Mastomys natalensis, the Natal multimammate mouse, were Mopeia virus, whereas Merino Walk virus was characterized as a novel virus in a separate study. The remaining 4 isolates from 3 rodent species potentially constitute novel viruses pending full characterization.We conducted a survey for group-specific indirect immunofluorescence antibody to mammarenaviruses by using Lassa fever and Mopeia virus antigens on serum specimens of 5,363 rodents of 33 species collected in South Africa and Zimbabwe during 1964–1994. Rodents were collected for unrelated purposes or for this study and stored at −70°C. We found antibody to be widely distributed in the 2 countries; antibody was detected in serum specimens of 1.2%–31.8% of 14 species of myomorph rodents, whereas 19 mammarenavirus isolates were obtained from serum specimens and viscera of 4 seropositive species. Phylogenetic analysis on the basis of partial nucleoprotein sequences indicates that 14 isolates from  In response to the emergence of Lassa fever, Marburg virus, and Ebola virus in Africa, a Biosafety Level 4 laboratory was constructed at the National Institute for Communicable Diseases (NICD) in Johannesburg, South Africa, and became operational in 1980 (https://www.thermofisher.com) for nonmyomorph species. Polyclonal control antiserum was prepared by intraperitoneal inoculation of mice with live virus and exsanguination 6 weeks later. We screened serum specimens at dilutions of 1:8 and 1:16, titrated positive samples to endpoint, and confirmed the result by ELISA with MOPV antigen.We prepared antigen slides to screen for group-specific antibody activity to mammarenaviruses by indirect immunofluorescence (IF) with Mopeia virus (MOPV) AN20410 and Lassa virus (LASV) Josiah  grown inhttps://www.seracare.com). In the absence of control data, we recorded reactions as positive where the net optical density of test serum specimens at 1:100 was >2.5 times the mean optical density of a panel of serum specimens from specific pathogen-free laboratory mice. Monoclonal antibodies to LASV and MOPV were obtained from the US Centers for Disease Control and Prevention  or prepared at NICD as described elsewhere for Crimean-Congo hemorrhagic fever virus (Cell lysate antigen for the ELISA was prepared and assays conducted as described previously for Ebola virus (er virus  (6).Mastomys natalensis mice) were collected in northeastern South Africa during 1984–1994 specifically for the investigation of mammarenaviruses, 764 rodent samples collected in Zimbabwe in 1974 were remnants of a study on Rift Valley fever virus  and examined after incubation for 7–10 days at 37°C by IF with pooled mouse antiserum to MOPV and LASV. We passed samples 3 times before recording them as negative. The 5 original isolates of MOPV from We performed phylogenetic analysis on 48 isolates by using an ≈912-nt (299–304-aa) fragment of the nucleocapsid protein (NP) gene, consisting of 15 isolates from this study, 5 MOPV isolates from Mozambique . We omithttps://www.roche.com) and performed reverse transcription PCR with primers 19C (5′-CGCACAGTGGATCCTAGGC-3′) (2 (5′-TTCTTCATAAGGGTTCCTTTCACC-3′)  by using the Titan One Tube reverse transcription PCR kit (Roche) to amplify an ≈1,000-bp fragment of the NP gene. The 19C primer is complementary to a conserved sequence at the 3′ terminus of the S RNA segment and the OWA2 primer corresponds to nucleotide positions 2402–2424 relative to LASV 391 (GenBank accession no. X52400). We designed a degenerate reverse primer, Arena A (5′-ATRTARGGCCAWCCSTCTCC-3′), corresponding to nucleotide positions 2357–2376 relative to LASV 391 and 2401–2420 relative to MOPV AN20410 (GenBank accession no. NC006575), to amplify the region of interest for isolates SPU94/88/235 and SPU86/485. Cycling conditions were 50°C for 30 min, 94°C for 2 min, 30 cycles of 94°C for 30 s, 47°C for 30 s, and 68°C for 90 s, plus extension at 68°C for 7 min. We purified PCR products with the Wizard SV Gel and PCR clean-up kit , sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific), purified on Centrisep columns , and ran testing on an ABI Prism 377 DNA Sequencing Unit. We aligned nucleotide and predicted amino acid sequences using ClustalW  incorporated in MEGA7 (https://www.megasoftware.net), performed phylogenetic analysis by the neighbor-joining method with 1,000 bootstrap iterations, and calculated sequence diversities (p-distances)  of IF antibody to mammarenaviruses; no clear tendency to cluster was noted, except that the reactions were detected in locations where antibody was prevalent in other species. Although IF titers were generally low in these species (geometric mean titers [GMT] 19.3–57.9), the few >16 tended to be supported by ELISA reactions, but samples cultured yielded no virus. Anomalous high IF titers of 8,192 supported by ELISA reactions were recorded in 2 serum samples from R. norvegicus rats collected from a location where antibody prevalence of 29.1% was recorded in serum specimens from O. irroratus rats, but no isolates were obtained.No mammarenavirus antibody or virus was found in 14 of the myomorph rodent species , and altA. chrysophilus, M. namaquensis, M. natalensis, O. unisulcatus, and O. irroratus—had mammarenavirus IF antibody prevalence of 4.2%–31.8%; positive reactions tended to cluster and reached 30%–50% prevalence at some trapping sites. The IF titers ranged from 8–16,384 (GMT 99.1–219.5), and titers >16 were supported by positive ELISA reactions. A total of 19 mammarenavirus isolates were obtained from 4 of these species  healthcare workers from 66 secondary hospitals that receive patient referrals from district hospitals who were tested for evidence of nosocomial infection, plus a study of 2,041 long-term (>5 years) rural residents and workers in the livestock and wildlife industries who were investigated for evidence of exposure to zoonotic viruses . An overall prevalence of 1.0%  of IF antibody to MOPV antigen was recorded at titers of 8–2,048, gmt 33.0; higher prevalences of 10%–15% occurred in a few widely separated locations near the eastern border, but no histories of disease considered indicative of mammarenavirus infection were obtained. To the west, in Free State, Northern Cape, North West, and Gauteng Provinces, no antibody to mammarenaviruses was detected in rural residents and workers in the livestock industry despite the isolation of mammarenaviruses from rodents. Aliquots of human serum samples collected during the original investigations in Mopeia village, Mozambique, in 1972 remained available at NICD, and IF antibody to MOPV antigen was detected at a prevalence of 16.1% (32/199) with titers ranging from 8 to 8,192, gmt 229.6, similar to the findings initially recorded when no disease associations were identified .Unpublished serosurveys conducted on humans in South Africa during 1984–1988 in parallel with the rodent survey included a study of 7,665 long-term .Among routine diagnostic samples submitted to NICD, resting IF titers of 128 and 256 of IgG to MOPV antigen were detected in 2 patients from South Africa, but no etiologic significance could be attached to these findings. A single case of fatal LASV infection was diagnosed in a patient from Nigeria who was evacuated to a hospital in South Africa in 2007 . The only other human arenavirus infections diagnosed within South Africa were in 2 patients referred successively from Zambia in 2008 who were infected with the novel Lujo virus and 3 local healthcare workers who acquired nosocomial infection from those patients (M. natalensis mouse of sub-Saharan Africa consists of 6 matrilineages that fall into 2 clades, AI-III and BIV-VI, on the basis of the mitochondrial cytochrome b marker (>1 mammarenavirus, ranging from LASV in lineage AI in West Africa to MOPV and Luna virus in lineage BVI in southern Africa (M. natalensis mice in southern Africa, where this rodent is sympatric with M. coucha mice in northeastern South Africa and in Zimbabwe. However, the distribution of M. coucha mice extends westwards into the drier interior of South Africa; the low prevalence of MOPV antibody found in this species could represent spillover of infection from other rodents, rather than the harboring of a mammarenavirus (M. natalensis mice in the mesic east are peridomestic, indigenous rodents tend to be sylvatic and less closely associated with human dwellings in the xeric west, where no evidence of infection was detected in humans.The widely distributed enavirus . WhereasM. natalensis mice represent exemplar isolates of MOPV, and Merino Walk virus is clearly distinct. Although the apparent sharing of rodent hosts mitigates against species recognition within the mammarenaviruses (The isolates from this study are provisionally named for their locations of origin , but theO. unisulcatus rat, of the Omdraaivlei isolates. However, O. unisulcatus rats reportedly comprise a coastal lowland group that is located where the host of Merino Walk virus was collected and a central interior group that covers the area where the hosts of the Omdraaivlei isolates were obtained, although the low sequence divergences did not warrant recognition of subspecies (M. natalensis mice from a farm near Masvingo was not related to the nonfatal illness of a former farm resident who was hospitalized in South Africa.The phylogenetic relationships between 8 rodents from which mammarenaviruses were isolated in this study and reference taxonomic voucher sequences from GenBank are compatible with the concept of cospeciation of arenaviruses and their rodent hosts , except O. irroratus rats and possibly Lemniscomys rosalia mice, which were underrepresented in this survey; the presence of Luna-related and Lunk-related viruses that were identified in Zambia in M. natalensis and M. minutoides rodents should also be investigated (Further research on mammarenaviruses in rodents in South Africa should include attempts to isolate virus from


Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish ( Until recently, knowledge of invertebrate viruses was limited mostly to viral pathogens causing high mortalities and arboviruses (arthropod-borne viruses), which are vectored by arthropods and cause disease in humans and other vertebrate species . This viFor understanding the diversity of invertebrate viruses, arthropods, including crustaceans, may be of particular importance, as they account for over 80% of the total animal diversity , are gloAphanomyces astaci, while other disease agents, including fungi, bacteria, and viruses, are significantly understudied [Crayfish, belonging to a diverse order of decapod crustaceans, are a keystone species of freshwater ecosystems, and their populations can reach high densities. They are an important component of freshwater food webs and ecosystem engineers due to their larger size and longer life span compared to other benthic macroinvertebrates, as well as due to their bioturbation and burrowing activity . Crayfisrstudied .Cherax quadricarinatus [Phenuiviridae family that was associated with a massive disease outbreak in the population of the endangered white-clawed crayfish Austropotamobius pallipes [Most of the knowledge on crustacean pathogenic viruses comes from the study of viral diseases in aquaculture . For insarinatus , althougarinatus . Despitearinatus , and relarinatus . Howeverarinatus . For inspallipes .Pacifastacus leniusculus, , one of the most successful freshwater invaders in Europe. The signal crayfish is present in over 29 EU countries [Pontastacus leptodactylus, Eschscholtz, 1823) [The aim of our study was to analyze the virome of the signal crayfish, ountries , reachesountries , and is ountries ,28,29, aountries . Recentlz, 1823) . Host poz, 1823) ,31.In this study, we sampled the signal crayfish individuals at four locations along its invasion range, including upstream and downstream invasion cores and invasion fronts. We sequenced and analyzed signal crayfish samples to: (1) identify putative viral sequences in the signal crayfish hepatopancreas, as this is the tissue most often associated with viral infections in freshwater crayfish ; (2) desPantastacus leptodactylus, Eschscholtz, 1823) are present, with 2.5–12.9 higher abundances of the native P. leptodactylus compared to the signal crayfish [P. leptodactylus [Research was carried out in the continental part of Croatia, in the Korana River. Here, the signal crayfish was illegally introduced in the lower section of the river, and is spreading both upstream and downstream , with itcrayfish . Populatdactylus ,29, as dCrayfish were collected in the period of increased crayfish activity (early autumn 2018) using baited LiNi traps . A totalThe total RNA was extracted from hepatopancreas tissue using the RNeasy Lipid Tissue Mini Kit . Before RNA extraction, equal amounts of tissue (30 mg) of each of the 25 individuals collected at the same location were pooled together, resulting in four composite pools corresponding to four sampling locations. After homogenizing tissue in liquid nitrogen, RNA extraction, followed by on-column DNase digestion step , was performed as recommended by the manufacturer. The eluted RNA from all four samples was sent for ribosomal RNA depletion (Illumina Ribo-Zero Plus rRNA Depletion Kit), sequencing library preparation (NEBNext Ultra RNA Library Prep Kit), and shotgun sequencing  to CD Genomics, USA.Sequencing datasets were quality checked, filtered, and analyzed as described below. The overview of the bioinformatic analyses performed is schematized in To identify longer virus-like sequences, reads were used for de novo assembly using SPAdes  , and theNudiviridae family) and possible endogenized virus-like sequences , as well as viral sequences belonging to bacteriophages , plant infecting viruses , and sequences identified as laboratory contamination  were excluded from further analysis. The final selection of virus-like contigs included only sequences of potential invertebrate viruses longer than 300 nts. The differences between the abundance of reads corresponding to selected virus-like contigs detected in individual samples from different locations were identified by mapping the reads to virus-like contig sequences in CLC Genomics Workbench 20 , and the percentage of mapped reads was calculated  and following cycling conditions: 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s; followed by 5 min of final extension at 72 °C. Amplicons were detected after electrophoresis in 1% agarose gel stained with SYBR Safe DNA Gel Stain .Based on the presence of RNA-dependent RNA polymerase (RdRp) domain, contig length, and average read coverage, we selected four viral contigs for further analysis; virus-like contig 4 , virus-like contig 139 , virus-like contig 1 , and virus-like contig 141 . To confirm their presence in the samples, primer pairs  amplifyiFor selected virus-like contigs 4, 139, 1, and 141, the most similar homologous sequences with corresponding RdRp sequences were retrieved from the NCBI using blastp search against NCBI nr database (May 2021), and used in the phylogenetic analysis. The RdRp protein sequences of recognized viral species belonging to reo-like, hepe-like, picorna-like and toti-like virus groups selected according to previous studies , and aliTo compare the possible differences in the diversities of populations of selected viruses detected at all sampling locations, we determined the nucleotide diversities of populations of three signal crayfish-associated viruses, which were present at all sampling locations with an average read coverage higher than 40. For these analyses, read data was additionally filtered. First, reads with low nucleotide quality (<20) were discarded using a FASTQ Quality Filter (FASTX-toolkit). Reads from individual samples were then mapped to selected viral contigs . Before mapping, the read datasets were subsampled to amount for the same read coverage of individual contig sequence in different samples . Single-nucleotide polymorphism (SNP) calling was performed using a Low Frequency Variant Detection tool in the CLC Genomics Workbench 20 . SNP tables were used as an input for SNPGenie software , which wTo identify viral sequences, we performed sequencing of ribosomal RNA depleted total RNA resulting in high numbers (100–164 M) of reads per sample . First, Sequences belonging to potential invertebrate viruses exhibited relatively low levels of similarity to known viral sequences, with sequence identities ranging from 25% to 84% according to blastx results. Many virus-like contigs representing novel, highly divergent RNA viral sequences were most similar to reo-like, hepe-like, toti-like, picorna-like, tombus-like, chu-like, and partiti-like viruses. A small fraction of viral reads and the dominance of previously unknown RNA viruses in aquatic arthropod hosts samples is in line with previous studies ,5,45. MaSome viral contig sequences  discovered in this study likely correspond to full-length genomes, given that the length and genomic organization is similar to the annotated closest hits from the blastx analysis. High average read coverage values were obtained for some virus-like contigs , which mBased on the presence of the RdRp domain and contig length, we selected four distinct viral contigs for further analyses. They represent putative novel virus species belonging to reo-like (virus-like contig 4), hepe-like (virus-like contig 139), toti-like (virus-like contig 141), and picorna-like (virus-like contig 1) virus clades. These viral sequences showed similarities to viruses previously described in crustacean hosts, suggesting that they were likely infecting signal crayfish rather than being associated with the environment or co-infecting microorganisms. The presence of signal crayfish-associated reo-like virus 1, hepe-like virus 1, and toti-like virus 1 sequences was confirmed in all four analyzed samples by obtaining the products of the expected size after RT-PCR , while tCherax quadricarinatus) from Australia [In our study, signal crayfish-associated reo-like virus 1  had the highest average read coverage in all samples . A varieustralia . This reustralia . Notablyustralia . Thus, fMacrobrachium rosenbergii with growth retardation, however, it could not be determined as the causative agent of the disease [Signal crayfish-associated hepe-like virus 1 , detected in all samples, is represented by near complete genome sequence with four open reading frames (ORF), including ORF1—nonstructural polyprotein with RdRp domain . In blas disease .Charybdis crab host [Litopenaeus vannamei) [Cherax quadricarinatus) [The signal crayfish-associated toti-like virus 1 , containing four ORFs, was clustered with the Wenzhou crab virus 5 (deposited sequence is 8691 nts long) in a phylogenetic analysis . It had rab host  and was annamei) , and therinatus) . Howeverrinatus) . CGV wasrinatus) ,53.Scylla paramamosain) [Distinct contigs similar to previously described picorna-like viruses, a loosely defined broad group of viruses, were detected mostly in UF, with the highest blastx similarities to different crustacean picorna-like viruses , such asamosain) . Other sActinia equine [Further viral contigs potentially associated with signal crayfish, often detected in only one sample and with low average read coverage, belonged to tombus-like, chu-like, partiti-like, sobemo-like, and narna-like virus groups. Sequences similar to tombus-like viruses were previously associated with plant hosts, however, numerous tombus-related viruses have recently been described in non-plant hosts, such as marine and terrestrial invertebrates. Based on the phylogenetic analyses, it was suggested that this group of viruses was primarily associated with invertebrates, and was later horizontally transferred from aquatic invertebrates to plant hosts ,6. Aparta equine . Signal a equine . These tMivirus genus from the recently discovered negative-strand RNA Chuviridae family, characterized by HTS analysis only [Halichondria panacea) [At all locations, different contigs similar to the Beihai hermit crab virus 3 [sis only ,58. Divesis only . Additiopanacea) . AccordiWe have compared the abundance of viral sequences as well as the nucleotide diversity of selected virus populations at different sampling locations in order to obtain insights into the possible patterns of variations along the invasion range. Based on the observed patterns, we could postulate hypotheses about how the observed patterns could be connected with ecological conditions at different sample sites, which represent a baseline for future research work on the virus ecology of crayfish and/or invasive alien species.In our study, an abundance of virus-like reads, distinct virus-like contigs, and sequences mapping to signal crayfish-associated virus-like contigs varied between locations along the invasion range, with virus-like reads constituting between 0.0016% of the total reads in UF and 0.0174% in DC . When coThe differences in signal crayfish abundance along the invasion range could influence the abundance of virus sequences detected, according to the classical epidemiological theory that links larger populations with higher contact rates with an increased likelihood of viral transmission ,61,62. TP. leptodactylus is also present with 2.5–12.9 higher abundances than the signal crayfish [Despite the lower total number of virus-like reads in the UF sample, the highest number of distinct putative virus-like contigs similar to unclassified RNA viruses  was founcrayfish , potenticrayfish .For the three viruses, which were present at all four locations with relatively high average read coverage, we determined the diversities of their populations by calculating nucleotide diversities. This enabled us to estimate how the diversity of each discovered viral species population might be changing along its host invasion range. For all analyzed viruses, we observed differences in nucleotide diversity between populations at the invasion cores and invasion fronts. We observed two contrasting patterns. For signal crayfish-associated reo-like virus 1, the nucleotide diversity was decreased at the invasion fronts, while for signal crayfish-associated hepe-like virus 1 and toti-like virus 1, the nucleotide diversity was higher at the invasion fronts .P. leptodactylus with higher density at the fronts) to populations of low signal crayfish abundance at invasion fronts. Successful inter-specific transmission relies on physical opportunities for transmission, such as occupying the same environment at the same time [Observed differences in nucleotide diversities of viral populations of the three investigated viruses from samples collected along the signal crayfish invasion range might result from different processes, and several hypotheses can be put forward to explain the observed patterns. For instance, higher nucleotide diversity might indicate a higher prevalence of a particular virus, e.g., reo-like virus in the core sites. However, since we were analyzing composite hepatopancreas samples, we are limited in our estimations of the prevalence of individual viruses. Furthermore, we speculate that the estimated nucleotide diversity (π) values potentially reflect transmission dynamics of analyzed viruses, with a higher core nucleotide diversity of signal crayfish-associated reo-like virus 1 suggesting the transmission of the virus from longer established core populations with high signal crayfish abundance to recently established front populations of low signal crayfish abundance. The signal crayfish-associated reo-like virus 1 might be an endemic virus of signal crayfish, introduced to the new habitat along with its host, which could be confirmed/rejected by detecting the signal crayfish-associated reo-like virus 1 sequence in signal crayfish from other geographic regions. On the contrary, a higher nucleotide diversity of signal crayfish-associated toti-like virus 1 and hepe-like virus 1 in samples from invasion fronts could potentially reflect the introduction of these viruses from other host populations .Finally, the results regarding the comparison of different locations can be confounded by other factors, which we were not able to elucidate using our experimental design (sequencing of samples pooled by location), and further analysis of individual samples would be needed to advance the understanding and test the postulated hypotheses.We reported, for the first time, the virome of signal crayfish hepatopancreas tissue and found a high diversity of novel divergent viral sequences most similar to different unclassified RNA viruses.We identified putative novel RNA viruses, including near complete genome sequence of signal crayfish-associated hepe-like virus 1 and toti-like virus 1, and the partial genomes of signal crayfish-associated reo-like virus 1 and picorna-like viruses. We identified additional tombus-like, partiti-like, and chu-like virus sequences potentially representing novel crayfish viruses. This pioneer study represents a baseline for the future research of a signal crayfish virome, e.g., to confirm the association of novel viruses with signal crayfish host, and to investigate their potential involvement in the observed necrotizing hepatopancreatitis.P. leptodactylus as a co-occurring and phylogenetically related native species.We speculate that the differences in the signal crayfish population density along the invasion range, non-random dispersal, and possibilities of inter-specific viral transmissions may have an effect on the diversity and abundance of signal crayfish-associated viral sequences. Different hypotheses can be postulated to explain these patterns, and this study represents a baseline for the further research of virus transmission dynamics as a result of the invader’s fast dispersal, including inter-species transmission between the signal crayfish as an invader and


Arthropod-borne viruses (arboviruses) cause morbidity and mortality in humans and domestic animals worldwide. The percentage of population immunity or susceptibility to these viruses in Ecuador is unknown.To investigate the proportion of Ecuadorian populations with IgG antibodies (Abs) (past exposure/immunity) and IgM Abs (current exposure) against flaviviruses and alphaviruses and to study the activity of these viruses in Ecuador.During 2009-2011, we conducted a serosurvey for selected arboviruses in humans , equines (n=149), and sentinel hamsters (n=84) at two coastal locations and one in the Amazon basin (Eastern Ecuador) using enzyme-linked immunosorbent assay and hemagglutination inhibition test.From 20.63% to 63.61% of humans showed IgG-antibodies for the flaviviruses: Dengue virus (DENV), yellow fever virus (YFV) Saint Louis encephalitis virus, and West Nile virus (WNV); from 4.67% to 8.63% showed IgG-Abs for the alphaviruses: Venezuelanequine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus. IgM-Abs were found for DENV and WNV. Equines and hamsters showed antibodies to alphaviruses in all locations; two hamsters seroconverted to YFV in the Amazonia.The results show a YFV vaccination history and suggest the activity of arboviruses not included in the current surveillance scheme. Enhanced arbovirus and mosquito surveillance, as well as continued YFV vaccination and evaluation of its coverage/ effectiveness, are recommended. They include the Saint Louis encephalitis virus (SLEV), the West Nile virus (WNV), and the Ilheus virus (ILHV) belonging to the Ministerio de Salud Pública - MSP  are focused on the diagnosis of DENV, YFV, CHIKV, and ZIKV, mainly using nucleic acid detection; other arboviruses are surveyed using generic primers, a method with intrinsic limitations that may lead to missing arboviruses of medical concern. In this study, we aimed to investigate the proportion of three Ecuadorian populations with IgG Abs (past exposure/immunity) against selected flaviviruses and alphaviruses. This population included conscripts of a military fort of the Ecuadorian Amazonia recently vaccinated against YFV with the “YFV-17D” vaccine. We also determined the percentage of IgM Abs (recent exposure) against DENV and WNV in febrile and healthy individuals to look for clinical and subclinical infections. Finally, we emphasize the importance of widening and strengthening arbovirus surveillance in Ecuador.Serosurveys are a key tool for determining the proportion of individuals exposed and of those with immunity to infectious agents, as well as for the detection of subclinical infections Instituto Nacional de Higiene y Medicina Tropical “Dr. Leopoldo Izquieta Pérez” (INHMTLI) now Instituto Nacional de Investigación en Salud Pública. Human samples were collected under verbal informed consent. Domestic animals were sampled given owner consent and supervision. Hamsters were used as sentinel species in the field and suckling mice “Balb/C4” were required for inoculation of hamster tissue and mosquito pools. Hamsters and mice were grown and housed at the laboratory animal facility of INHMTLI, which has adequate infrastructure to guarantee animal welfare and dedicated personnel for cage cleaning and animal feeding. After exposure/inoculation, the animals were euthanized using chloroform as an inhaled anesthetic. All these procedures were performed in strict accordance with the institutional guidelines for the care and use of laboratory animals. We followed the ethical guidelines of the Declaration of Helsinki and the “Public Health Service Policy on Humane Care and Use of Laboratory Animals” published by the National Institute of Health of the United States of America.This project was approved by the institutional review board of the This study comprised serum samples of humans and equines for arbovirus seroprevalence analysis, serum samples of hamsters for arbovirus seroconversion analysis, mosquito pools, and hamster tissues for viral isolation. Sampling was performed from 2009 to 2011 at three locations in Ecuador, two in the coastal region  and one in the Amazonia basin (Pastaza Province- Puyo) . Manglaret al. The procedure for sampling humans and domestic animals is described by Beaty, 2Mosquitoes were trapped with CDC light traps supplemented with COet al.Seroprevalence/seroconversion to arbovirus was evaluated mainly through the analysis of IgG Abs. For humans, each sample was analyzed against a battery of arboviruses comprising DENV, YFV, SLEV, WNV, EEEV, WEEV, and VEEV. Only monotypic results were considered positive for the corresponding virus. IgG Abs against DENV and WNV were evaluated by commercial ELISA methods of PANBIO  and FOCUS , respectively. IgG Abs against YFV, SLEV, EEEV, WEEV, and VEEV were evaluated with in-house ELISAs following Johnson, et al.Técnicas de laboratorio para el diagnóstico y la caracterización de los virus del dengue-Equine and hamster IgG Abs were evaluated with the hemagglutination inhibition (HI) test on kaolin-adsorbed sera according to the technique of Clarke, Mosquitoes were identified, classified, and grouped by species. Spearman correlation was carried out using SPSS to test the relationship between the number of mosquitoes captured by species and location and the proportion of human samples showing IgG Abs reactive against the arboviruses tested. This nonparametric test was chosen given that the proportion of samples having Abs was calculated on categorical data (yes/no). Mosquitoes pooled by species in groups of 50 were crushed in a sterile environment and processed for intracerebral (IC) inoculation in suckling mice Balb/C4 and RT-PCR analysis.et al.et al.RT-PCR for flaviviruses and alphaviruses was conducted on samples from febrile humans, hamster tissues, and mosquito pools according to Ayers, Analysis of IgG Abs against selected arboviruses was performed on 1,842 human sera. We found Abs for all the viruses investigated: DENV, YFV, SLEV, WNV, EEEV, WEEV, and VEEV. The percentages of Abs against different arboviruses in 1,263 samples (without including conscripts) are shown in The results of IgG analysis by location are shown in The percentage of conscripts with IgG Abs against arbovirus is shown in A total of 102 individuals were registered as febrile and analyzed for IgM Abs against WNV and DENV, and the detailed results are shown in A total of 1,343 samples of nonfebrile individuals (including conscripts) were analyzed for IgM Abs against DENV of which 5.06% (18/356) in Vinces, 2.42% (12/496) in Manglaralto, and 7.94% (39/491) in Puyo showed Abs. A total of 893 samples of nonfebrile individuals (including conscripts) analyzed for IgM Abs against WNV demonstrated 0.20% (1/496) positivity from Manglaralto and 0.20% (1/491) positivity from Puyo.The HI results are shown in A total of 84 hamsters were used as sentinels. After exposure in the field, 22.62% of them demonstrated Abs to EEEV , 20.24% showed Abs to VEEV , and 2.38% seroconverted to YFV (22.22% -2/9 in Puyo) . HemagglCulex (Mel) spp. (N=63.057). The most abundant by location were Culex (Mel) spp. in Vinces (N=62.736), Aedes (Stg) aegypti in Manglaralto (N=912), and Coquillettidia venezuelensis in Puyo (N=160). Correlation results are shown in table S6. There was a very strong negative correlation  between Psorophora ferox and samples with Abs against DENV, SLEV, WNV, EEV, WEEV, and between C. venezuelensis or A. albimanus and samples showing Abs for YFV (ρ=-1 p≥0.1). A strong positive correlation was found between Mansonia pseudotitilians/indubitans or Anopheles (Nys) triannulatus and the proportion of individuals showing Abs against VEEV . There was a positive correlation between A. aegypti and DENV, SLEV, WNV, EEV, and WEEV, but it was not significant. A list of mosquito species identified in this study and the arboviruses they harbor according to viral isolation studies elsewhere is shown in A total of 73,505 mosquitoes were captured. The number by locality and species is described in Flavivirus or Alphavirus RNA.RT-PCR analysis of 128 mosquito pools and 119 hamster tissues did not lead to the amplification of We inoculated 807 suckling Balb/C mice, 576 with hamster tissue and 231 with suspensions of polled mosquitoes. No virus was isolated.Serosurvey of arboviruses is constrained by Ab cross-reactivity because of antigenic similarities between members of the same virus family cocirculating in the same region. The plaque reduction neutralization test (PRNT) is the gold standard for the specific detection of Abs, but it is difficult to implement in resource-limited countries. ELISA and HIA tests are valuable alternatives when monotypic results are accounted for. Using these techniques, we report the percentage of individuals with IgG Abs reactive against DENV, YFV, WNV, SLEV, EEEV, WEEV, and VEEV and with IgM Abs reactive against DENV and WNV. We found monotypic results for WNV, SLEV, and each of the encephalitis viruses evaluated while the frequency was found in individuals of different ages. We also demonstrated the seroconversion of naive sentinel hamsters to the EEEV and VEEV in all sampling locations and for YFV in Amazonia. Mosquito genera recognized as vectors of arboviruses were found in all locations studied. We discuss our findings in the context of arboviruses whose activity has been confirmed in Ecuador and with the current surveillance schemes.Culex nigripalpus mosquitoes (isolated 76 V-1177 and 24684) collected in Huertas Negras-Tenguel/Guayas and Puerto Nuevo- Vinces/Los Ríos and sentinel hamsters (isolated 78V-5682) exposed in Playas/Guayas (20). From 2001 to 2004, specific IgM Abs against this virus were found in febrile patients sampled from Ecuadorian Amazonia Monotypic IgG Abs against WNV were found in Manglaralto-Santa Elena Province and Vinces-Los Ríos Province . IgM Abs against this virus were also found in two IgG-positive samples, one from Manglaralto and one from Vinces. The result in Vinces is consistent with the finding of specific IgG Abs against this virus in equines sampled during 2007-2009 by Coello ,A higher percentage of Abs was found against YFV, which was probably due to YFV vaccination history. Overall, the civilian population showed ~60% Abs against YFV, with Puyo showing a higher percentage of civilian people with YFV Abs (52.75%). In conscripts, the percentage of Abs was between 81.31% and 85.05%, which is not optimal but agrees with vaccine efficacy percentages Alphavirus, we found IgG Abs reactive against VEEV, EEEV, and WEEV in humans and equines and Abs to VEEV and EEEV in naive hamsters. All these viruses were isolated in Ecuador by Calisher, et al. between 1974 and 1978 ,,Madariaga virusRegarding Culex species are vectors of WNV, SLEV, and VEEV, and the presence of these mosquitoes in all locations is consistent with the finding of Abs against the viruses they transmit. A. aegypti, the main vector of DENV, was found only in Manglaralto, although Abs against dengue were found in all locations. The absence of this mosquito species in the other locations studied could be due to sampling bias; in Manglaralto, traps were placed near human settlements and in Puyo and Vinces they were placed in more forested areas. Correlation data between species and the proportion of human individuals showing IgG Abs against arbovirus should be interpreted with caution because few locations were analyzed (N=3). Anopheles nuneztovari has not been reported in Ecuador before, but this species is difficult to identify and will need further confirmation. Anopheles are not usual vectors for arboviruses, but the finding of unreported species highlights the importance of carrying out more studies on mosquitoes.As regards mosquitoes, most of the species/genera identified in this study are known vectors of arboviruses elsewhere . For exaBy 2009-2012, arboviral surveillance in Ecuador comprised DENV and YFV through ELISA and nucleic acid detection. After the arrival of ZIKV and CHIKV and the confirmation of OROV, the surveillance was extended to these viruses. Following our results, a percentage of negative samples are currently analyzed for VEEV, EEEV, and MAYV by RT-PCR. Given the emergence and reemergence of arboviral diseases worldwide and their continuous spread, we strongly recommend widening the analysis and surveillance of other arboviruses, especially those isolated previously or detected in febrile patients in the country. These must include the arbovirus surveyed here but also the ILHV and the Playas virus. This last virus, isolated in Ecuador in 1974-1978, is genetically similar to the Cache Valley virus and the Maguari virus, which have been reported to cause human disease. Likewise, we emphasize the need for updated studies and surveillance of mosquito ecology and their vectorial capacity for arbovirus transmission.-et al.We also urge for the establishment of a set of recommendations given by the Global Arbovirus Group of Experts (GAGE)


The aim of this study was to analyze the relationship between the concentration of hormones in the seminal plasma, the bull maintenance system in the insemination station, and the regularity of sperm donation and the response to the phantom (libido level). An additional goal was to determine whether there is a relationship between the hormonal profile in the blood, the sperm plasma, the oxidative and antioxidant profile in the blood of bulls and the biometry of their testicles and scrotum, as well as the quality of their sperm in both different seasons and intensities of reproductive use. For the study, 220 healthy and sexually mature Polish Holstein–Friesian bulls were used. They all had normal libido and were fed equally. The animals were grouped according to the scheme: young (16–20 month/n = 60) and old (26–30 month/n = 60) including: individually housed (n = 30) and group housed (n = 30) young, old individually housed (n = 30) and group housed (n = 30) ; young animals donating semen once a week (every Thursday) (n = 25) and sporadically (once every two months on a random day of the week) (n = 25), old animals donating semen once a week (every Thursday) (n = 25 ) and sporadic donors (once every two months on a random day of the week) (n = 25) . When analyzing the results of this study, it should be stated that regular use has a positive effect on the secretion of sex hormones in bulls. Higher levels of testosterone and lower levels of estradiol and prostaglandins resulted in higher sexual performance, expressed by a stronger response to the phantom. The differences in favor of regular use were independent of the bull's age. The results of our research illustrate that the quality of semen and its freezing potential may depend on the season and frequency of its collection, as well as on the age of the males. The rush to introduce very young individuals into the production system occurred along with the dynamic development of the 'genomic era', which undoubtedly contributed to even greater problems with sexual immaturity in these animals. Moreover, there is a significant loss of time and an increased risk taken by technical personnel to stimulate bulls with a lower libido. Moreover, in such cases a much higher labor input is observed2. In the work of Bryant3 it was noted that libido is a response to endogenous or exogenous stimuli. This response may be mediated by various physiological mechanisms, male motivation and the experience acquired by the bulls. This parameter can be used to measure the reproductive competences of bulls4. This parameter is defined as the time elapsed from exposure to stimuli to the first service, and is calculated using the reaction time5. Ejaculation efficiency and semen quality are modified by the level of sexual arousal and performance8. High sex drive in bulls leads to better erection, penile protrusion, ejaculation thrust, libido, and female mating ability9.Insemination stations are losing income every day due to the growing number of males showing problems with sperm donation due to poor libido. Lack of semen means that the station will not produce insemination straws, and a bull showing symptoms of libido disorders is directed to slaughter, which generates even greater losses for the station. It has been observed that almost one quarter of breeding bulls maintained at bull stations are affected by poor libido10. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) hormones are also responsible for male fertility. These hormones have the ability to modulate testosterone synthesis in Leydig cells. The pituitary-hypothalamus-gonadal axis regulates the secretion of GnRH, which is responsible for the secretion of gonadotrophins and testosterone needed for spermatogenesis, sperm maturation and reproductive behavior. In addition, adiponectin, which is a pleiotryopic regulator of many biological functions, may increase testosterone production, which contributes to an increase in libido. Adiponectin plays an important role in the structure and function of sperm and may also be involved in the fusion of the sperm with the oocyte11. Estrogen is the end product of the irreversible transformation of androgens by the aromatase enzyme. Although the influence of seasonality on sperm characteristics and male libido has been extensively studied in various breeds15, there are no reports on the influence of the intensity of reproductive use of bulls kept in insemination stations on their libido level and the concentration of hormones in the semen plasma. In many studies, the relationship between testosterone in the seminal plasma and sperm quality and sperm motility has not been proven17. However, there is contradictory information on fertility and testorerone levels in bulls17. Jeved et al.18 found a relationship between higher concetrations of testosterone in semen plasma and bull semen quality including parameters such as pH, motility and sperm count. Moreover, in the case of low libido in bulls, it has been suggested that this is due to low testosterone levels17.Testosterone is the major androgen necessary for spermatogenesis in the testes and is mainly responsible for the maintenance of secondary sexual characteristics and libido and thus promotes sperm production in bullsThis study aimed to analyze the relationship between the concentration of hormones in the semen plasma, the bull maintenance system in the insemination station, and the regularity of sperm donation and the response to the phantom (libido level). An additional goal was to determine whether there is a relationship between the hormonal profile in the blood, in sperm plasma, oxidative and antioxidant profile in the blood of bulls and the biometry of their testicles and scrotum, as well as the quality of their sperm in different seasons and different intensities of reproductive use.The experiment was performed as part of routine activities during the current semen production in the reproductive station and did not require the approval of the ethics committee. These experiments were performed in the Breeding and Insemination Centre ‘MCB’ .For the study, 220 healthy, sexually mature and equally-fed Polish Holstein–Friesian bulls with normal libido were used. The animals were grouped according to the scheme: young (16–20 months/n = 60), old (26–30 months/n = 60); including: individually housed (n = 30) and group housed (n = 30) young, old individually housed (n = 30) and group housed (n = 30) ; young animals donating semen once a week (every Thursday) (n = 25) and sporadically (once every two months on a random day of the week) (n = 25), old animals donating semen once a week (every Thursday) (n = 25 ) and sporadic donors (once every two months on a random day of the week) (n = 25) . The trial was conducted in two phases over summer (May-Oct), and winter (Nov—April) season. The entire experience was conducted over 2 years.Two ejaculates were collected from each bull at 7 a.m. using an artificial vagina and the standard phantom intended for cattle. The ejaculates were then centrifuged to separate seminal plasma from sperm cells and the supernatant was used for the hormonal assay. Commercial ELISA kits were used to evaluate the endocrine profile of seminal plasma. The analyzes were performed according to the manufacturer's instructions. Testosterone was determined in seminal plasma of crossbred cattle bulls by Bovine Testosterone (T) ELISA Kit . The estrogen was detected using a Bovine Estradiol ELISA kit . Prostaglandin E2 was determined using Prostaglandin E2 ELISA kit .To assess the reaction to the phantom, a two-point scale was adopted depending on the speed of crossing the yellow line applied to the floor at a distance of 1.5 m from the phantom; low libido > 3 s, high libido < 2.9 s. Only jumps on the phantom after crossing the indicated line by the bull ended with ejaculation were considered correct. Before starting the timing, each male has the opportunity to sniff the phantom and mount it for the same amount of time (‘warm-up exercise’).Blood samples were collected by puncture of the jugular vein into heparin tubes (20 IU heparin per ml of blood) at four-week intervals at various times of the year. Blood samples were collected on the same day and at the same time from all animals.g for 15 min at 4 °C. Serum samples were separated, removed by pipette, labeled and stored at − 20 °C in a freezer until further analysis.Blood samples were centrifuged at 120019 and Moura et al.20.Serum LH and FSH concentrations were determined by the dual antibody RIA method, while testosterone and estriadol concentrations were determined by the solid-phase RIA method as previously described by Dance et al.21.The antioxidant status of the bulls' serum was determined by the analysis of reduced glutathione (GSH), urid acid (UA), total protein (TP) and albumin (ALB). The obtained values of the abovementioned parameters were expressed per liter of bloodThe intensity of the oxidative damage to the proteins was established by determining the concentration of the protein carbonyl content (PCC) and for the intensity of the oxidative stress was analysed blood serum lipid peroxidation assessment .The concentration of lipid peroxides was determined spectrophotometrically by measuring the concentration of malondialdehyde and other reactive products of thiobarbituric acid resulting from free radical peroxidation of unsaturated fatty acids at high temperature and low pH.22. An extinction coefficient of 21.0 m−1 cm−1 was used to calculate the carbonyl content per gram of protein.PCC content was determined spectrophotometrically using 2,4 dinitrophenyl hydrazine (DNPH) according to the method of Levine et al.The volume of the ejaculate was measured by reading the graduated tube and the concentration was calculated spectrophotometrically.23.The fresh undiluted semen was then evaluated microscopically  for mass motility. Subsequently, the semen was extended with animal protein–free commercial BIOXcell® extender  to a final concentration of 120 × 106 spermatozoa/mL and rated in terms of motile sperm percentage, progressive motility and viability. Semen was automatically packed  into polyvinyl chloride (PVC) straws (0.25 mL)  which were filled and equilibrated for 1.5 h at 4 °C. After equilibration, the straws were frozen in liquid nitrogen vapor using a computer controlled automatic freezer from 4 to − 15 °C at the rate of − 3 °C /min and from − 15 to − 80 °C at the rate of − 10 °C /min After reaching − 80 °C, semen straws were plunged into liquid nitrogen and packaged in plastic goblets for 24 h of storage in the liquid nitrogen container. The straws were thawed in a water bath at 38 °C for 20 s and were then examined to evaluate the quality after thawing.24. The mass motility was scored into four scales: + no motion, ++ free spermatozoa moving without forming any waves, +++ vigorous movement with moderately rapid waves, ++++ very rapidly moving waves23.Mass motility was examined in 20 µL of semen which was placed on a prewarmed slide without any cover slip and analyzed under microscope  equipped with phase-contrast optics (100×)23.Total sperm motility and progressive movement were examined using a Sperm Class Analyzer , a light microscope (Nikon Eclipse E200). Just prior to analysis, semen was diluted 1:10 in a warm (25 °C) physiological solution (sodium chlorate 0.9%). Then, 2 μL of the prepared sample was placed in a Leja 4 analysis chamber  at a thickness of 20.0 μm. The slide was placed on a stage warmer (38 °C). A minimum of 500 cells were evaluated, and depending on sperm concentration, five analyses were performed per sample23.The double stain SYBR-14 with propidium iodide  using flow cytometer was applied . For this purpose, 50 µL of thawed semen was measured (37 °C for 20 s) and 940 µL NaCl (0.9%) and 5 µL SYBR14 were added. The whole was thoroughly mixed and then incubated (36 °C for 10 min) without light access. Subsequently, 5 µL of PI was remixed and incubated for 3 min without light, followed by a test24 by using the following formula for volume of an ellipsoid, i.e., 4/3πabc, where, c = length/2, a = thickness/2 and b = width/2.Testicular volume was estimated according to the method described by Love et al.25. The scrotum was measured with a caliper and a tape measure. The circumference of the scrotum was measured at its widest point with a taut measuring tape.Scrotal circumference was estimated with the method explained by the Society of TheriogenologyThe biometeorological factors, relative ambient temperature and relative humidity values were received from the meteorology station Institute of Meteorology and Water Management. Airport Meteorological Station, located at close proximity of the experimental station for estimation of temperature humidity index (THI) Table .Table 1CUsing the analysis of variance with the LSD test, the influence of such factors as bull's age, housing system, semen collection season, and frequency of collection on the concentration of hormones in the semen plasma were determined. Correlations were calculated between the frequency of sperm collecting and the strength of response to the phantom. Spearman correlations were calculated using the Corr SAS Enterprise Guide procedure. The significance of differences between the means was estimated using the T-test and Fisher's NIR (Least Significant Difference) test. Pearson's correlations were estimated for pairs of the analyzed random variables using the CORR Sas Enterprise Guide procedure .ijklm = µ + si + wbj + sbk + ejl + eijklm.Yijklm—analyzed feature.Yµ—expected value of the feature.i—season of sperm collecting i = 1, 2sj—bull age class, j = 1, 2wbk—housing system, k = 1, 2sbl—frequency of sperm collecting – l = reg, unreg.ejijklm—random error effect.eTable P < 0.01) were found in the testosterone concentration in the bulls’ seminal plasma used regularly and irregularly. These differences were highly significant (P < 0.01) regardless of the age of the bulls and the season of their use. This dependence is explained by a higher concentration of testosterone, and thus a higher libido, in substances used regularly. Considering the winter season—among the bulls used regularly, older animals showed a statistically higher concentration of testosterone (9.64 ng/mL) compared to younger animals (8.98 ng/mL) (C-D). Among the bulls used regularly, a significantly higher concentration of testosterone was also noted in older animals (8.21 ng/mL) compared to in younger siblings (8.02 ng/mL), the difference turned out to be statistically significant (e–f). Statistically, in the winter season a significantly higher testosterone concentration was observed in younger bulls, not used regularly (8.1 ng/mL) compared to the concentration of this hormone in older animals (7.52 ng/mL). In the winter season, the concentration of estradiol in the seminal plasma of bulls used regularly was lower than in bulls used in the irregular cycle, while for older bulls the concentration was statistically significantly higher (350.7 pg/mL) compared with irregularly used bulls (322.4 pg/mL). Comparing the age groups in terms of estradiol concentration, statistically significant differences were found between the estradiol concentration in the groups used regularly and in a variable manner. In the summer season, the concentration of estradiol was higher in bulls used irregularly and in younger animals, but these differences were statistically insignificant. Statistically, a significantly higher concentration of prostaglandins was recorded in the winter season in older animals that were used regularly. In the summer season, the concentration of prostaglandins was statistically higher in the seminal plasma of younger animals than in older animals, regardless of the regularity of sperm collection.There was a negative and statistically significant correlation (r = − 0.32) between the concentration of testosterone and estradiol in the bulls’ seminal plasma. The correlation coefficient between the concentration of testosterone and prostaglandins was 0.11, while the correlation between the concentration of estradiol and prostaglandins was negative but statistically insignificant (r = − 0.12). Pearson's correlation coefficients were calculated without division by the summer/winter production season. Highly significant differences (P < 0.01). The concentration of estradiol was statistically, significantly higher in the group of bulls with a poor reaction to the phantom, both used regularly and variably.When analyzing the behavior of bulls before jumping onto the phantom Table , their rWhen the animals donated sperm regularly, males kept in the group pen showed a statistically higher concentration of testosterone in the semen plasma (9.55 ng/mL) compared to the animals kept individually (8.22 ng/mL). The concentration of estradiol in the plasma of semen of bulls housed regularly (housed in a single pen) (333.10 pg/mL) was lower than that of bulls housed in a group pen (349.92 pg/mL). Prostaglandin concentration was higher in bulls kept in a single pen (22.76 pg/mL) than in bulls kept in a group pen (21.07 pg/mL).P < 0.01). Statistically significantly higher (P < 0.01) TBARS values were recorded for younger bulls in the spring-winter season (1.1 mmol/L and 1.2 mmol/L) compared to the summer season (0.5 mmol/L). Moreover, in older bulls, it was observed that the concentration of TBARS showed the opposite tendency of changes than in the case of young bulls, as in the winter-spring season it was the lowest (0.4 mmol/L), while in autumn and winter it increased (0.9 mmol/L and 1.0 mmol/L). There were no significant differences between the TBARS content in age groups for the summer-autumn seasons. The correlation between PCC and TBARS was 0.812 and was statistically, highly significant (P < 0.01).Statistically significant differences were observed Table  between P < 0.05) in the spring-winter seasons, and in the following seasons no significant differences between the age groups were found. In terms of ALB concentration, the results remained at a similar level throughout the study period, reaching the lowest values in winter and spring, respectively , and the highest in summer and in the fall . The lowest AU values in both bull age groups were observed in winter , and the highest in summer , whereby these differences were statistically significant (P < 0.05). A higher GSH value in terms of concentration was observed in older bulls compared to young bulls. The lowest concentration of GSH in both examined age groups was observed in summer, and in turn, the highest in spring   depending on the age of the animal and amounted to 2.66 mIU/mL for young bulls and 3.98 mIU/mL for old bulls, respectively. In the case of old animals, these values were higher. The LH concentration in the summer season, amounting to 2.66 mIU/mL, in young animals was significantly lower (P < 0.05) compared to those of all ages in the spring and winter seasons. The lowest concentration of LH was in the summer season in young animals (2.66 mIU/mL), while the highest concentration was in old animals in the same period (3.98 mIU/mL).Analyzing the concentration of luteinizing hormone (LH), it can be noticed that in the case of young animals these values decreased in the period from winter to summer, then in autumn they increased slightly, while in the case of older animals a reverse trend was observed and the concentration of LH increased from winter to summer, to then be lowered in the fall. There were no statistically significant differences in the concentrations of luteinizing hormone (LH) between young and old animals in winter, spring and autumn. The values of LH concentrations in summer were significantly different (P < 0.05) were observed in the concentration values between animals in terms of their age. The concentration of FSH in spring and winter was significantly higher (P < 0.05) in bulls of all ages compared to summer and autumn months and amounted to 26.11 ± 5.5 for young bulls and 29.18 mIU/mL for old specimens in winter. In the spring season concentration of FSH was on level 22.0 mIU/mL in young males and in old 25.34 mIU/mL. The lowest concentration of FSH was observed in young animals in summer (09.04 mIU/mL) and the highest in old animals in winter (29.18 mIU/mL).When analyzing the values of follicle stimulating hormone (FSH) concentrations, a downward trend can be noticed in the period from winter to autumn, except for young animals in the autumn period, because in their case the concentration increased compared to the previous season, i.e. in summer, and amounted to 13.13 mIU/mL. In any single study period, no significant differences (P < 0.01) was demonstrated in the fall in relation to the remaining seasons of the year, while in the autumn months younger bulls had statistically insignificantly higher testosterone levels than old animals. The lowest value of testosterone concentration was observed in old individuals in the fall (44.71 ng/mL), while the highest value was in old bulls in the summer period (156.90 ng/mL).Analyzing the testosterone concentration, an upward trend can be noticed in the older animals from winter to summer, followed by a decrease in the described values. There were no statistically significant differences in testosterone concentrations, both in young and old animals, between winter, spring and summer months. A highly significant statistical difference (P < 0.05) were, however, observed in the winter months, when the estradiol concentration was lower in both age groups compared to the rest of the year. The lowest estradiol concentration was found in winter in young animals and it is equal to 4.4 pg/mL. The highest concentration of estradiol was obtained in the spring months in older animals and was 10.8 pg/mL.In the case of estradiol levels, the age groups did not significantly differ statistically at specific times of the year. Significant differences (P < 0.01) correlations (Table P < 0.05) were found between: PCC and TP, TP and ALB, and ALB and UA. The other correlation coefficients were statistically insignificant. Highly significant (P < 0.01) and positive correlations were noted between blood testosterone level and blood LH level (0.36) and between LH and FSH (0.69). Highly significant (P < 0.01) but negative correlations were found between blood testosterone content and estradiol content (-0.41) and between estradiol and FSH (-0.25). The correlations between FSH and testosterone in the blood as well as estradiol and LU levels were not statistically significant. The values of the correlation between the content of hormones in the blood and analogous hormones in seminal plasma were highly significant (P < 0.01), positive and reached 0.98–0.999.Highly significant (ns Table  were notP < 0.01) highest in older bulls  and in bulls from both age groups, but used on a regular basis. The percentage of live sperm was higher in animals donating sperm regularly (in both age groups) by an average of 6.69% (young) and 8.54% (old). These differences were statistically, significantly (P < 0.05) higher in the group of bulls giving sperm regularly, regardless of the age of the male. The opposite tendency was observed for the percentage of dead sperm—in the group of regularly used there were significantly less (P < 0.05) dead sperm than in the group that donated irregularly. There was no significant difference between age groups in this parameter. Regularly used bulls, regardless of age, had better sperm motility results . Moreover, the semen of older bulls showed better total motility and progressive movement in comparison to younger bulls, regardless of the regularity of semen collection. The mobility of the sperm mass was higher (+++) in older bulls used regularly than in younger males (++). There were no differences in sperm mass motility in bulls used irregularly.Table P < 0.05) higher volume (7.1 mL) and concentration (2598 × 106/mm3) compared to young bulls (4.9 mL and 1646 × 106/mm3). In terms of the mobility and viability parameters of fresh and frozen semen, no significant differences were observed during the winter season within the studied age groups. In contrast, in the spring season, both the volume of fresh ejaculate, its concentration and the percentage of live and motile sperm were significantly (P < 0.05) higher in the group of older bulls. The frozen semen of the younger bulls was statistically of significantly (P < 0.05) lower quality in terms of viability than the older bulls and contained on average 8.04% less viable sperm than the frozen semen of older males. In the summer season, ejaculate volume and sperm concentration differed significantly between young and old bulls, 1.2 mL and 962 × 106/mm3 sperm, respectively. Moreover, in terms of sperm concentration, it was observed that in the summer season this value was the lowest among all analyzed seasons of use. Similarly, in terms of viability and sperm motility, the analyzed ejaculates were of the poorest quality in the summer season. However, in the autumn season, significant differences in terms of sperm mobility and viability were observed between older and younger males by analyzing their frozen semen. Older bulls were characterized by significantly (P < 0.05) better parameters of frozen semen than younger males. The highest mobility of sperm mass was observed in the winter and spring seasons.Table Table P < 0.01). In the winter season, negative and highly significant correlations were noted between the levels of estradiol and testosterone (-0.28) and estradiol and prostaglandins (− 0.13). A similar tendency was observed in the summer season.The testosterone level was negatively correlated with the level of estradiol (− 0.32) and positively with prostaglandin (0.11). The level of estradiol was also negatively correlated with the level of prostaglandins in the blood (− 0.12). The quoted correlations turned out to be statistically highly significant . In the winter season, negative and highly significant correlations were noted between the levels of estradiol and testosterone (− 0.28), and estradiol and prostaglandins (− 0.13). A similar tendency was observed in the summer season.The testosterone level was negatively correlated Table  with theP < 0.05).The values of the correlation coefficients between the level of hormones and fresh semen quality parameters were determined. The volume of semen was positively and highly significantly correlated with the level of testosterone. Testosterone was positively and statistically significantly correlated with the value of sperm concentration. There were no significant correlations between the level of estradiol and the volume of semen, while the level of sperm concentration was positively correlated with the content of prostaglandins and negatively with the content of estradiol in the blood. The above-mentioned correlations turned out to be statistically significant . The level of estradiol was negatively correlated with the motility, progressive movement and live spermatozoa, and positively correlated with the level of dead sperm. Positive and statistically significant correlations were found between the analyzed characteristics of sperm quality and prostaglandins.The testosterone level was highly significantly correlated with the area (0.82) and volume of the testicles (0.72). Similarly, highly significant, albeit negative, correlations were noted for the level of estradiol by area (− 0.42) and testicular volume (− 0.34). The prostaglandin level was positively correlated with the surface area (0.34) and testicular volume (0.24).After applying the division according to the semen collection season and regularity, very similar correlation results between the analyzed values were obtained.P < 0.05) differences in testicular volume were noted. Older bulls that regularly donate semen had a statistically significantly (P < 0.05) larger testicle volume by an average of 230.57 cm3 compared to younger males. This tendency was identical in the group of animals used irregularly, where the volume of testes in younger males was lower by an average of 564.34 cm3. Moreover, males used in an irregular manner were characterized by a greater circumference of the scrotum and a larger volume of testicles compared to males donating sperm regularly.In older bulls Table , The cir1.The poor libido of bulls used in insemination stations is the main source of several difficulties related not only to the handling of such animals, their delayed inclusion in the current production of insemination doses, but most of all the generation of great financial losses. This is because bulls whose reproductive use does not bring profit due to their disorders of sexual encounters, are slaughteredThe determination of hormones, and in particular of reproductive hormones in semen plasma, is an important diagnostic and therapeutic marker of male fertility disorders.18, while the testosterone to estradiol ratio is important in their libido26. In our study, we found that regular use of bulls was associated with testosterone levels in semen plasma. Males that were used irregularly had a lower level of the hormone than bulls that regularly ejaculated. Moreover, bulls with higher testosterone levels reacted faster, more effectively, and more strongly to the phantom, which in turn allows us to hypothesize that regular sperm donation may improve bulls’ libido levels. Furthermore, we showed that the male group maintenance system was associated with higher levels of testosterone in their semen plasma, suggesting that this system promotes the expression of normal sexual patterns. Perhaps the higher level of this hormone is also due to the fact that during the contact of the bull with other animals, it can "train" the climbing reflexes of other males, which helps to maintain a proper level of libido.Testosterone plays a key role in the expression of normal sexual patterns in bullsWe also showed that the concentration of estradiol was subject to seasonal fluctuations and was increasing with the passing of the winter season. Furthermore, the concentration of estradiol in the seminal plasma of bulls used regularly was lower than in bulls used in an irregular cycle. On the other hand, the bull's maintenance system was not related to the level of this hormone in their sperm plasma.27 by mediating an increase in intracellular calcium concentrations28. Moreover, increased concentration of this protein may lead to premature capacitation and acrosome reaction interfering with the fertilization process29. In this study, it was observed that the concentration of the above hormone in semen plasma was higher in the summer season than in the winter. Moreover, males used regularly were characterized by a higher concentration of this hormone in sperm plasma. There was also no relationship between the strength of the phantom response and the concentration of prostaglandins, similar to estradiol concentration.Prostaglandins present in the ejaculated semen acts through its receptors PTGER1 and PTGER3 and modulate sperm motility, capacitation, acrosome reaction, and fertilizing ability of spermatozoa30, who reported that both the age of males and the season of sperm collection have an impact on the biometric features of male testes.We proved that bull age and regularity of their use were related to the biometric features of the testicles and scrotum. Similar results were obtained by Perumal et al.31.Analyzing the levels of hormones in the peripheral blood of bulls, we showed that they change with the season of the year. These data were also confirmed by other researchers in their experimentsDetermining antioxidative and oxidative parameters in the serum of bulls may help in establishing the antioxidative and oxidative status of individual animals during different conditions such as season (temperature outside), housing system, regularity of sperm donating etc.32 have shown that younger bulls are more sensitive to elevated ambient temperatures in summer, when increased pro-oxidative processes in semen plasma and sperm cause a decrease in sperm motility and, consequently, worsen sperm quality. Other researchers34 report that younger specimens achieve poorer sperm quality parameters than older ones, and that the season of the year significantly affects sperm mobility and viability. The results obtained by us also indicate that the older bulls had both a higher concentration of sperm in the fresh ejaculate, a greater volume of semen and a better viability and mobility  compared to the young bulls.As the results of our research show, the quality of semen and its freezing potential may depend on the season and frequency of its collection, as well as on the age of the males. Balić et al.35 showed that bulls show high sensitivity to changes in climatic conditions in terms of the analyzed antioxidant and oxidation profile in the blood, which is also confirmed by the results of similar variability in the analyzed profiles.Žaja et al.The conducted research shows that regular use has a positive effect on the secretion of sex hormones in bulls. Higher levels of testosterone and lower levels of estradiol and prostaglandins resulted in higher sexual performance, expressed by a stronger response to the phantom. The differences in favor of regular use were independent of the bull's age. As the results of our research show, the quality of semen and its freezing potential may depend on the season and frequency of its collection, as well as on the age of the males.


To evaluate the feasibility of two-colour index maps containing combined diffusion and perfusion information from simplified intravoxel incoherent motion (IVIM) for liver lesion malignancy assessment.b values  estimated diffusion coefficient D′, perfusion fraction f′, and apparent diffusion coefficient (ADC) maps were calculated and analysed for regions of interest (ROIs). D′ and f′ cutoff values were determined by differentiating haemangiomas from other lesions and focal nodular hyperplasias from other lesions, respectively. Combined IDf index maps were generated with a voxel value set to 100, if both D′ and f′ voxel values were lower than their cutoff values , otherwise to 0. Moreover, IADC index maps were generated from ADC cutoff value  obtained by differentiating benign from malignant lesions. Discriminatory power was assessed for both IDf and IADC. Index maps were displayed as two-colour overlays to b-800 images and visually assessed within the translucent hyperintense areas.Diffusion-weighted data from a respiratory-gated 1.5-T magnetic resonance sequence were analysed in 109 patients with liver lesions. With three Df, the same diagnostic accuracy was achieved as for the combined use of parameters D′ and f′ (93.6%). Compared to IADC, IDf showed a higher diagnostic accuracy. Visual judgment of IDf yielded an accuracy (95.4%) similar to that of quantitative analysis (93.6%).For IDf provide similar diagnostic accuracy as ROI-based combination of estimated IVIM parameters D′ and f′ and are suitable for visual assessment of liver lesion malignancy.Voxel-wise combined two-colour index maps I Df can replace the combined use of D and f parameters.Index map Ib-800 overlay IDf enables a visual assessment of liver lesion malignancy.Two-colour Df showed comparable diagnostic accuracy.Visual judgment and quantitative analysis of IDiffusion-weighted imaging (DWI) is an important magnetic resonance imaging (MRI) technique for detection and differentiation of liver lesions not needing contrast agent administration and should be implemented in standard liver examination in routine clinical practice .b value (“black blood” images) provides high sensitivity for lesion detection [b values between 0 and 500–1000 s/mm2 is usually used for lesion characterisation [b values and thus relatively long acquisition times. Limited data quality due to signal variations caused by respiratory and cardiac motion and due to low signal-to-noise ratio may lead to unstable fitting results, measurement errors, and poor reproducibility [b values. Simplified IVIM turned out to be valuable for liver lesion characterisation and assessment of therapy in clinical routine [While DWI acquired with a low etection , 3, the risation , 5. The risation –8. The trisation . These rcibility . Improve routine –20.0 DWI images [For lesion assessment, voxel-wise evaluation and the creation of parameter maps are important. Still somewhat inconvenient for clinical use is the quantitative analysis of regions of interests (ROI) in the IVIM parameter maps. The use of colour-coded maps , 22 as oI images –16, 23 eI images , 25. SuiThe purpose of this study was to create and evaluate two-colour index maps, which combines diffusion and perfusion information obtained by simplified IVIM for convenient visual assessment of liver lesion malignancy.b value DWI sequence at 1.5 T, were reviewed. Data of 1350 examinations were not used because the patients had no liver lesions, only cysts, or lesions < 1 cm, or because it was not the first examination in the study time frame, so that data of 371 different patients with at least one focal liver lesion ≥ 1 cm other than cysts were included. Of these 371 patients, 262 (70.6%) were excluded due to lack of a definitive diagnosis based on histology or typical imaging characteristics (n = 46), local treatment of the liver (n = 143), insufficient image quality caused by motion artifacts (n = 27) or pixel misalignments (n = 5), unfavourable lesion location as close to prior biopsy or drainage tracts or at the edge of the liver (n = 6), partial volume of an adjacent slice (n = 10), or difficulties to identify the lesions on DWI (n = 5). In the presence of a combination of (non-cystic) benign lesions and malignant disease, patients were excluded because malignant disease may affect the appearance of benign liver lesions, e.g., due to thrombosis (n = 20). Finally, data of 109 patients were analysed  of patients, who received a 4 These patients had already been examined in an upcoming study by Mesropyan et al and in a previous study , where bCholangiocellular carcinomas (CCCs) were histologically proven. Hepatocellular carcinomas (HCCs) were either histologically proven or diagnosed according to the American Association for the Study for Liver Disease MRI criteria . Diagnosb values  was applied before contrast agent administration. Isotropic diffusion-weighted images were reconstructed by from the images with diffusion-sensitised gradients in three orthogonal directions on the MRI system.Imaging was performed on a clinical whole-body 1.5-T MRI system  equipped with powerful gradient system  and 32-channel abdominal coil with digital interface for signal reception. DWI with a respiratory-triggered single-shot spin-echo echo-planar imaging variant  were calculated offline using custom written software in MATLAB .b-800 images were excluded in an upcoming study by Mesropyan et al. After visually cross-checking for pixel misalignments between images with different b values, the ROI was analysed in the related parameter maps ADC, D′, and f′ and saved for later use (see below).Image analysis by ROIs was performed by a radiologist (N.M.) with 3 years of experience and checked by a radiologist (C.C.P.) with 10 years of experience in abdominal imaging and a physicist (P.M.) with more than 20 years of experience in DWI. All were blinded to clinical information. One reference lesion per lesion type was analysed. A two-dimensional ROI was placed centrally in each lesion on a single representative slice. This slice was largely unaffected by motion and susceptibility artifacts and pixel misalignments and not at the rim of the lesion to avoid partial volume effects. ROIs were drawn as large as possible using DWI with the highest contrast between lesion and normal tissue. Central necrosis, cystic components, and scars as found by hyperintensities on b0 images and/or hypointensities on ADC, ID, and If were constructed from suitable cutoff values for ADC, D′, and f′, respectively. The cutoff values were determined as previously introduced [D and If, the index map IDf was generated. For IDf, a voxel value was set to 100 if the corresponding voxel values of ID and If were both 100; otherwise, the voxel value was set to 0. Voxel values 0 and 100 were displayed in green and red, respectively, indicating benign and malignant structures. These index maps were displayed as overlay over the DWI b-800 images.Two-colour index maps Itroduced , 15: thetroduced , 28 and troduced , 15, 29,ADC, ID, and If, the same ROC analysis was performed as for the related original parameter (see the "Construction of two-colour index maps" section). AUC values were compared pairwise. Second, to compare the diagnostic performance of ADC, D′, and f′ as well as of IDf, IADC, and ADC, all maps were quantitatively analysed using ROIs . ROC analyses of the benign and malignant lesion groups were then performed. AUC values were compared with each other.First, to ensure that the voxel-wise consideration of the cutoff values does not worsen diagnostic performance compared to ROI-wise, for IADC and IDf index maps were evaluated visually by one investigator (P.M.). The visual assessment was restricted to areas of translucent hyperintensity from DWI b-800 images, whereby necrosis, cystic components, and scars identified as hyperintense areas on b-0 images and/or hypointense areas on b-800 images were excluded. A four-point scale was used, as follows: (1) definitely malignant, if the red voxels dominated definitely; (2) probably malignant, if red voxels dominated only slightly; (3) probably benign, if green voxels dominated only slightly; and (4) definitely benign, if green voxels dominated definitely.Third, the IADC and IDf for lesion differentiation by visual assessment was determined and compared with each other and with ADC.The accuracy of IThe assessment was repeated after 4 months by the same investigator (P.M.) and by a second independent investigator (C.C.P.).p < 0.05) between groups (independent samples) were tested in SPSS  by using Student t test or non-parametric Mann–Whitney U test, depending on whether the data were normally distributed or not. In order to differentiate between two groups, ROC analysis was performed using pROC package in R  [intra) and of the two different investigators (ICCinter).Statistically significant differences (License) . Youden’License) . The intExamples of DWI and two-colour index maps are given in Figs. ADC and ADC in discriminating benign from malignant lesions , nor between ID and D′ in discriminating haemangiomas from all other lesions , nor between If and f′ in discriminating FNHs and all other lesions .ROC analysis of ROI-based analysed index maps and related original parameter maps revealed similar AUC values Table . The come.g., for ADC 1,124 ± 180 × 10-6 mm2/s (mean ± standard deviation) versus 1,692 ± 313 × 10-6 mm2/s (p < 0.001). Accordingly, for all index values, the numbers of red voxels were significantly higher for malignant than for benign lesions, e.g., for IADC 80% ± 21% versus 17% ± 25% (p < 0.001) and for IDf 76% ± 17% versus 20% ± 18% (p < 0.001).All parameter values were significantly lower for malignant lesions than for benign Table , e.g., fversus 0.902, p = 0.001) and f′ , AUC of D′ was significantly higher than that of f’ . By ADC, 89.9% of the lesions were correctly identified as malignant and benign . By using the combination of D′ and f′, 93.6% of the lesions were correctly identified , which was an improvement compared to ADC.As can be seen in Table Df and IADC, larger values were found for IDf than for IADC (0.975 versus 0.945), but differences were not significant (p = 0.168). The diagnostic accuracy was higher for IDf than for IADC. With IDf 93.6% of the lesions  were correctly identified as benign and malignant, with IADC 88.1% . Falsely identified cases by IDfversus IADC were 1 versus 2 FNHs, 1 versus 1 haemangiomas, 4 versus 3 HCCs, 0 versus 1 CCCs, and 1 versus 6 metastases. IDf was superior to IADC especially in case of metastases identifying 5 cases correctly as malignant, which were falsely assigned as benign by IADC.Comparing the AUC values of IDf and IADC maps within translucent hyperintensity from DWI b-800 images (Table Df and 90.8% instead of 88.1% for IADC). As in the quantitative analysis, the reached diagnostic accuracy was higher for IDf than for IADC. With IDf 95.4% of the lesions were correctly identified, with IADC 90.8%. The assignment was “definite” in 87.2% for IDf and in 89.9% for IADC and “probable” in 12.8% for IDf and in 10.1% for IADC. “Probable” assignment by IDf and IADC was mainly found for FNHs  and HCCs  and only rarely for haemangiomas , CCCs , and metastases . Falsely identified cases by IDfversus IADC were 2 versus 5 FNHs, 1 versus 1 haemangiomas, 1 versus 3 HCCs, 0 versus 0 CCCs, and 1 versus 1 metastasis. IDf was superior to IADC especially in case of FNHs and HCCs identifying 3 FNHs and 2 HCCs correctly, which were falsely assigned by IADC. Examples are given in Fig. Df was superior especially in case of HCCs identifying 4 HCCs correctly, which were falsely assigned by quantitative analysis. Visual judgment of IADC was superior especially in case of metastases but inferior in case of FNHs identifying 5 metastases correctly, which were falsely assigned by quantitative analysis, and 3 FNHs falsely , which were correctly identified by quantitative analysis.By visual judgment of Ies Table , a similintra 0.992 for IADC and 0.989 for IDf; ICCinter 0.986 for IADC and 0.977 for IDf).The repeated analysis by the same investigator and by the independent investigator . Visual judgment of the IDf index map as two-colour overlay to b-800 images showed comparable diagnostic accuracy than quantitative analysis of IDf.In this study, simplified IVIM was used to create combined two-colour index maps Iversus 82.1%) and for combined D′ and f′ (93.6% versus 85.6%). In the present study, one reference ROI per lesion type and patient was included, in the previous study up to 5 lesions per lesion type and patient were included and averaged for analysis . Necrotic areas, liquids, and scares were excluded from ROIs in both studies, but can also be excluded retrospectively by automatically selecting voxels with low diffusion coefficients with the help of histogram analysis of D′ .In previous simplified IVIM studies on liver lesions at 1.5 and 3.0 T it was found that ADC is the best single parameter to discriminate between malignant and benign liver lesions but that improved discriminatory power could be reached by combined use of D′ and f′ , 15. ThiDf, which combine the information from D′ and f′, use only two colours, and are presented as overlay to b-800 in order to be able to assess only the vital tumour areas by translucent hyperintensity and to exclude necrosis, cystic components, and scars from assessment. Up to now, colour-coded maps with more than two colours have been used for the different IVIM parameters [Df was not clear in advance. Perfusion and diffusion restrictions do not necessarily have to occur in the same voxels. But the fact that IDf provided identical diagnostic accuracy than combined use of D′ and f′ (93.6% versus 93.6%) means that the ROI-wise obtained cutoff values of the parameters can be applied voxel-wise in the index maps.New in the present work is the creation and evaluation of the index maps Irameters , 22, 32,rameters , 15. WheDf higher accuracy was reached than for IADC, by quantitative analysis (93.6% versus 88.1%) and by visual judgment (95.4% versus 90.8%). The relative good performance of IADC is due to the fact that for liver lesion differentiation diffusion and perfusion influences act in the same direction.For IDf (95.4% versus 93.6%) and IADC (90.8% versus 88.1%). The assignment malignant/benign was “definite” in about 90% of the cases and “probable” in about 10%, for IDf and IADC. Some of the FNHs showed relatively high numbers of red voxels on IDf with scattered distribution caused by heterogeneous perfusion as can be seen on If index map. Those FNHs looked similar to typical HCCs  and the determination of D* [e.g., patients with hemochromatosis or fatty liver), small lesions , or presence of artifacts. Due to motion influences artificially enlarged D′ and reduced f′ values may be measured, especially for the left liver lobe and on slices close to the heart. It is important to check the surrounding liver in the b-800 image for signal loss (Fig. Df were checked on the same patients who provided the cutoff points for generation in order to enable a direct comparison with the combined use of the parameter maps D′ and f′. A validation study is planned on a larger patient cohort, which also includes rarer and atypical lesions as well as lesions difficult to identify in DWI. It is interesting to compare the use of the two-colour index maps IDf with full set of conventional protocol in terms of reading time and reader confidence.General concerns regarding the simplified IVIM approach as for example the b value choice have already been addressed in the previous studies , 15. Onlon of D* , 15. Simb value 50 s/mm2 Df and the ROI-based combination of D′ and f′ parameters provide concordant diagnostic accuracy for the differentiation of malignant and benign liver lesions. The IDf index map used as two-colour overlay to b-800 images can be considered as a new tool for visual assessment of liver lesion malignancy.In conclusion, the voxel-wise combined index maps I