observed in the chestnut sample comprising 50% in foals, 54% in mares and 62% in breeding stallions. The mean inbreeding of homozygous carrier animals reached 4.10%, whereas non-carrier horses were characterized by an inbreeding coefficient of 3.48%. Lowest GYS1 carrier frequencies were observed in the leopard spotted Noriker subpopulation. Here the mean carrier frequency reached 15% in foals, 17% in mares and 4% in stallions and inbreeding decreased from 3.28% in homozygous non-carrier horses to 2.70% in heterozygous horses and 0.94% in homozygous carriers. This study illustrates that lineage breeding and specified mating strategies result in genetic substructures, which affect the frequencies of the GYS1 gene mutation.

[Comparative Analysis of the Effectiveness of STR and SNP Markers for Intraspecific and Interspecific Differentiation of the Genus Ovis].

A comparative study of the informativeness of SNP and STR markers for interspecific and intraspecific differentiation of the two species of the genus Ovis, snow sheep (O. nivicola) and domestic sheep (O. aries), was conducted. Eleven STR loci combined into two multiplex panels were examined. SNP analysis was performed with the DNA microarray OvineSNP50K BeadChip featuring 54241 SNPs. The possibility of clear differentiation of the studied Ovis species with both types of genetic markers was demonstrated. The advantages of SNP markers for intraspecific differentiation of the O. aries breeds and O. nivicola geographical groups were revealed. The areas of application of the studied types of DNA markers are discussed.

Whole genome SNP scanning of snow sheep (Ovis nivicola).

This is the first report performing the whole genome SNP scanning of snow sheep (Ovis nivicola). Samples of snow sheep (n = 18) collected in six different regions of the Republic of Sakha (Yakutia) from 64° to 71° N. For SNP genotyping, we applied Ovine 50K SNP BeadChip (Illumina, United States), designed for domestic sheep. The total number of genotyped SNPs (call rate 90%) was 47796 (88.1% of total SNPs), wherein 1006 SNPs were polymorphic (2.1%). Principal component analysis (PCA) showed the clear differentiation within the species O. nivicola: studied individuals were distributed among five distinct arrays corresponding to the geographical locations of sampling points. Our results demonstrate that the DNA chip designed for domestic sheep can be successfully used to study the allele pool and the genetic structure of snow sheep populations.

Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning.

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

New genomic insights into the conformation of Lipizzan horses.

Conformation traits are important selection criteria in equine breeding, as they describe the exterior aspects of the horse (height, joint angles, shape). However, the genetic architecture of conformation is not well understood, as data of these traits mainly consist of subjective evaluation scores. Here, we performed genome-wide association studies on two-dimensional shape data of Lipizzan horses. Based on this data, we identified significant quantitative trait loci (QTL) associated with cresty neck on equine chromosome (ECA)16 within the MAGI1 gene, and with type, hereby differentiating heavy from light horses on ECA5 within the POU2F1 gene. Both genes were previously described to affect growth, muscling and fatty deposits in sheep, cattle and pigs. Furthermore, we pin-pointed another suggestive QTL on ECA21, near the PTGER4 gene, associated with human ankylosing spondylitis, for shape differences in the back and pelvis (roach back vs sway back). Further differences in the shape of the back and abdomen were suggestively associated with the RYR1 gene, involved in core muscle weakness in humans. Therefore, we demonstrated that horse shape space data enhance the genomic investigations of horse conformation.

Role of the oviduct in early embryo development.

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

Assessment of actin cytoskeleton and nuclei in bovine blastocysts developed under different culture conditions using a novel computer program.

This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro-to-in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir-sourced ovaries were matured in vitro and allocated to four groups: IVP-group embryos developed up to blastocyst stage in vitro. Gamete intra-fallopian transfer (GIFT)-group oocytes were co-incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2-7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)-group embryos were obtained by superovulating and inseminating heifers; the heifers' genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2-7 transfer did not differ significantly from each other. Gamete intra-fallopian transfer- and MOET-group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment.

The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.

Interleukin 6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover, IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B cells, T cells, keratinocytes, neuronal cells, osteoclasts, and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific IL-6 receptor (IL-6R) and a signal transducing subunit (gp130). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as acquired immune deficiency syndrome, rheumatoid arthritis, and others. The function of the soluble (s)IL-6R in vivo is unknown. Since human (h)IL-6 acts on human and murine target cells, but murine IL-6 on murine cells only, we constructed transgenic mice expressing the hsIL-6R. We report here that in the presence of hsIL-6R, mice are hypersensitized towards hIL-6, mounting an acute phase protein gene induction at significantly lower IL-6 dosages compared to control animals. Furthermore, in hsIL-6R transgenic mice, the detected acute phase response persists for a longer period of time. The IL-6/IL-6R complex prolongs markedly the Il-6 plasma half-life. Our results reinforce the role of the hsIL-6R as an agonistic protein, help to understand the function of the hsIL-6R in vivo, and highlight the significance of the receptor in the induction of the acute phase response.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility.

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

The effect of long-term in vivo culture in bovine oviduct and uterus on the development and cryo-tolerance of in vitro produced bovine embryos.

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short- vs long-term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP-Group), or after IVF and 2-3 days of IVC, 4-8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo-Group) or IVM oocytes were co-incubated with spermatozoa for 3-4 h and transferred into the oviducts of synchronized heifers (GIFT-Group). Embryos of the In Vivo-Group and the GIFT-Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa-medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP-Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo-survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo-tolerance. However, the duration of in vivo culture crucially influenced the cryo-tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.

Review: Environmental impact on early embryonic development in the bovine species.

Assisted reproduction techniques (ARTs) provide access to early stage embryos whose analysis and assessment deliver valuable information. The handling of embryos, including the in vitro production of bovine embryos, is a rapidly evolving area which nonetheless exposes the embryos to unnatural conditions for a period of time. The Fallopian tube provides innumerable quantitative and qualitative factors, all of which guarantee the successful development of the embryo. It is well known that the Fallopian tube can be bypassed, using embryo transfer, resulting in successful implantation in the target recipient animal and the birth of calves. However, the question arises as to whether such circumvention has a negative impact on the embryo during this sensitive development period. First crosstalk between the embryo and its environment confirms mutual recognition activities and indicate bilateral effects. Nowadays, in vitro production of bovine embryos is a well-established technology. However, it is still evident that in vitro generated embryos are not qualitatively comparable to embryos obtained ex vivo. To counteract these differences, comparative studies between in vitro and ex vivo embryos are advantageous, as embryos grown in their physiological environment can provide a blueprint or gold standard against which to compare embryos produced in vitro. Attempts to harness the bovine oviduct were sometimes very invasive and did not result in wide acceptance and routine use. Long-term development and refinement of transvaginal endoscopy for accessing the bovine oviduct has meanwhile been routinely applied for research as well as in practice. Comparative studies combining in vitro development with development in the cattle oviduct revealed that the environmental conditions to which the embryo is exposed before activation of the embryonic genome can have detrimental and lasting effects on its further development. These effects are manifested as deviations in gene expression profiles and methylation signatures as well as frequency of whole chromosomal or segmental aberrations. Furthermore, it was shown that hormonal superstimulation (multiple ovulation and embryo transfer), varying progesterone concentrations as well as metabolic disorders caused by high milk production, markedly affected embryo development in the postpartum period. Assisted reproductive techniques that allow the production and handling of extra numbers of generated embryos promise to have a very high impact on scientific and practical application. Any influence on the early embryonic life, both in animals and in vitro, is accompanied by a sensitive change in embryonic activity and should be assessed in vivo on the basis of physiological conditions before being used for ART.

Development of a 25-plex SNP assay for traceability in cattle.

Single nucleotide polymorphisms (SNPs) are amenable to automation and therefore have become the marker of choice for DNA profiling. SNaPshot, a primer extension-based method, was used to multiplex 25 SNPs that have been previously validated as useful for identity control. Detection of extended products was based on four different fluorochromes and extension primers with oligonucleotide tails of differing lengths, thus controlling the concise length of the entire chromatogram to 81 bases. Allele frequencies for Holstein, Simmental, Limousin, Angus, Charolais and Tux Cattle were estimated and significant positive Pearson-correlation coefficients were obtained among the analysed breeds. The probability that two randomly unrelated individuals would share identical genotypes for all 25 loci varied from 10(-8) to 10(-10) for these breeds. For parentage control, the exclusion power was found to be 99.9% when the genotypes of both putative parents are known. A traceability test of duplicated samples indicated a high genotyping precision of >0.998. This was further corroborated by analysis of 60 cases of parent-sib pairs and trio families. The 25-plex SNaPshot assay is adapted for low- and high-throughput capacity and thus presents an alternative for DNA-based traceability in the major commercial cattle breeds.

Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation.

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.

Endoscopic recovery of early preimplantation bovine embryos: effect of hormonal stimulation, embryo kinetics and repeated collection.

The collection of extra numbers of bovine embryos by superstimulation of donors underlies variation concerning yield of morulae and blastocysts. Our study aimed at establishing a correlation between hormonal treatment and embryo development during oviductal passage including repeated flushing. A transvaginal endoscopic procedure was used to flush the oviducts at six different time intervals (beginning at 24 h until 105 h) after artificial insemination. In total, 119 animals were superovulated using either FSH or eCG. The hormonal treatment resulted in the stimulation of 2076 follicles of which 77% (1590 CL) ovulated. The bilateral flushing resulted in the collection of 1411 complexes (collection rate: 89%), of which 78% (1098) were assessed as viable embryos. The use of FSH resulted in significantly more stimulated follicles and ovulation sites compared with eCG (p < 0.001). Generally, the embryo kinetics were similar among the FSH and eCG treated animals. However, the embryo cleavage of the eCG treated animals was ahead of that of the FSH group comparing the different collection time points. The overall proportions of non-viable embryos in both groups were similar. Regarding the embryo collection intervals in the eCG group, this proportion significantly increased during 51-105 h compared to 24-50 h (p < 0.05), whereas FSH delivered constant results. It was shown that the repeated endoscopic collection of oviductal stage embryos had no negative influence on the collection parameters. It is concluded that the introduced transvaginal endoscopic technique could have main impact on further studies focusing on early embryo development.