Morphometric analysis of the skull base and palatal regions for gender identification using CBCT: a retrospective study.

Objectives: The objectives of this study were to evaluate the accuracy of morphometry of skull base and palate in gender discrimination using cone beam computed tomography (CBCT) scanning and to assess the accuracy of the results among a sample of the Arab population. Materials & Methods: Using CBCT scans, a cross-sectional analysis was conducted on 142 consented patients who underwent various dental procedures at the University Dental Hospital, Sharjah (UDHS). Of these patients, 70 were females and 72 were males, with respective means of 38.5 and 36.2 years. Eleven parameters related to skull base and palatal region were measured on the CBCT scans by two expert radiologists followed by statistical analysis. Results: There was significant gender-based difference in the mean palatal width (PW) (p = 0.001), mean palatal height (PH) (p = 0.005). Among other skull base region parameters that were significant in term of gender-based difference like; the clivus length (CL) (p < 0.001), occipital condyle height (OCH) (p < 0.001), basal angle (BA) (p = 0.006) and transverse diameter of foramen magnum (p = 0.003). Only palate variables showed a significant age difference. Discriminant analysis related to gender showed that occipital condyle height was the most accurate and best discriminator among the skull base region parameters. Conclusion: The use of discriminant analysis in CBCT based on skull base and palatal region variables provides an efficient method for determining gender, which is particularly valuable in forensic science and anthropological research. Significance of study: Accurate gender identification is crucial in forensic investigations, and the skull base region, being a stable and sexually dimorphic anatomical feature, can serve as a reliable marker for this purpose.

Inter and intra-island genetic structure and differentiation of the endemic Bolle's Laurel Pigeon (Columba bollii) in the Canary archipelago.

Islands provide excellent settings for studying the evolutionary history of species, since their geographic isolation and relatively small size limit gene flow between populations, and promote divergence and speciation. The endemic Bolle's Laurel Pigeon Columba bollii is an arboreal frugivorous bird species distributed on laurel forests in four islands of the Canary archipelago. To elucidate the population genetics, we genotyped ten microsatellite loci using DNA obtained from non-invasive samples collected across practically all laurel forest remnants, and subsequently grouped into eight sampling sites. Analyses including F-statistics, Bayesian clustering approaches, isolation by distance tests and population graph topologies, were used to infer the genetic diversity and the population differentiation within and among insular populations. Additionally, we evaluated the effect of null alleles on data analysis. Low genetic diversity was found in all populations of Bolle's Laurel Pigeon, with no significant differences in diversity among them. However, significant genetic differentiation was detected among all populations, with pigeons from La Palma and El Hierro exhibiting the closest affinity. Bayesian clustering supported population separation between islands, and also detected fine-scale structure within the Tenerife and La Gomera populations. Our results suggest that, despite columbids have a high movement ability, they can show signature of genetic divergence among populations, particularly on oceanic islands. Geological history of the islands and distribution range of habitats could have close influence on the evolutionary trajectories of these birds. This approach can provide practical tools to implement appropriate conservation measures for range-restricted species and their habitat.

Evaluation of the Effectiveness of Sex Determination in the Great Cormorant (Phalacrocorax carbo) Based on External Measurements.

The possibility of sex identification of birds has substantial importance for studies on different aspects of bird ecology and behaviour. Using discriminant functions is becoming increasingly popular in studies of bird species that are monomorphic in plumage characteristics because they are cheap, hardly invasive and may be applied to data collected in the past. In this paper, we provide a discriminant function to sex great cormorants using external measurements. Males were larger than females in all linear body measurements, but there were no significant differences between adults and juveniles. Thus, data on juveniles and adults within a sex was combined. Discriminant equations with the most commonly used linear measurements, wing length and bill length, were provided. If identifying birds with discriminant function values D2 < -1.256 as females and those with D2 > 0.916 as males, 99% of birds will be correctly sexed. The method presented here makes it possible to account for sex-specific patterns in ecological studies of the great cormorant and may be applied to data collected in the past. The cross-application of discriminant functions developed for other populations of the great cormorant produces a 5.4% and 7.5% misclassification rate for birds from northern Poland using discriminant equations developed for populations in Greece and the Netherlands, respectively.

Non-destructive prediction of fertility and sex in chicken eggs using the short wave near-infrared region.

The objective of this study was to evaluate the ability of a handheld near-infrared device (900-1600 nm) to predict fertility and sex (male and female) traits in-ovo. The NIR reflectance spectra of the egg samples were collected on days 0, 7, 14 and 18 of incubation and the data was analysed using principal component analysis (PCA), linear discriminant analysis (LDA) and support vector machines classification (SVM). The overall classification rates for the prediction of fertile and infertile egg samples ranged from 73 % to 84 % and between 93 % to 95 % using LDA and SVM classification, respectively. The highest classification rate was obtained on day 7 of incubation. The classification between male and female embryos achieved lower classification rates, between 62 % and 68 % using LDA and SVM classification, respectively. Although the classification rates for in-ovo sexing obtained in this study are higher than those obtained by chance (50 %), the classification results are currently not sufficient for industrial in-ovo sexing of chicken eggs. These results demonstrated that short wavelengths in the NIR range may be useful to distinguish between fertile and infertile egg samples at days 7 and 14 during incubation.

Comparison three primer pairs for molecular sex determination in Eurasian pygmy owls (Glaucidium passerinum).

Bird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum). We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing. The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively. The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls' laboratory sexing by qPCR with CHD1F/CHD1R primers only.

Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential.

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions.

The global expansion of Aedes albopictus has stimulated the development of environmentally friendly methods aiming to control disease transmission through the suppression of natural vector populations. Sterile male release programmes are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation which can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic Wolbachia-free Ae. albopictus line. We then measured the penetrance of cytoplasmic incompatibility and life-history traits of both transinfected lines, selected the wPip-IV line and combined it with the genetic sexing strain. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Two novel PCR-based assays for sexing of Silene latifolia and Silene dioica plants.

Silene latifolia and S. dioica are model systems in studies of plant reproduction, chromosome evolution and sexual dimorphism, but sexing of plants based on morphology is only possible from flowering stage onwards. Both species show homogametic females (XX) and heterogametic males (XY).•Here we developed two assays (primer pairs ss816 and ss441) for molecular sexing of S. latifolia and S. dioica, targeting length polymorphisms between the X and Y-linked copies of the spermidine synthase gene.The two assays were successful in identifying known (flowering-stage) males and females from UK and Spanish populations, with an error rate of 3.1% (ss816; successful for both species) and 0% (ss441, only successful for S. latifolia). Our assays therefore represent novel tools for rapid, robust and simple determination of the genotypic sex of S. latifolia and S. dioica.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing.

The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.

Molecular sexing of birds using quantitative PCR (qPCR) of sex-linked genes and logistic regression models.

The ability to sex individuals is an important component of many behavioural and ecological investigations and provides information for demographic models used in conservation and species management. However, many birds are difficult to sex using morphological characters or traditional molecular sexing methods. In this study, we developed probabilistic models for sexing birds using quantitative PCR (qPCR) data. First, we quantified distributions of gene copy numbers at a set of six sex-linked genes, including the sex-determining gene DMRT1, for individuals across 17 species and seven orders of birds (n = 150). Using these data, we built predictive logistic models for sex identification and tested their performance with independent samples from 51 species and 13 orders (n = 209). Models using the two loci most highly correlated with sex had greater accuracy than models using the full set of sex-linked loci, across all taxonomic levels of analysis. Sex identification was highly accurate when individuals to be assigned were of species used in model building. Our analytical approach was widely applicable across diverse neognath bird lineages spanning millions of years of evolutionary divergence. Unlike previous methods, our probabilistic framework incorporates uncertainty around qPCR measurements as well as biological variation within species into decision-making rules. We anticipate that this method will be useful for sexing birds, including those of high conservation concern and/or subsistence value, that have proven difficult to sex using traditional approaches. Additionally, the general analytical framework presented in this paper may also be applicable to other organisms with sex chromosomes.

In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848.

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos.

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 µL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

Method for the molecular and quantitative identification of oocytes and their developmental stage in teleost fish gonads.

Fish display diverse reproductive strategies and their gametogenesis is influenced by numerous genetic, physiological and environmental factors. The analysis of 5S rRNA expression levels in gonads has been proposed as useful method for the molecular identification of the presence of oocytes in fish tissues. The present method provides an easy and unbiased approach to analyse the expression of tRNAs and 5S rRNA in teleost gonads and stablish the presence and developmental stage of oocytes. Total RNA extracted from gonads is analysed through capillary electrophoresis in a Bioanalyzer 2100 (Agilent Technologies) using Small RNA Assays. Electropherograms allow quantifying the concentrations of tRNAs, 5S rRNA and 5.8S rRNA per sample and calculate their tRNA/5.8S rRNA and 5S/5.8S rRNA indices. Both indices clearly differentiate ovaries from testes and can be used to identify testes that present oocytes due to exposure to environmental xenoestrogens. The tRNA/5.8S and 5S/5.8S indices show the highest values in ovaries in previtellogenic stage, values decreasing as they advance towards maturity.•Detailed molecular method to sex fish and quantitatively identify the maturity stage of females.•tRNA levels in gonads can help in the study of teleost reproduction (female fecundity assessment, molecular gonad sexing) and environmental health assessment.

Accurate Bayesian inference of sex chromosome karyotypes and sex-linked scaffolds from low-depth sequencing data.

The identification of sex-linked scaffolds and the genetic sex of individuals, i.e. their sex karyotype, is a fundamental step in population genomic studies. If sex-linked scaffolds are known, single individuals may be sexed based on read counts of next-generation sequencing data. If both sex-linked scaffolds as well as sex karyotypes are unknown, as is often the case for non-model organisms, they have to be jointly inferred. For both cases, current methods rely on arbitrary thresholds, which limits their power for low-depth data. In addition, most current methods are limited to euploid sex karyotypes (XX and XY). Here we develop BeXY, a fully Bayesian method to jointly infer the posterior probabilities for each scaffold to be autosomal, X- or Y-linked and for each individual to be any of the sex karyotypes XX, XY, X0, XXX, XXY, XYY and XXYY. If the sex-linked scaffolds are known, it also identifies autosomal trisomies and estimates the sex karyotype posterior probabilities for single individuals. As we show with downsampling experiments, BeXY has higher power than all existing methods. It accurately infers the sex karyotype of ancient human samples with as few as 20,000 reads and accurately infers sex-linked scaffolds from data sets of just a handful of samples or with highly imbalanced sex ratios, also in the case of low-quality reference assemblies. We illustrate the power of BeXY by applying it to both whole-genome shotgun and target enrichment sequencing data of ancient and modern humans, as well as several non-model organisms.

Assessing the Effectiveness of Sex-Linked Molecular Markers to Identify Neomale Breeders for the Production of All-Female Progenies of Rainbow Trout.

A cultured stock of masculinized rainbow trout was diagnosed with Y-linked markers (sdY and OmyY1) aiming to detect neomales before their use at the production level. To achieve a reliable diagnosis, the following steps were considered: (1) PCR amplification of the housekeeping ß-actin gene to determine the DNA quality of samples, (2) validation of the Y-linked markers by their PCR amplification in male and female samples with known sex, and (3) molecular sexing of the masculinized juveniles based on male-specific (XY genotype) and neomale-specific (XX genotype) PCR product band patterns visualized on agarose gel. The validity and concordance of the markers were assessed. The housekeeping gene identified samples with negative PCR amplification revealing a poor DNA quality. The OmyY1 marker presented a more distinctive PCR product band pattern between males and females than the sdY marker and identified a higher proportion of true males (sensitivity = 1.0 and 0.91, respectively). The OmyY1 marker accurately identified 105 neomales of the 198 masculinized individuals on account their consistent and distinctive PCR product band pattern. Among both markers, there was a medium high positive concordance (γ index = 0.7). It is concluded that the OmyY1 marker shows the best performance to reliably detect neomales, a step that is essential to have certified breeders for the production of all-female progenies in fish farming.

Sustainable animal production: exploring the benefits of sperm sexing technologies in addressing critical industry challenges.

The sex of the animals is of paramount importance in many animal production systems. This is particularly evident in the production of milk or in breeding programs focused on the production of female animals. In some cases, slaughter or euthanasia of animals of the unwanted sex becomes the only solution, highlighting ethical and economic concerns. As global demand for food continues to rise, the importance of addressing these issues becomes more evident. Reproductive technologies, such as sperm sexing techniques, may hold the key to addressing both animal welfare and the sustainability of animal production. The use of semen enriched with sperm capable of producing offspring of the desired sex can serve as a valuable tool for producers to exert greater control over production outcomes, not only helping to mitigate welfare issues related to the unnecessary premature death of unwanted offspring but also providing a possible ally in the face of stricter animal welfare guidelines. In addition, sexed semen can also contribute to financial gains and reduce greenhouse gas emissions and food waste associated with the less profitable part of the herd. This paper explores the positive impacts that sperm sexing can have on animal welfare, economy, and environment. It also discusses currently available options and strategies for more successful implementation of sexed semen. Partnerships between companies and scientists will be essential to find innovative ways to adapt current production systems and develop sperm sexing technologies that apply to most livestock industries.

Genotyping blastoderms of avian eggs.

Undeveloped eggs occur frequently in birds and are often considered infertile, and discarded. However, the majority of undeveloped eggs may in fact have been fertilised and embryos might have died at an early stage. Such eggs contain valuable information, for example about offspring sex and paternity, and level of inbreeding. Obtaining such information may also give insight into the patterns and causes of early embryo mortality. Here we describe a simple technique for removing embryo cells from the blastoderm to obtain DNA to genotype the offspring and unequivocally ascertain fertilisation status, while retaining the overlying perivitelline layer (PVL) for sperm counts over the entire membrane. We tested this method on freshly collected eggs (high-quality material), as well as on eggs from abandoned clutches and unhatched eggs (potentially deteriorated material) of blue tits (Cyanistes caeruleus). We sampled a total of 707 eggs from a wild population of blue tits, extracted DNA from the eggs' blastoderm using a Qiagen kit, and genotyped the samples with 14 polymorphic microsatellite markers, plus one sexing marker. Overall, we successfully genotyped 97% of all eggs. Our study is the most extensive dataset of genotyped undeveloped eggs to date and demonstrates that one can reliably genotype undeveloped fertile eggs as well as retain the PVL for observations of sperm and embryo cells.

Minimally Invasive Sampling Methods for Molecular Sexing of Wild and Companion Birds.

Birds are highly social and must be paired in order to increase their welfare. Most bird species are monomorphic; therefore, molecular sexing helps provide appropriate welfare for birds. Moreover, early sex determination can be of great value for bird owners. The aim of this study was to demonstrate that sex identification in birds achieved using molecular methods and samples collected via minimally invasive methods is fast, efficient, and accurate. A total of 100 samples (29 paired samples of feathers and oral swabs and 14 tripled samples of feathers, oral swabs, and blood) from 43 birds were included in this study, as follows: wild birds (Falconiformes, Accipitriformes, landfowl-Galliformes, waterfowl-Anseriformes) and companion birds (Passeriformes, Psittaciformes-large-, medium-, and small-sized parrots). Amplification of CHD1-Z and CHD1-W genes was performed via conventional PCR. The results obtained from feathers were compared to those obtained from oral swabs and to those obtained from blood samples, where applicable. The obtained results show that all types of samples can be used for molecular sexing of all studied bird species. To the best of our knowledge, the present study reports, for the first time, molecular sex identification in Red Siskin (Carduelis cucullata) and Goldfinch (Carduelis carduelis major). For higher accuracy, our recommendation is to use minimally invasive samples (oral swabs and feathers) and to test both types of samples for each bird instead of blood samples.

Long-term female bias in sex ratios across life stages of Harpy Eagle, a large raptor exhibiting reverse sexual size dimorphism.

The primary (PSR), secondary (SSR) and adult (ASR) sex ratios of sexually reproducing organisms influence their life histories. Species exhibiting reversed sexual size dimorphism (RSD) may imply a higher cost of female production or lower female survival, thus generating biases in PSR, SSR and/or ASR towards males. The Harpy Eagle is the world's largest eagle exhibiting RSD. This species is found in the Neotropical region and is currently threatened with extinction. We used molecular markers to determine the sex of 309 Harpy Eagles spanning different life stages-eaglets, subadults and adults-from 1904 to 2021 within the Amazon Rainforest and Atlantic Forest. Sex ratios for all life stages revealed a female-biased deviation across all periods and regions. Our results suggest that the population bias towards females is an evolutionary ecological pattern of this species, and SSR and ASR likely emerged from the PSR. This natural bias towards females may be compensated by an earlier sexual maturation age of males, implying a longer reproductive lifespan and a higher proportion of sexually active males. A better understanding of the Harpy Eagle's life history can contribute to understanding sex-role evolution and enable more appropriate conservation strategies for the species.

Identification of sex-linked SNP markers in wild populations of monomorphic birds.

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z- and W-linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W-linked SNP and are heterozygous or homozygous for the Z-linked SNP, while females exhibit both Z- and W-linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex-linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques.