Investigating age-related differences in muscles of Kazakh horse through transcriptome analysis.

This study conducted transcriptome sequencing on the skeletal muscles of three different anatomical locations across various growth stages to investigate the impact of ages on crucial candidate genes and molecular mechanisms associated with muscle development in Kazakh horses. Sixteen Kazakh horses were selected, and they were divided into four age groups, each with four biological replicates. Tissue samples from the longest dorsal muscle, abdominal muscle, and diaphragm muscle were collected for analysis. The results revealed differential mRNA expression in the longest dorsal muscle between the eight-month group (Group O) and the 10-year group (Group F), with 434 up-regulated and 322 down-regulated genes. In the abdominal muscle, there were 125 up-regulated and 127 down-regulated genes, while in the diaphragm muscle, there were 73 up-regulated and 70 down-regulated genes. In this study, GO enrichment analysis focused on biological processes. KEGG pathway analysis highlighted the Oxidative Phosphorylation pathway for the longest dorsal muscle, annotating 37 differentially expressed genes (DEGs), including ATP5PF, NDUFB8, and ATP5MG, all of which were down-regulated. For the abdominal muscle, the ECM-receptor interaction pathway was enriched, annotating 7 DEGs such as COL4A2, COL4A1, and ITGA5. In the diaphragm muscle, the Hippo signaling pathway was enriched, annotating 6 DEGs, including SERPINE1, RASSF1, and FZD10. This study provides robust data support and a theoretical foundation for a comprehensive understanding of the influence of age on skeletal muscle development in horses.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Effects of steam-flaked grains on foals' growth and faecal microbiota.

BACKGROUND: There is little objective information concerning the effect of steam-flaked grains on foal's growth performance and faecal microbiota. To determine the effects of steam-flaked grains on foal's growth performance and faecal microbiota, faecal samples were collection from 18 foals which had been fed either corn, oat or barley diets over the 60 days of the experiment. Body weight and conformation measurements were collected. Next-generation sequencing of the V3 + V4 region of the 16 S rRNA gene was used to assess the microbial composition of faeces. Alpha diversity, Venn graph, Relative abundance and beta diversity are presented. RESULTS: There was a significantly higher larger increase in the body weight of those foals fed barley compared to either corn or oats. There were also significant changes in the Alpha diversity of the gut microbiota. The Shannon and Simpson indices were significantly higher in the barley fed group than those fed corn or oats. The Chao1 index was significantly higher in the oat fed group than the corn or barley fed groups. There were significant changes in the relative abundance of bacteria in the microbiota in terms of phylum, family and genus. The histogram of LDA value distribution showed that the 12 statistically different biomarkers of the bacteria were present. Tax4Fun function annotation clustering heat map showed that functional information was detected from 26 species of bacteria in faecal samples from the foals. CONCLUSIONS: Differences by starch sources were found in overall growth of the foals and in the faecal microbiota if either supplementary corn, oat or barley was fed. Further studies are required to determine the potential impact of the changes in the microbiota on the health and development of foals fed cereal starch of different sources.

Research article expression of surfactant protein-A and D, and CD9 in lungs of 1 and 30 day old foals.

BACKGROUND: Respiratory diseases are a major cause of morbidity and mortality in the horses of all ages including foals. There is limited understanding of the expression of immune molecules such as tetraspanins and surfactant proteins (SP) and the regulation of the immune responses in the lungs of the foals. Therefore, the expression of CD9, SP-A and SP-D in foal lungs was examined. RESULTS: Lungs from one day old (n = 6) and 30 days old (n = 5) foals were examined for the expression of CD9, SP-A, and SP-D with immunohistology and Western blots. Western blot data showed significant increase in the amount of CD9 protein (p = 0.0397) but not of SP-A and SP-D at 30 days of age compared to one day. Immunohistology detected CD9 in the alveolar septa and vascular endothelium but not the bronchiolar epithelium in the lungs of the foals in both age groups. SP-A and SP-D expression was localized throughout the alveolar septa including type II alveolar epithelial cells and the vascular endothelium of the lungs in all the foals. Compared to one day old foals, the expression of SP-A and SP-D appeared to be increased in the bronchiolar epithelium of 30 day old foals. Pulmonary intravascular macrophages were also positive for SP-A and SP-D in 30 days old foals and these cells are not developed in the day old foals. CONCLUSIONS: This is the first data on the expression of CD9, SP-A and SP-D in the lungs of foals.

SNPs analysis of height traits in Ningqiang pony.

Ningqiang pony is one of the five pony breeds in China and is listed as a rare species. It is, therefore, meaningful to conduct stature research on this breed for its efficient reproduction, utilization and protection. This study is based on four SNP variants of adjacent LCORL/NCAPG, HMGA2, ZFAT and LASP1 genes, which have been found to be associated with horse height, and then, the 4 SNP loci in 22 Ningqiang ponies were analyzed. The results showed that there were genetic variations at the four loci in Ningqiang pony, there was a SNP on LCORL/NCAPG, HMGA2 and ZFAT gene all including T allele and C allele and LASP1 including A allele and G allele. Further data analysis revealed that LCORL/NCAPG variant harbor the allele T and LASP1 variant harbor the allele G were significantly related to body height (p < 0.05). These findings suggested that the two SNP mutations within the LCORL/NCAPG and LASP1 genes could be considered as a screening marker for the body height of Ningqiang pony and could provide a new strategy for breeding and protect the rare germplasm resources.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

Concentração de minerais no tecido queratinizado podal de equinos jovens da raça Crioula em diferentes fases de crescimento / [Mineral concentrations in the keratinized tissue on the feet of young Crioulo horses at different stages of growth]

O presente estudo avaliou o perfil mineral de Cálcio (Ca), Cobre (Cu) e Zinco (Zn) no estojo córneo pré e pós-desmame e sua associação com pigmentação, gênero e idade de potros da raça Crioula criados em pastagens nativas no Rio Grande do Sul. Foram coletadas amostras do casco de potros da raça Crioula 41 dias pré e 28 dias pós-desmame. Os teores de Ca, Cu e Zn variaram no casco fetal (571,0±39,4; 14,5±7,6 e 130,0±30,5mg/kg, respectivamente; P<0,05) e no definitivo (653,0±169,2; 33,8±11,5 e 69,3±36,8mg/kg, respectivamente; P<0,05), no pré (620,0±184; 17,2±21,2 e 103,0±75,5mg/kg, respectivamente; P<0,05) e no pós-desmame (517,5±181; 0 e 79,0±41,7mg/kg, respectivamente; P<0,05). Houve associação positiva (P<0,05) com a faixa etária no pré-desmame para Ca (r=0,5) e Cu (r=0,57), e negativa para Zn (r=-0,69; P<0,05). No pós-desmame, houve associação positiva (P<0,05) para Ca (r=0,36) e Zn (r=0,64) e negativa para Cu (r=-0,39; P<0,05). Tanto a pigmentação quanto o gênero não afetaram o perfil mineral. Conclui-se que há diferenças nas concentrações de Ca, Cu e Zn na epiderme do casco de potros da raça Crioula no pré e pós-desmame, independentemente da pigmentação e do gênero, onde as concentrações de Ca e Cu aumentam com a idade, enquanto as de Zn diminuem.(AU)

The aim of this study was to evaluate the mineral profile of Ca, Cu and Zn in the hoof horny capsule at pre and post-weaning and its association with pigmentation, gender and age range of Crioulo foals raised in native pastures in RS. Samples were collected from the epidermis of the Crioulo foal's hoof at two times, 41 pre and 28 post-weaning. The contents of Ca, Cu, Zn Varied in the fetal hoof ( 571.0±139.4, 14.5±7.6 and 130.0±30.5mg/kg, respectively, P<0.05) and definitive (653.0±169.2, 33.8±11.5 and 69.3±36.8mg/kg, respectively, P<0.05), in the pre (620.0±184, 17.2±21.2 and103.0±75.5mg/kg, respectively, P<0.05) and post-weaning (517.5±181, 0.1 and 79.0±41.7mg/kg, respectively, P<0.05).There was a positive association (P<0.05) with a preweaning age for Ca (r=0.5) and Cu (r=0.57) and negative for Zn (r=-0.69, P<0.05). In the post weaning there was a positive association (P<0.05) for Ca (r=0.36) and Zn (r=0.64) and negative for Cu (r=-0.39; P<0.05). Neither pigmentation nor gender affected the mineral profile. It is concluded that there are differences in Ca, Cu, Zn concentrations in the epidermis of foals of the crioulo breed at the pre and post weaning, regardless of pigmentation and sex, where Ca and Cu concentrations increase with age, while Zn concentrations decrease.(AU)

Aspectos epidemiológicos e clínico-patológicos de um surto de miopatia nutricional em potros ˗ relato de caso / [Epidemiological and clinical-pathological aspects of an outbreak of nutritional myopathy in young horses ˗ case report]

A miopatia nutricional é uma doença degenerativa que pode afetar os músculos esqueléticos e cardíaco, causada pela deficiência dietética de selênio e/ou vitamina E. Objetivou-se relatar a ocorrência de miopatia nutricional em dois potros Puro Sangue Inglês, criados de forma extensiva, com baixa disponibilidade de forragem e sem suplementação mineral. De um lote de nove animais de diferentes idades (cinco éguas adultas, uma potra lactente de três meses,uma mula e dois potros de 16 e 17 meses), apenas os dois últimos foram afetados. Clinicamente, havia decúbito lateral ou esternal, taquicardia, taquipneia, desidratação e dor à palpação muscular na região dos glúteos.O diagnóstico presuntivo de miopatia nutricional foi realizado por meio da associação dos sinais clínicos aos dados epidemiológicos.O animal 1 foi eutanasiado cinco dias após o atendimento devido à piora gradativa do quadro, e o animal 2 recuperou-se com o tratamento adotado. O diagnóstico foi confirmado com base nos exames complementares, nos achados de necropsia e na dosagem da concentração hepática de selênio. Este relato de caso alerta para o risco da ocorrência de miopatia nutricional em equinos, sobretudo jovens, sem acesso a pastagens ou feno de boa qualidade e sem suplementação mineral adequada.(AU)

Nutritional myopathy is a degenerative disease caused by the dietary deficiency of selenium and/or vitamin E that can affect skeletal and cardiac muscles. The objective of this study was to report the occurrence of nutritional myopathy in two Thoroughbred young horses, raised in pastures with low forage availability and no mineral supplementation. From a herd of nine animals of different ages (five adult mares, one three-month-old nursing foal, one mule and two 16- and 17-month-old horses), only the last two were affected. Clinical signs were lateral or sternal recumbency, tachycardia, tachypnea, dehydration and muscle pain. Presumptive diagnosis of nutritional myopathy was made associating clinical signs and epidemiological data. Animal 1 was euthanized five days after the first evaluation due to clinical signs gets worse, and animal 2 recovered with the established treatment. Diagnosis was confirmed with complementary exams, necropsy findings and levels of hepatic selenium. This paper alerts to the risk of nutritional myopathy occurrence in horses, mainly young animals, which are not ingesting good quality hay or green forage and do not receive appropriate mineral supplementation.(AU)

Endothelial cells and angiogenesis in the horse in health and disease-A review.

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.

Analysis of the miRNA transcriptome during testicular development and spermatogenesis of the Mongolian horse.

Numerous studies have shown that microRNAs (miRNAs) are essential for testicular development and spermatogenesis. In order to further characterise these physiological processes, three immature and three mature testes of the Mongolian horse were collected and six libraries were established. Using small RNA sequencing technology, 531 mature miRNAs were identified, including 46 novel miRNAs without previously ascribed functions. Among the 531 miRNAs, 421 were expressed in both immature and mature libraries, 65 miRNAs were found solely in immature testis libraries and 45 miRNAs were found solely in mature testis libraries. Furthermore, among the miRNAs that were identified in both immature and mature libraries, 107 were significantly differentially expressed (corrected P value (padj)<0.05). Among the miRNAs that were only expressed in immature testes, two miRNAs were differentially expressed, whereas among the miRNAs that were only expressed in mature testes, nine miRNAs were differentially expressed. Comprehensive analysis of miRNA and mRNA expression profiles predicted 107 miRNA-mRNA interaction sites. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted target genes suggested roles of the differentially expressed miRNAs in testicular development and spermatogenesis. These findings identify miRNAs as key factors in the development of the testes and spermatogenesis in the Mongolian horse, which may also help us to understand the mechanisms of fertility in related mammalian species.

Genomic Divergence in Swedish Warmblood Horses Selected for Equestrian Disciplines.

The equestrian sport horse Swedish Warmblood (SWB) originates from versatile cavalry horses. Most modern SWB breeders have specialized their breeding either towards show jumping or dressage disciplines. The aim of this study was to explore the genomic structure of SWB horses to evaluate the presence of genomic subpopulations, and to search for signatures of selection in subgroups of SWB with high or low breeding values (EBVs) for show jumping. We analyzed high density genotype information from 380 SWB horses born in the period 2010-2011, and used Principal Coordinates Analysis and Discriminant Analysis of Principal Components to detect population stratification. Fixation index and Cross Population Extended Haplotype Homozygosity scores were used to scan the genome for potential signatures of selection. In accordance with current breeding practice, this study highlights the development of two separate breed subpopulations with putative signatures of selection in eleven chromosomes. These regions involve genes with known function in, e.g., mentality, endogenous reward system, development of connective tissues and muscles, motor control, body growth and development. This study shows genetic divergence, due to specialization towards different disciplines in SWB horses. This latter evidence can be of interest for SWB and other horse studbooks encountering specialized breeding.

Development of the equine gut microbiota.

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

Physics of animal health: on the mechano-biology of hoof growth and form.

Global inequalities in economic access and agriculture productivity imply that a large number of developing countries rely on working equids for transport/agriculture/mining. Therefore, the understanding of hoof conditions/shape variations affecting equids' ability to work is still a persistent concern. To bridge this gap, using a multi-scale interdisciplinary approach, we provide a bio-physical model predicting the shape of equids' hooves as a function of physical and biological parameters. In particular, we show (i) where the hoof growth stress originates from, (ii) why the hoof growth rate is one order of magnitude higher than the proliferation rate of epithelial cells and (iii) how the soft-to-hard transformation of the epithelium is possible allowing the hoof to fulfil its function as a weight-bearing element. Finally (iv), we demonstrate that the reason for hoof misshaping is linked to the asymmetrical design of equids' feet (shorter quarters/long toe) together with the inability of the biological growth stress to compensate for such an asymmetry. Consequently, the hoof can adopt a dorsal curvature and become 'dished' overtime, which is a function of the animal's mass and the hoof growth rate. This approach allows us to discuss the potential occurrence of this multifaceted pathology in equids.

Genome-Wide Association Studies Based on Equine Joint Angle Measurements Reveal New QTL Affecting the Conformation of Horses.

The evaluation of conformation traits is an important part of selection for breeding stallions and mares. Some of these judged conformation traits involve joint angles that are associated with performance, health, and longevity. To improve our understanding of the genetic background of joint angles in horses, we have objectively measured the angles of the poll, elbow, carpal, fetlock (front and hind), hip, stifle, and hock joints based on one photograph of each of the 300 Franches-Montagnes (FM) and 224 Lipizzan (LIP) horses. After quality control, genome-wide association studies (GWASs) for these traits were performed on 495 horses, using 374,070 genome-wide single nucleotide polymorphisms (SNPs) in a mixed-effect model. We identified two significant quantitative trait loci (QTL) for the poll angle on ECA28 (p = 1.36 × 10-7), 50 kb downstream of the ALX1 gene, involved in cranial morphology, and for the elbow joint on ECA29 (p = 1.69 × 10-7), 49 kb downstream of the RSU1 gene, and 75 kb upstream of the PTER gene. Both genes are associated with bone mineral density in humans. Furthermore, we identified other suggestive QTL associated with the stifle joint on ECA8 (p = 3.10 × 10-7); the poll on ECA1 (p = 6.83 × 10-7); the fetlock joint of the hind limb on ECA27 (p = 5.42 × 10-7); and the carpal joint angle on ECA3 (p = 6.24 × 10-7), ECA4 (p = 6.07 × 10-7), and ECA7 (p = 8.83 × 10-7). The application of angular measurements in genetic studies may increase our understanding of the underlying genetic effects of important traits in equine breeding.

Identification of piRNAs and piRNA clusters in the testes of the Mongolian horse.

P-element induced wimpy testis-interacting RNAs (piRNAs) are essential for testicular development and spermatogenesis in mammals. Comparative analyses of the molecular mechanisms of spermatogenesis among different organisms are therefore dependent on accurate characterizations of piRNAs. At present, little is known of piRNAs in non-model organisms. Here, we characterize piRNAs in the Mongolian horse, a hardy breed that reproduces under extreme circumstances. A thorough understanding of spermatogenesis and reproduction in this breed may provide insights for the improvement of fecundity and reproductive success in other breeds. We identified 4,936,717 piRNAs and 7,890 piRNA clusters across both testicular developmental stages. Of these, 2,236,377 putative piRNAs were expressed in the mature samples only, and 2,391,271 putative piRNAs were expressed in the immature samples only. Approximately 3,016 piRNA clusters were upregulated in the mature testes as compared to the immature testes, and 4,874 piRNA clusters were downregulated. Functional and pathway analyses indicated that the candidate generating genes of the predicted piRNAs were likely involved in testicular development and spermatogenesis. Our results thus provide information about differential expression patterns in genes associated with testicular development and spermatogenesis in a non-model animal.

Extracellular vesicles in synovial fluid from juvenile horses: No age-related changes in the quantitative profile.

Extracellular vesicle (EV) concentration, characteristics and function in equine synovial fluid (SF) during normal growth and development has not previously been studied. Isolation of EVs was performed in SF from three healthy foals and two adult horses by differential ultracentrifugation (10,000g and 200,000g); EVs were purified by sucrose density gradient floatation and analysed by high-resolution flow cytometry (FCM), buoyant density and western blotting. Additionally, repeated biomarker analysis of sulphated glycosaminoglycans (GAG), matrix metalloproteinase (MMP), C-terminal crosslinked telopeptide type II collagen (CTX-II), collagenase cleaved neopeptide type II collagen (C2C) was performed in SF from 10 foals and six adult horses. In contrast with the quantitative EV profile, the biomarker profile in SF from juvenile joints was substantially different from that in SF from adult animals. However, there were qualitative differences in the high-resolution FCM scatter plots. Future in-depth functional analyses may reveal differences between juvenile and mature EVs in SF.

Differences in third metacarpal trabecular microarchitecture between the parasagittal groove and condyle at birth and in adult racehorses.

BACKGROUND: The aetiology of equine metacarpal condylar fractures is not completely understood and a developmental cause has been postulated. OBJECTIVES: To investigate the subchondral bone trabecular microarchitecture of the lateral parasagittal groove and condyle in equine neonates and its adaptation with maturation and athletic activity. STUDY DESIGN: Ex vivo observational study. METHODS: Distal metacarpi of neonates, yearlings and adult racehorses (n = 24) were harvested. Dorsal and palmar frontal histological sections, containing the lateral parasagittal groove and condyle, were studied. The sections were digitalised and subchondral trabecular bone quantity and quality parameters and trabecular orientation in the frontal plane were measured. RESULTS: Trabecular spacing and length were greater (P = 0.004 and P = 0.0005 respectively) whereas bone fraction, trabecular number and connectivity were all lower (P = 0.0004, P = 0.0001 and P = 0.001 respectively) in the lateral parasagittal groove compared with the condyle in neonatal foals. Trabecular thickness and bone fraction increased with age in racehorses and trabecular spacing decreased. The predominant trabecular orientation had a consistent pattern in neonates and it changed with maturity and the cumulative effect of racing at all the ROIs except for the palmar lateral parasagittal groove that retained a more 'immature' pattern. MAIN LIMITATIONS: Samples were investigated in 2D. 3D processing could have provided more information. CONCLUSIONS: Already at birth there are striking differences in the subchondral bone trabecular microarchitecture between the lateral parasagittal groove and condyle in foals. Adaptation of trabeculae is confirmed with maturity in racehorses, with the greatest adaptation measured in bone quantity parameters. The trabecular orientation had a unique and more immature orientation pattern in the lateral palmar parasagittal grooves in adult racehorses and may reflect a weaker structure at this site.

Sequence and functional variability of Toll-like receptor 9 gene in equines.

Significant structural differences in the extracellular domain of toll-like receptor 9 (TLR9) account for species-specific recognition of its ligand CpG-ODN sequences. TLR9 is extensively studied in human, mice and some domestic animals. The recognition ability appears to be utilized differently by various species and breeds, but so far no comprehensive study exists about the equine TLR9 gene. We characterized TLR9 sequences of Marwari and Zanskari breeds of horses and Poitu donkey. We sequenced and identified the protein coding regions of equine TLR9 and compared with other animals and human beings. Furthermore, we also analyzed the amino acid substitutions and their likely implications on functions. The analysis revealed 14% evolutionary divergence between equine and human TLR9, while it was 1% between the Equus caballus and Equus asinus and less than 1% within Equus caballus. In phylogenetic analysis of predicted amino acids, the indigenous equines grouped with thoroughbred Equus caballus, while human, cattle, dog, sheep, mice, and buffalo formed separate clades. Furthermore, we also analyzed the amino acid substitutions and their likely implications on functions by sorting intolerant from tolerant (SIFT) analysis and predicted two substitutions of amino acids (D80N and S822P) in Marwari horses in leucine rich repeat 1 (LRR1) without any functional effects. The substitutions (V214A and Y579C) in LRR 3 and LRR11 in Zanskari horses were predicted to have functional consequences. Out of overall 8 substitutions, three substitutions (I420V, S970R and R1001C) were found in Equus asinus in LRR7, LRR 13, and toll interleukin receptor (TIR) domains, while the substitution G649S is observed in Poitu donkey only. We report for the first time that despite the conserved residues, the striking effect of substitutions, found within the TLR9 genes of different equine breeds/species may have possible implications.

Zinc silicate mineral-coated scaffold improved in vitro osteogenic differentiation of equine adipose-derived mesenchymal stem cells.

In current study we aimed to coat the PLLA scaffold with zinc (Zn) silicate mineral nanoparticles. Then, using equine adipose-derived stem cells (ASCs) we intended to compare the osteogenic induction potency of Zn silicate mineral-coated PLLA scaffold with uncoated PLLA scaffold and tissue culture plastic (TCPS). Adipose tissues were collected from 3 horses, and isolation of ASCs was achieved by enzymatic digestion. PLLA scaffold was successfully prepared using a phase separation method and coated with Zn silicate mineral nanoparticles. The coating efficiency was then characterized by scanning electron microscopy and further evaluated with the application of fourier transform infrared microscopic imaging. Viability and growth characteristics of ASCs on TCPS, uncoated and coated PLAA scaffolds were investigated by MTT assay. Alizarin Red staining was performed for determination of calcium deposition following the osteogenic induction. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content, as well as osteogenic (Runx2, ALP, osteonectin, and collagen I) marker genes were also evaluated. Our data showed that Zn silicate mineral nanoparticles was coated successfully on PLLA scaffold and such scaffold had no detrimental effect on cell growth rate as indicated by MTT assay. Moreover, ASCs that differentiated on Zn silicate mineral-coated PLLA scaffold indicated higher ALP activity, more calcium content, and higher expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. Adequate proliferation rate and higher expression of osteogenic markers of stem cells, provides this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.

A regression model including fetal orbit measurements to predict parturition in Standardbred mares with normal pregnancy.

In the mare, foaling is a critical unpredictable event due to a wide range of gestational length and the absence of clear signs of impending parturition. To predict foaling, pH, inversion sodium potassium and increase of calcium concentration in mammary secretions are used. The aim of this study was to find how many days are left until parturition knowing mare's age (A) and parity (P) combined with ultrasonographic measurements of the fetal orbit in Standardbred mares with normal pregnancy. Eighty healthy Standardbred mares with normal pregnancy were hospitalized for attended delivery. Information about mare's age, parity and breeding date were recorded at admission. Transrectal ultrasonography were routinely performed at admission and every 10 days until parturition using a B-mode real time portable unit equipped with a 5-7.5 MHz linear transducer. The images of the fetal orbit were acquired when cornea, anterior and posterior chamber, vitreous body, lens and optic nerve were visible. Longitudinal diameter (LD) was considered as the distance between the two ocular poles, within the vitreous body; transverse diameter (TD), perpendicular to LD and bisecting the lens, was measured as the distance between cornea and retina. At delivery, length of pregnancy and gestational age at each exam were registered. For each ultrasound examination, days before parturition (DBP) were calculated. Seventy-eight Standardbred mares with normal pregnancies were included in the study. Mares' mean age was 9 ±â€¯5 years old (range 4-20 years) and mean gestation length was 341 ±â€¯7 days (range 327-366 days). Thirty-three mares were primiparous and 45 mares were multiparous. Data were analyzed using a regression tree: P, A, LD and TD were used as covariates. DBP was used as the variable of interest. Nine terminal nodes were identified based on the selected covariates. The first split is produced by the TD: fetuses with TD greater or equal than 2.97 cm are further split according to LD, with a threshold of 3.28 cm. The next split is dictated by A; after a further split on LD, the first terminal node is built, containing 34 fetuses with average DBP equal to 10 days. If the A is ≥ 9.5 years a further split is on P: when mares are multiparous, the TD built two different nodes. Since prediction of mare's foaling date is an important factor in stud farm management, the regression model developed may help the veterinarian to estimate the DBP in Standardbred mares with normal pregnancy.