Morphometric dataset of Varanus salvator for non-invasive sex identification using machine learning.

Reliable sex identification in Varanus salvator traditionally relied on invasive methods like genetic analysis or dissection, as less invasive techniques such as hemipenes inversion are unreliable. Given the ecological importance of this species and skewed sex ratios in disturbed habitats, a dataset that allows ecologists or zoologists to study the sex determination of the lizard is crucial. We present a new dataset containing morphometric measurements of V. salvator individuals from the skin trade, with sex confirmed by dissection post- measurement. The dataset consists of a mixture of primary and secondary data such as weight, skull size, tail length, condition etc. and can be used in modelling studies for ecological and conservation research to monitor the sex ratio of this species. Validity was demonstrated by training and testing six machine learning models. This dataset has the potential to streamline sex determination, offering a non-invasive alternative to complement existing methods in V. salvator research, mitigating the need for invasive procedures.

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.

Determinação do sexo a partir da morfometria geométrica e mensurações em imagens tridimensionais de dentes caninos / Sex determination from geometric morphometry and measurements in three-dimensional images of canine teeth

O presente estudo teve como objetivo avaliar a determinação do sexo por meio da análise geométrica e de mensurações a partir de imagens tridimensionais de dentes caninos, advindas de escaneamento intraoral, no Autodesk MeshMixer, bem como elaborar um roteiro prático para servir de guia na manipulação e coleta de dados nesse software. Trata-se de um estudo do tipo cego e transversal, dividido em duas etapas. Inicialmente, foram selecionadas imagens de 127 caninos (65 femininos e 63 masculinos) para a análise morfométrica geométrica, utilizando a marcação de 50 pontos, distribuídos em 27 pontos para contorno e 23 pontos para delimitação da forma das superfícies vestibular e lingual. Cada ponto de referência recebeu um valor para as coordenadas x, y e z, representando a sua posição. Na análise estatística dos dados foi utilizado um software específico para análise morfométrica geométrica, o MorphoJ, no qual foram importados os dados e submetidos à Análise Ajustada de Procrustes, Análise Discriminante e Análise da Variação Canônica. Não foram exibidas diferenças significativas para distribuição do sexo nas analises anteriores. Ainda, os dados relativos ao tamanho do centroide gerado no MorphoJ foram importados para o IBM SPSS Statistics 22.0 e submetidos ao teste t student, resultando em uma diferença estatística para o sexo. Para a aplicabilidade das mensurações digitais, foram utilizadas imagens de 345 caninos (191 femininos e 154 masculinos). Foram realizadas medidas das distâncias mesiodistais, vestibulolinguais, cervicoincisais (altura) e distâncias intercaninas de 5 pontos estabelecidos em cada canino (cúspide, cervical por vestibular e lingual, ponto máximo mesial e lingual). Todas as médias foram maiores para o sexo masculino e, com exceção da altura do elemento 33, apresentaram diferenças significativas para o sexo (p<0,05). As mensurações mesiodistais, vestibulolinguais e intercaninas (com exceção do ponto cervical por lingual), apresentaram uma boa acurácia, representadas por valores de áreas sobre a curva ROC maiores do que 0,7. O Autodesk MeshMixer apresentou-se como um ótimo auxiliar e de grande contribuição para análises dentro da Antropologia Forense. Apesar dos caninos serem elementos altamente dimórficos, para a análise morfométrica, não foram exibidas diferenças estatísticas na análise discriminante. Porém, o tamanho do centroide apresentou diferenças estatísticas e com médias maiores para o sexo masculino. As medidas realizadas nos dentes caninos apresentaram diferenças estatísticas para o sexo, com boas classificações na análise discriminante e com boas predições sexuais de acordo com os valores de especificidade e sensibilidade. A aplicabilidade de técnicas digitais de mensuração exibiu bons resultados, concordando esses dados obtidos com estudos que utilizaram as técnicas manuais de medida.

SRY gene isolation from teeth for forensic gender identification-An observational study.

Personal identification in forensics is possible with gender determination using DNA (deoxyribonucleic acid) analysis. DNA isolation from teeth samples subjected to extreme temperatures has been shown to predict the gender of the deceased. However, the literature lacks studies on DNA extracted from tooth samples exposed to freezing temperatures. This study aimed to isolate the SRY gene from the extirpated pulp of teeth that were subjected to varying temperatures for gender identification. Thirty teeth with vital pulps, divided into 3 groups were included in the study. Each group consisted of 5 male and 5 female tooth samples. The groups were exposed to diverse environmental factors for three weeks. Group 1: room temperature (R group); Group 2: high temperature (H group) and Group 3: freezing temperature (F group). Later, DNA was isolated from the pulp tissue, and the SRY gene was amplified using PCR (Polymerase Chain Reaction). The Sensitivity and Specificity of the results were analyzed. SRY gene detected in the study samples identified accurate gender with a 46.70% Sensitivity and 93.30% Specificity. Significant difference was found in the correlation between gene expression and gender among the three groups (p = 1.000). The study validates that dental pulp tissue can be a reliable source for DNA extraction. And SRY gene amplification from teeth exposed to diverse environmental conditions. Further investigations are required to validate its application in forensics.

Palatal rugae as an unique and stable marker in personal identification-An interracial pilot study.

Background: In post-mortem scenarios, often it is a very difficult process to establish a person's identity. Rugae are unique in that they are protected from trauma as they are insulated from heat by tongue and buccal pad of fat unlike fingerprint or lip print that is prone to destruction. Aim and Objectives: This study was aimed to compare the palatal rugae among people of different races. The sole objectives of the study were to assess the predominant pattern in the selected groups, reliability of rugae pattern in personal identification, to evaluate reliability of sex determination and to compare the total number of rugae on right and left sides of the palate among the males and females. Study Design: A total of 90 subjects were enrolled into the study and divided into three groups that are African, Dravidian and Mongoloid population. Shapes of rugae present were analyzed according to the classification given by Kapali et al. (1997) and Thomas & Kotze (1983). Result: The predominant rugae shape in African and Dravidian population was wavy pattern, whereas Mongoloid race was predominant in curve pattern. African and Dravidian males were predominant in wavy pattern when compared to Mongoloid males where unification type was more predominant. Conclusion: A statistically significant association between the rugae shape in three populations exists, although subtle yet definite.

A review of the recent advances for the in ovo sexing of chicken embryos using optical sensing techniques.

The culling of day-old male chicks has caused ethical and economic concerns. Traditional approaches for detecting the in ovo sex of chicken embryos involve opening the eggshell and inner membrane, which are destructive, time-consuming, and inefficient. Therefore, noncontact optical sensing techniques have been examined for the in ovo sexing of chicken embryos. Compared with traditional methods, optical sensing can increase determination throughput and frequency for the rapid sexing of chicken embryos. This paper presented a comprehensive review of the different optical sensing techniques used for the in ovo sexing of chicken embryos, including visible and near-infrared (Vis-NIR) spectroscopy, hyperspectral imaging, Raman spectroscopy, fluorescence spectroscopy, and machine vision, discussing their advantages and disadvantages. In addition, the latest research regarding different detection algorithms and models for the in ovo sexing of chicken embryos was summarized. Therefore, this paper provides updated information regarding the optical sensing techniques that can be used in the poultry industry and related research.

SNP Panel and Genomic Sex Identification in Atlantic Halibut (Hippoglossus hippoglossus).

The ability to identify sex is necessary in population biology for a proper understanding of the dynamics of a population. In Atlantic halibut, phenotypic sex identification is not possible due to the lack of significant external morphological differences. We developed an Illumina SNP panel for Atlantic halibut with 4000 SNPs spread evenly throughout the genome with a minor allele frequency MAF ≥ 0.4, except for N = 249 SNPs located in a sex-determining region on chromosome 12, N = 176 of these SNPs were selected to genetically identify male and female individuals using a DAPC analysis. The genomic identification of sex allows for non-lethal sex determination and validation of sex identification in the field. The SNP panel is a new genomic resource for Atlantic halibut that will make it possible to generate the genotypic data for the large number of individuals needed to estimate population abundance using genomics and the Close Kin Mark Recapture (CKMR) approach, an emerging component of fisheries management and stock monitoring.

Bioimpedance-Measurement-Based Non-Invasive Method for In Ovo Chicken Egg Sexing.

Day-old male chick culling is one of the world's most inhumane problems in the poultry industry. Every year, seven billion male chicks are slaughtered in laying-hen hatcheries due to their higher feed exchange rate, lower management than female chicks, and higher production costs. This study describes a novel non-invasive method for determining the gender of chicken eggs. During the incubation period of fourteen days, four electrodes were attached to each egg for data collection. On the last day of incubation, a standard polymerase chain reaction (PCR)-based chicken gender determination protocol was applied to the eggs to obtain the gender information. A relationship was built between the collected data and the egg's gender, and it was discovered to have a reliable connection, indicating that the chicken egg gender can be determined by measuring the impedance data of the eggs on day 9 of incubation with the four electrodes set and using the self-normalization technique. This is a groundbreaking discovery, demonstrating that impedance spectroscopy can be used to sex chicken eggs before they hatch, relieving the poultry industry of such an ethical burden.

Utilization of a Gender-Based Sorting Machine for Crustacean Selection in Bioconcentration Studies with the Freshwater Amphipod Hyalella azteca.

Bioconcentration factors (BCFs) are determined by fish flow-through tests performed according to Organisation for Economic Co-operation and Development test guideline 305. These are time-consuming and expensive and use a large number of animals. An alternative test design using the freshwater amphipod Hyalella azteca for bioconcentration studies has been recently developed and demonstrated a high potential. For bioconcentration studies using H. azteca, male amphipods are preferred compared with female organisms. Manual sexing of male adult amphipods is, however, time-consuming and requires care and skill. A new fully automatic sorting and dispensing machine for H. azteca based on image analysis has recently been developed by the company Life Science Methods. Nevertheless, an anesthesia step is necessary prior to the automatic selection. In the present study, we show that a single-pulse of 90 min of tricaine at the concentration of 1 g/L can be used and is recommended to select H. azteca males manually or automatically using the sorting machine. In the second part, we demonstrate that the machine has the ability to select, sort, and disperse the males of a culture batch of H. azteca as efficiently as manual procedures. In the last part of the study, BCFs of two organic substances were evaluated using the H. azteca bioconcentration test (HYBIT) protocol, with an anesthetizing step and robotic selection compared with manual selection without an anesthetizing step. The different BCF values obtained were in accordance with those indicated in the literature and showed that an anesthetizing step had no effect on the BCF values. Therefore, these data validated the interest in this sorting machine for selecting males to perform bioconcentrations studies with H. azteca. Environ Toxicol Chem 2023;42:1075-1084. © 2023 SETAC.

Highly sensitive and quick in ovo sexing of domestic chicken eggs by two-wavelength fluorescence spectroscopy.

The in ovo sexing of chicken eggs is a current task and a prerequisite to overcome the mass killing of male day-old chicks from laying lines. Although various methods have been developed and tested in recent years, practicable methods for sex determination are still missing which can be applicated in poultry hatcheries before the chicken embryo is capable of nociception and pain sensation. Optical spectroscopic methods enable an early determination of the sex. In this study, a novel method based on two-wavelength in ovo fluorescence excitation is described. More than 1600 eggs were examined. In ovo fluorescence was sequentially excited at 532 nm and 785 nm. The fluorescence intensities of the spectral regions behave inversely with respect to sex. It is shown that the observed sex-related differences in the fluorescence intensities are based on the embryonic hemoglobin synthesis. The accuracy of sex determination is 96% for both sexes. The hatching rate is not reduced compared to an equivalent reference group.

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene.

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples. Previous studies mainly used sex differences in an intron of the amelogenin gene. However, this region is highly polymorphic, and there are cases where accurate sexing cannot be performed because of genetic mutations in the target region. Thus, for reliable sex determination, it is desirable to select loci with as few non-sexual polymorphisms as possible. In this study, we focused on the exon 1 region of the amelogenin gene, which has very little polymorphism other than sex differences. We developed a primer set for sex determination and compared it with the GlobalFiler™ PCR Amplification Kit (GF), which is widely used for forensic DNA typing. The results showed that the amount of DNA required for accurate sex determination was 25 pg for both methods, achieving equivalent sensitivity. Next, we compared the two methods using ancient human skeletons and found that the present method with its shorter amplicon was considerably superior to GF. The present method is simple, rapid, inexpensive, and suitable for analyzing highly degraded samples. Therefore, this method is expected to contribute to forensic sciences and physical anthropology.

Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed Lizard.

Non-avian reptiles, unlike mammals and birds, have undergone numerous sex determination changes. Casque-Headed Lizards have replaced the ancestral XY system shared across pleurodonts with a new pair of XY chromosomes. However, the evolutionary forces that triggered this transition have remained unclear. An interesting hypothesis suggests that species with intermediate states, with sex chromosomes but also thermal-induced sex reversal at specific incubation temperatures, could be more susceptible to sex determination turnovers. We contrasted genotypic data (presence/absence of the Y chromosome) against the histology of gonads of embryos from stages 35-37 incubated at various temperatures, including typical male-producing (26°C) and female-producing (32°C) temperatures. Our work apparently reports for the first time the histology of gonads, including morphological changes, from stages 35-37 of development in the family Corytophanidae. We also observed that all embryos developed hemipenes, suggesting sex-linked developmental heterochrony. We observed perfect concordance between genotype and phenotype at all temperatures. However, analysis of transcriptomic data from embryos incubated at 26°C and 32°C identified transcript variants of the chromatin modifiers JARID2 and KDM6B that have been linked to temperature-dependent sex determination in other reptiles. Our work tested the validity of a mixed sex determination system in the family Corytophanidae. We found that XY chromosomes are dominant; however, our work supports the hypothesis of a conserved transcriptional response to incubation temperatures across non-avian reptiles that could be a reminiscence of an ancestral sex determination system.

Automação da determinação do sexo a partir de pontos anatômicos em radiografias panorâmicas / Automation of sex determination from anatomical points on panoramic radiographs

Introdução: A automação da determinação do sexo em radiografias panorâmicas pode auxiliar na prática pericial, tornando as rotinas mais fáceis de serem aplicadas nos exames realizados pelos odontolegistas. Objetivo: Criar algoritmos que realizem a automação da determinação do sexo através de pontos anatômicos visíveis em radiografias panorâmicas e também verifique a reprodutibilidade das medidas mensuradas com intuito de propor método de identificação pessoal. Método: Foi realizado um estudo em duas etapas. Na primeira, para verificar a confiabilidade do método, foram selecionadas 25 radiografias, foram calculados o coeficiente intra-classe (ICC), e a técnica estatística de Bland-Altiman. Na segunda etapa, para criar o algoritimos utilizando Machine Learning foram utilizadas 200 radiografias, de serviço radiológico privado, sendo 100 do sexo feminino e 100 do sexo masculino. Foram analisadas 4 medidas lineares, que serviram de base para as análises que foram mensuradas em pixel. Foram verificadas a adesão dos dados à curva de normalidade foi testada utilizando o teste estatístico de Shapiro-Francia. Para a realização da automação foi utilizado o algoritmo e para verificar a acurácia do método foi utilizada a regressão logística. Foi utilizado o R studio, o R, Orange e Medcalc. Resultados: O ICC foi acima de 0.90 para todas as medidas. Na análise de Bland-Altiman, as medidas estavam dentro dos intervalos de confiança fixados. Em relação à predição do sexo, as variáveis que apresentaram diferenças estatísticas foram as medidas 5, 6, 7, 8, para o sexo feminino o acerto foi de 70,4%, já para o sexo masculino a predição foi de 69,6%, utilizando o algoritmo da regressão logística. A curva ROC do algoritmo de regressão logística e o valor encontrado foi de 0,460. Conclusões: A estimativa de sexo automatizada pode ser realizada por pontos anatômicos visíveis em radiografias panorâmicas e pode ser um bom método auxiliar de identificação em desastres em massa.

A qPCR-duplex assay for sex determination in ancient DNA.

Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.

Differential proteomics between unhatched male and female egg yolks reveal the molecular mechanisms of sex-allocation and sex-determination in chicken.

There is a huge demand to identify the sex of unhatched fertilized eggs for laying industry and to understand the differences between male and female eggs as early as possible. Then the molecular mechanisms of sex determination and sex allocation in chicken were revealed. Therefore, TMT proteomic was applied to characterize the variation of molecular matrix between unhatched male and female egg yolks. A total of 411 proteins were identified and 35 differentially expressed proteins (DEPs), including 375332005, 015809562, 763550308 (upregulated, UPs) and 1337178851, 89000557, 89000581 (downregulated, DPs), etc. were confirmed between them. Gene ontology analyses showed that DEPs were mainly involved in response to stimulus, distributed in the extracellular region and participated in binding; KEGG analyses showed that few DPs were participated in cell growth and death, transport and catabolism, signaling molecules, interaction and were enriched in ubiquitin mediated proteolysis, endocytosis, ferroptosis, etc. metabolic pathways. Moreover, most of the DEPs and related metabolic pathways were associated with sex hormones. More importantly, this study supports maternal sex-allocation theory and extends our understanding of the molecular mechanism of sex determination and differentiation in avian. Which also provides a powerful evidence for ovo sexing of unhatched fertilized domestic chicken eggs by nondestructive approach and will be of great significance to eggs processing and production.

Something Fishy about Siamese Fighting Fish (Betta splendens) Sex: Polygenic Sex Determination or a Newly Emerged Sex-Determining Region?

Fishes provide a unique and intriguing model system for studying the genomic origin and evolutionary mechanisms underlying sex determination and high sex-chromosome turnover. In this study, the mode of sex determination was investigated in Siamese fighting fish, a species of commercial importance. Genome-wide SNP analyses were performed on 75 individuals (40 males and 35 females) across commercial populations to determine candidate sex-specific/sex-linked loci. In total, 73 male-specific loci were identified and mapped to a 5.6 kb region on chromosome 9, suggesting a putative male-determining region (pMDR) containing localized dmrt1 and znrf3 functional sex developmental genes. Repeat annotations of the pMDR revealed an abundance of transposable elements, particularly Ty3/Gypsy and novel repeats. Remarkably, two out of the 73 male-specific loci were located on chromosomes 7 and 19, implying the existence of polygenic sex determination. Besides male-specific loci, five female-specific loci on chromosome 9 were also observed in certain populations, indicating the possibility of a female-determining region and the polygenic nature of sex determination. An alternative explanation is that male-specific loci derived from other chromosomes or female-specific loci in Siamese fighting fish recently emerged as new sex-determining loci during domestication and repeated hybridization.

New Sex Chromosomes in Lake Victoria Cichlid Fishes (Cichlidae: Haplochromini).

African cichlid fishes harbor an extraordinary diversity of sex-chromosome systems. Within just one lineage, the tribe Haplochromini, at least 6 unique sex-chromosome systems have been identified. Here we focus on characterizing sex chromosomes in cichlids from the Lake Victoria basin. In Haplochromis chilotes, we identified a new ZW system associated with the white blotch color pattern, which shows substantial sequence differentiation over most of LG16, and is likely to be present in related species. In Haplochromis sauvagei, we found a coding polymorphism in amh that may be responsible for an XY system on LG23. In Pundamilia nyererei, we identified a feminizing effect of B chromosomes together with XY- and ZW-patterned differentiation on LG23. In Haplochromis latifasciatus, we identified a duplication of amh that may be present in other species of the Lake Victoria superflock. We further characterized the LG5-14 XY system in Astatotilapia burtoni and identified the oldest stratum on LG14. This species also showed ZW differentiation on LG2. Finally, we characterized an XY system on LG7 in Astatoreochromis alluaudi. This report brings the number of distinct sex-chromosome systems in haplochromine cichlids to at least 13, and highlights the dynamic evolution of sex determination and sex chromosomes in this young lineage.

In Silico Analysis of Seven PCR Markers Developed from the CHD1, NIPBL and SPIN Genes Followed by Laboratory Testing Shows How to Reliably Determine the Sex of Musophagiformes Species.

Sex determination in birds, due to the very common lack of sexual dimorphism, is challenging. Therefore, molecular sexing is often the only reliable way to differentiate between the sexes. However, for many bird species, very few genetic markers are available to accurately, quickly, and cost-effectively type sex. Therefore, in our study, using 14 species belonging to the order Musophagiformes, we tested the usefulness of seven PCR markers (three of which have never been used to determine the sex of turacos), developed based on the CHD1, NIPBL, and SPIN genes, to validate existing and develop new strategies/methods of sex determination. After in silico analysis, for which we used the three turaco nuclear genomes available in GenBank, the suitability of the seven selected markers for sexing turacos was tested in the laboratory. It turned out that the best of the markers tested was the 17th intron in the NIPBL gene (not previously tested in turacos), allowing reliable sex determination in 13 of the 14 species tested. For the one species not sexed by this marker, the 9th intron in the CHD1 gene proved to be effective. The remaining markers were of little (4 markers developed based on the CHD1 gene) or no use (marker developed based on the SPIN gene).