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Topic:Genetics

Genetics in horses encompasses the study of hereditary traits and the genetic makeup that influences various characteristics and health conditions in equine populations. This field involves the analysis of genes and their functions, inheritance patterns, and the impact of genetic variations on traits such as coat color, performance ability, and susceptibility to diseases. Research in equine genetics employs techniques such as genome mapping, sequencing, and genetic testing to identify specific genes and mutations associated with these traits. This page gathers peer-reviewed research studies and scholarly articles that explore the genetic basis of equine traits, the methodologies used in genetic research, and the implications for breeding, health management, and conservation of horse breeds.
Differential expression of microRNAs in bovine papillomavirus type 1 transformed equine cells.
Veterinary and comparative oncology    April 4, 2016   Volume 15, Issue 3 764-774 doi: 10.1111/vco.12216
Terron-Canedo N, Weir W, Nicolson L, Britton C, Nasir L.Bovine papillomavirus (BPV) types 1 and 2 play an important role in the pathogenesis of equine sarcoids (ES), the most common cutaneous tumour affecting horses. MicroRNAs (miRNAs), small non-coding RNAs that regulate essential biological and cellular processes, have been found dysregulated in a wide range of tumours. The aim of this study was to identify miRNAs associated with ES. Differential expression of miRNAs was assessed in control equine fibroblasts (EqPalFs) and EqPalFs transformed with the BPV-1 genome (S6-2 cells). Using a commercially available miRNA microarray, 492 mature miRNAs we...
Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.
Theriogenology    April 4, 2016   Volume 86, Issue 4 1081-1091 doi: 10.1016/j.theriogenology.2016.03.040
Gambini A, De Stéfano A, Jarazo J, Buemo C, Karlanian F, Salamone DF.The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as en...
Claudin-1, -2, -4, and -5: comparison of expression levels and distribution in equine tissues.
Journal of veterinary science    April 1, 2016   Volume 17, Issue 4 445-451 doi: 10.4142/jvs.2016.17.4.445
Lee B, Kang HY, Lee DO, Ahn C, Jeung EB.Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenl...
Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.
Reproduction in domestic animals = Zuchthygiene    April 1, 2016   Volume 51, Issue 3 378-385 doi: 10.1111/rda.12689
Orsztynowicz M, Pawlak P, Podstawski Z, Nizanski W, Partyka A, Gotowiecka M, Kosiniak-Kamysz K, Lechniak D.Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four ...
New test for endothelin receptor type B (EDNRB) mutation genotyping in horses.
Molecular and cellular probes    March 30, 2016   Volume 30, Issue 3 182-184 doi: 10.1016/j.mcp.2016.03.005
Ayala-Valdovinos MA, Galindo-García J, Sánchez-Chiprés D, Duifhuis-Rivera T.Lethal white foal syndrome (LWFS) is an autosomal recessive disease of neonatal foals characterized by a white hair coat and a functional intestinal obstruction. Traditional techniques for identifying the dinucleotide mutation (TC→AG) of the endothelin receptor B gene (EDNRB) associated with LWFS are time-consuming. We developed a new technique based on mutagenically separated polymerase chain reaction (MS-PCR) for simple detection of the EDNRB genotype in horses.
Fertility Assessment in Sorraia Stallions by Sperm-Fish and Fkbp6 Genotyping.
Reproduction in domestic animals = Zuchthygiene    March 28, 2016   Volume 51, Issue 3 351-359 doi: 10.1111/rda.12686
Kjöllerström HJ, do Mar Oom M, Chowdhary BP, Raudsepp T.The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm-FISH and molecular analysis of FKBP6 - a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 6...
Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.
Veterinary microbiology    March 26, 2016   Volume 188 16-24 doi: 10.1016/j.vetmic.2016.03.019
Gressler LT, Bordin AI, McQueen CM, Cohen ND, de Vargas AC.Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-d...
Genome Editing in Large Animals.
Journal of equine veterinary science    March 25, 2016   Volume 41 1-6 doi: 10.1016/j.jevs.2016.03.008
West J, Gill WW.Genome editing in large animals has tremendous practical applications, from more accurate models for medical research through improved animal welfare and production efficiency. Although genetic modification in large animals has a 30 year history, until recently technical issues limited its utility. The original methods - pronuclear injection and integrating viruses - were plagued with problems associated with low efficiency, silencing, poor regulation of gene expression, and variability associated with random integration. With the advent of site specific nucleases such as TALEN and CRISPR/Cas9...
Bayesian Inference of Natural Selection from Allele Frequency Time Series.
Genetics    March 23, 2016   Volume 203, Issue 1 493-511 doi: 10.1534/genetics.116.187278
Schraiber JG, Evans SN, Slatkin M.The advent of accessible ancient DNA technology now allows the direct ascertainment of allele frequencies in ancestral populations, thereby enabling the use of allele frequency time series to detect and estimate natural selection. Such direct observations of allele frequency dynamics are expected to be more powerful than inferences made using patterns of linked neutral variation obtained from modern individuals. We developed a Bayesian method to make use of allele frequency time series data and infer the parameters of general diploid selection, along with allele age, in nonequilibrium populati...
Heritability of semen traits in German Warmblood stallions.
Animal reproduction science    March 21, 2016   Volume 170 10-14 doi: 10.1016/j.anireprosci.2016.03.004
Gottschalk M, Sieme H, Martinsson G, Distl O.The objectives of the present study were to evaluate genetic parameters for semen quality traits of 241 fertile German Warmblood stallions regularly employed in artificial insemination (AI). Stallions were owned by the National Studs Celle and Warendorf in Germany. Semen traits analyzed were gel-free volume, sperm concentration, total number of sperm, progressive motility and total number of progressively motile sperm. Semen protocols from a total of 63,972 ejaculates were collected between the years 2001 and 2014 for the present analysis. A multivariate linear animal model was employed for es...
Inability of FMDV replication in equine kidney epithelial cells is independent of integrin αvβ3 and αvβ6.
Virology    March 21, 2016   Volume 492 251-258 doi: 10.1016/j.virol.2016.01.025
Wang Y, Mao Q, Chang H, Wu Y, Pan S, Li Y, Zhang Y.Integrins can function as receptors for foot-and-mouth disease virus (FMDV) in epithelium. Horses are believed to be insusceptible to this disease, but the mechanism of resistance remains unclear. To detect whether FMDV can use integrin to attach to equine epithelial, we compared the utilities of αvβ3 and αvβ6 between bovine and equine kidney epithelial cells (KECs). Equine KECs showed almost equal efficiency to those of bovine. Further, the integrin αv, β3, and β6 subunits from bovine and equine were cloned and vectors were transfected into SW480 cells and COS-1 cells alone or together...
Prevalence, risk factors and genetic parameters of cresty neck in Pura Raza Español horses.
Equine veterinary journal    March 19, 2016   Volume 49, Issue 2 196-200 doi: 10.1111/evj.12569
Sánchez MJ, Azor PJ, Molina A, Parkin T, Rivero JL, Valera M.Cresty neck is a relatively common morphological defect in Pura Raza Español horses and other Baroque type horse breeds, which adversely affects the breeding industry. Objective: To establish the within-breed prevalence, possible associated factors and heritability of cresty neck in Pura Raza Español horses. Methods: Cross-sectional analysis of a large worldwide database of Pura Raza Español horses. Methods: The database included evaluations of 10,929 Pura Raza Español horses from 24 countries. Cresty neck score, on a 9 point scale, girth-to-height ratio, height at the withers, length of n...
Characterisation of the epidemic strain of H3N8 equine influenza virus responsible for outbreaks in South America in 2012.
Virology journal    March 19, 2016   Volume 13 45 doi: 10.1186/s12985-016-0503-9
Alves Beuttemmüller E, Woodward A, Rash A, Dos Santos Ferraz LE, Fernandes Alfieri A, Alfieri AA, Elton D.An extensive outbreak of equine influenza occurred across multiple countries in South America during 2012. The epidemic was first reported in Chile then spread to Brazil, Uruguay and Argentina, where both vaccinated and unvaccinated animals were affected. In Brazil, infections were widespread within 3months of the first reported cases. Affected horses included animals vaccinated with outdated vaccine antigens, but also with the OIE-recommended Florida clade 1 strain South Africa/4/03. Methods: Equine influenza virus strains from infected horses were isolated in eggs, then a representative stra...
Simulating forensic casework scenarios in experimental studies: The generation of footwear marks in blood.
Forensic science international    March 18, 2016   Volume 264 34-40 doi: 10.1016/j.forsciint.2016.03.023
McElhone RL, Meakin GE, French JC, Alexander T, Morgan RM.A study was designed to investigate the effects of external variables, including blood type, flooring surface, footwear tread depth and blood dryness, on the appearance of blood-based footwear marks, with particular reference to simulating a specific casework scenario. Results showed that footwear marks left in human blood tended to be of greater quality than those in equine blood, highlighting a potential issue in applying data generated with equine blood to human bloodstains in casework. Footwear tread effects were also dependent on blood type, but the type of flooring surface did not affect...
Genetic variation between and within Triodontophorus brevicauda and Triodontophorus nipponicus revealed by analyses of mtDNA and rDNA gene sequences.
Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis    March 17, 2016   Volume 28, Issue 4 570-574 doi: 10.3109/24701394.2016.1157867
Duan H, Gao JF, Su X, Fu X, Yue DM, Gao Y, Qiu JH, Wang CR.Triodontophorus spp. parasitizes the large intestine of equine, causing strongylid diseases. The present study assessed genetic variation in five gene regions within and between Triodontophorus brevicauda and Triodontophorus nipponicus from Heilongjiang Province and the Inner Mongolia Autonomous region. The five gene markers were three mitochondrial (mt) genes, cytochrome c oxidase subunit I (cox1), NADH dehydrogenase subunit 5 (nad5), cytochrome b (cytb); and two ribosomal RNA genes, the internal transcribed spacer 1 (ITS1) and the internal transcribed spacer 2 (ITS2). Partial (p) sequences o...
Quantitative analysis of short- and long-distance racing performance in young and adult horses and association analysis with functional candidate genes in Spanish Trotter horses.
Journal of animal breeding and genetics = Zeitschrift fur Tierzuchtung und Zuchtungsbiologie    March 16, 2016   Volume 133, Issue 5 347-356 doi: 10.1111/jbg.12208
Negro Rama S, Valera M, Membrillo A, Gómez MD, Solé M, Menendez-Buxadera A, Anaya G, Molina A.The association of five candidate genes with sporting performance in young and adult Spanish Trotter horses (STHs) was performed according to a previous selection based on quantitative analysis of the trait time per kilometre (TPK). A total of 334 516 records of TPK from 5958 STHs were used to estimate the estimated breeding values (EBVs) at different age groups (young and adults horses) throughout the range of distances (1600-2700 m) using a bicharacter random regression model. The heritability estimated by distance ranged from 0.16 to 0.40, with a different range for the two age groups. Co...
The concentration of iodine in horse serum and its relationship with thyroxin concentration by geological difference.
Environmental monitoring and assessment    March 14, 2016   Volume 188, Issue 4 226 doi: 10.1007/s10661-016-5221-7
Mochizuki M, Hayakawa N, Minowa F, Saito A, Ishioka K, Ueda F, Okubo K, Tazaki H.In this study, iodine and thyroxin (T4) concentrations in the serum of 69 horses were investigated. Higher iodine concentrations were obtained from the horses housed in Chiba Prefecture. In contrast, T4 concentrations of horses at Shizuoka Prefecture were higher than those of horses at Chiba Prefecture. There was a significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations of horses at Saitama and Shizuoka prefectures. Although a significant correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefec...
Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease.
BMC microbiology    March 12, 2016   Volume 16 41 doi: 10.1186/s12866-016-0653-3
Roy Chowdhury P, DeMaere M, Chapman T, Worden P, Charles IG, Darling AE, Djordjevic SP.Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfe...
Enterocytozoon bieneusi Genotypes in Grazing Horses in China and their Zoonotic Transmission Potential.
The Journal of eukaryotic microbiology    March 8, 2016   Volume 63, Issue 5 591-597 doi: 10.1111/jeu.12308
Qi M, Wang R, Wang H, Jian F, Li J, Zhao J, Dong H, Zhu H, Ning C, Zhang L.In present study, 262 fecal specimens were collected from 12 groups of grazing horses in the Xinjiang Uyghur Autonomous Region of China. The specimens were subjected to PCR and sequencing analyses of the ribosomal internal transcribed spacer (ITS). The overall prevalence of E. bieneusi in horses was 30.9% (81/262). No significant differences in prevalence were observed between horses of different ages or sexes. Nineteen genotypes were identified: 15 known genotypes (BEB6, CHG19, CM6, CM7, CM8, CS-1, CS-4, D, EpbA, EbpC, G, horse1, horse2, O, and Peru8) and four new genotypes (XJH1-XJH4). Six ...
Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.
Veterinary parasitology    March 6, 2016   Volume 221 24-29 doi: 10.1016/j.vetpar.2016.03.003
Liu Q, Meli ML, Zhang Y, Meili T, Stirn M, Riond B, Weibel B, Hofmann-Lehmann R.A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genus-specific probe. Of these, two hybridized also to the Theileria equi-specific probe. The oth...
Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma.
Journal of photochemistry and photobiology. B, Biology    March 3, 2016   Volume 158 89-98 doi: 10.1016/j.jphotobiol.2016.02.025
Kumar CS, Sivaramakrishna D, Ravi SK, Swamy MJ.Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
Correction: Focal Experimental Injury Leads to Widespread Gene Expression and Histologic Changes in Equine Flexor Tendons.
PloS one    March 1, 2016   Volume 11, Issue 3 e0150823 doi: 10.1371/journal.pone.0150823
Jacobson E, Dart AJ, Mondori T, Horadogoda N, Jeffcott LB, Little CB, Smith MM.No abstract available
Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).
Animal genetics    March 1, 2016   Volume 47, Issue 3 334-344 doi: 10.1111/age.12426
Ghosh S, Das PJ, McQueen CM, Gerber V, Swiderski CE, Lavoie JP, Chowdhary BP, Raudsepp T.We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive as...
Skeletal variation in Tennessee Walking Horses maps to the LCORL/NCAPG gene region.
Physiological genomics    March 1, 2016   Volume 48, Issue 5 325-335 doi: 10.1152/physiolgenomics.00100.2015
Staiger EA, Al Abri MA, Pflug KM, Kalla SE, Ainsworth DM, Miller D, Raudsepp T, Sutter NB, Brooks SA.Conformation has long been a driving force in horse selection and breed creation as a predictor for performance. The Tennessee Walking Horse (TWH) ranges in size from 1.5 to 1.7 m and is often used as a trail, show, and pleasure horse. To investigate the contribution of genetics to body conformation in the TWH, we collected DNA samples, body measurements, and gait/training information from 282 individuals. We analyzed the 32 body measures with a principal component analysis. Principal component (PC)1 captured 28.5% of the trait variance, while PC2 comprised just 9.5% and PC3 6.4% of trait vari...
Cathepsin K inhibition renders equine bone marrow nucleated cells hypo-responsive to LPS and unmethylated CpG stimulation in vitro.
Comparative immunology, microbiology and infectious diseases    February 27, 2016   Volume 45 40-47 doi: 10.1016/j.cimid.2016.02.005
Hussein H, Boyaka P, Dulin J, Bertone A.Cathepsin K (CatK) is an important enzyme regulating bone degradation and has been shown to contribute to the immune response. We have studied two inflammatory models in equine bone marrow nucleated cells (BMNCs); the LPS and the unmethylated CpG stimulation with the following objectives to: 1.determine whether CatK inhibition will alter the cytokine secretion by stimulated BMNCs; specifically IL-1β, IL-6, and TNF-α, and 2.determine the changes in BMNCs surface markers' expression and MHC II molecule under CatK inhibition. Cathepsin K inhibition promoted BMNCs viability and reduced cell apop...
Demographics of Australian horses: results from an internet-based survey.
Australian veterinary journal    February 26, 2016   Volume 94, Issue 3 52-59 doi: 10.1111/avj.12411
Smyth GB, Dagley K.To obtain information on the types of Australian horses, how they are kept and their activities. Methods: An invitation to participate in an opt-in, internet-based survey was sent to 7000 people who had registered an email address to receive information from the Australian Horse Industry Council Inc. Results: There were 3377 (48%) useable responses from owners of 26,548 horses. Most horses were kept on small properties (usually 2-8 ha) in paddocks in rural areas of Queensland, New South Wales and Victoria. Most horses were female or geldings and the most common of 54 different activities was...
Spinal Cord Hamartomatous Myelodysplasia in 2 Horses With Clinical Neurologic Deficits.
Veterinary pathology    February 25, 2016   Volume 53, Issue 4 844-846 doi: 10.1177/0300985815622971
Taylor KR, MacKay RJ, Nelson EA, Stieler AL, Roberts JF, Castleman WL.Two horses euthanized for neurologic deficits were diagnosed with hamartomatous myelodysplasia of the spinal cord. One was a 5-week-old Holsteiner colt exhibiting spasms of muscle rigidity in the extensor muscles of the limbs and epaxial muscles, and the other was a 3-year-old Thoroughbred colt exhibiting progressive ataxia and hypermetria in the pelvic limbs. Each had focal disorganization of the white and gray matter of the spinal cord forming a mass interspersed with neurons, glial cells, and disoriented axon bundles. In the Holsteiner colt, the mass was at the level of C5 and included isla...
Micromanipulation of equine blastocysts to allow vitrification.
Reproduction, fertility, and development    February 25, 2016   doi: 10.1071/RD15389
Hinrichs K, Choi YH.Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embr...
Fourier transform infrared spectroscopic analysis of sperm chromatin structure and DNA stability.
Andrology    February 24, 2016   Volume 4, Issue 3 430-441 doi: 10.1111/andr.12166
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Evidence for a PGF2α auto-amplification system in the endometrium in mares.
Reproduction (Cambridge, England)    February 23, 2016   Volume 151, Issue 5 517-526 doi: 10.1530/REP-15-0617
Kozai K, Tokuyama S, Szóstek AZ, Toishi Y, Tsunoda N, Taya K, Sakatani M, Takahashi M, Nambo Y, Skarzynski DJ, Yamamoto Y, Kimura K, Okuda K.In mares, prostaglandin F2α (PGF2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In t...
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