Sêmen ovino e caprino imunossexado e diluído em meio de conservação à base de água de coco em pó (ACP101/102c) / Ram and goat semen immunosexed and diluted in powdered coconut water-based preservation medium (ACP101/102c)

Sperm sexing aims to separate sperm populations in carriers of the "X" or "Y" chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c. Ejaculates from five rams and five goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with "Y" chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species. The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.(AU)

A meta-analytic review for the patella sexual dimorphism assessment / Una revisión meta-analítica para la evaluación del dimorfismo sexual de la patela

Sexual dimorphism is one of the most important ways to identify bone remains in mass disasters. Many of them have been used for this purpose; mainly skull, pelvis and long bones. However, only a few studies using the patella have been done and, to our knowledge, there are no assessments of previous results in the literature. Meta-analysis provides a useful strong tool to test, in a systematic way, the most relevant information about a given research field. The aim of this study is to apply the meta-analytic technique to assess the major studies concerning sexual dimorphism in the patella by measuring classical metric traits: maximum height and maximum width, with different techniques, such as caliper, radiography, tomography and magnetic resonance. The 17 papers found, involving a total sample size higher than 2600 patellae, showed a very high heterogeneity- around 93 % of I2 value, for height and width measurements when all the studies were analyzed together. Homogeneity increased when each study was classified according to the techniques used. In this case, a statistical difference appeared, among the several subgroups of techniques for the two measurements, suggesting the importance of the methodology used. Maximum height and maximum width were all showed to be statistically relevant in distinguishing both sexes.

El dimorfismo sexual es una de las formas más importantes para identificar restos óseos en desastres masivos. Se han utilizado huesos como cráneo, pelvis y huesos largos para la diferenciación sexual. Sin embargo, solo se han realizado unos pocos estudios con la patela y, hasta donde sabemos, no hay evaluaciones de resultados anteriores en la literatura. El meta-análisis proporciona una herramienta sólida y útil para probar, de manera sistemática, la información más relevante sobre un cierto campo de investigación. El objetivo de este estudio consiste en aplicar la técnica metaanalítica para evaluar los principales estudios sobre dimorfismo sexual en la patela midiendo los rasgos métricos clásicos: altura máxima y anchura máxima, con diferentes técnicas: calibre, radiografía, tomografía y resonancia magnética. Los 17 de documentos encontrados, con un tamaño de muestra total superior a 2600 patelas, mostraron una heterogeneidad muy alta, alrededor del 93 % del valor de I2, para mediciones de altura y anchura cuando todos los estudios se analizaron juntos. La homogeneidad aumentó cuando cada estudio se clasificó de acuerdo con las técnicas utilizadas. En este caso, se observó diferencias estadísticas, entre los subgrupos de técnicas para las dos mediciones, lo que sugiere la importancia de la metodología utilizada. La altura máxima y la anchura máxima mostraron ser estadísticamente relevantes para distinguir ambos sexos.

Can human maxillary premolars discriminate between sexes in South Asian populations? / ¿Pueden los premolares maxilares humanos discriminar entre sexos en las poblaciones del sur de Asia?

The studies have illustrated odontometric analysis can be used to determine the sexual dimorphism effect on size of the teeth in various populations. The main aim of the study was to identify the inter-cuspal-, bucco-lingual -dimensions and weight of human upper-arch pre-molars in males and females of different South Asian populations. These metrics can distinguish sex which can have application in mass disasters, archaeology of mingled human remains and the in unidentified or several ancestry. The sample size consisted of 60 orthodontically extracted maxillary pre-molars from Pakistani and Saudi Arabian populations respectively. For male and female groups of each population fifteen first and second maxillary premolars were collected respectively, stored in PBS solution. The weight of the individual teeth was recorded. Later, digitally pictures were captured parallel to the occlusal surface to measure maximal bucco-lingual and inter-cuspal dimensions using Image-J software. The dimensions and weights were compared using Students' t-test between males and females respective Pakistani and Saudi Arabian first (P1) and second (P2) maxillary pre-molars. The dimensions for male P1 and P2 were statistically significantly larger than that for females in both populations. Furthermore, wet-weight of pre-molars in males is significantly greater than females in both populations. The findings demonstrate maxillary pre-molars can discriminate between the sexes in various populations.

Las investigaciones han ilustrado que el análisis odontométrico se puede utilizar para determinar el efecto del dimorfismo sexual en el tamaño de los dientes en varias poblaciones. El objetivo principal del estudio fue identificar las dimensiones y el peso entre cúspides, buco-linguales y el peso de los premolares de la arcada superior humana en hombres y mujeres de diferentes poblaciones del sur de Asia. Estas medidas pueden distinguir el sexo y ser importante en desastres masivos, arqueología de restos humanos entremezclados y en ancestros no identificados. El tamaño de la muestra consistió en 60 premolares maxilares extraídos ortodóncicamente de las poblaciones de Pakistán y Arabia Saudita, respectivamente. Para los grupos de hombres y mujeres de cada población, se recogieron quince primeros y segundos premolares superiores respectivamente, almacenados en solución de PBS. Se registró el peso de los dientes individuales. Posteriormente se capturaron imágenes digitales paralelas a la superficie oclusal para medir las dimensiones máximas buco-linguales e intercúspides utilizando software Image-J. Las dimensiones y los pesos se compararon mediante la prueba t de Student entre lo premolares maxilares (P1) y segundos (P2) de hombres y mujeres paquistaníes y saudíes. Las dimensiones para P1 y P2 de los hombres fueron estadísticamente significativos mayores que para las mujeres en ambas poblaciones. Además, el peso húmedo de los premolares en los varones era significativamente mayor que el de las mujeres en ambas poblaciones. Los hallazgos demuestran que los premolares maxilares pueden discriminar entre los sexos en varias poblaciones.

Development of an Extraction Protocol for Fetal Sexing from the Fourth Week of Gestation by Real Time PCR

Abstract Low number of fetal cells in maternal blood limited the use of fetal materials in diagnostic and clinical applications. This research developed a technology which allowed the extraction of fetal DNA by a non-invasive method that offers no risk to the mother or fetus. A total of 132 pregnant women participated in this inquiry. The DNA extraction was performed employing an in-house method based on guanidine thiocyanate and magnetic bead. For the amplification it was used the Quantifier Y Kit™. The fetal sexing analysis of the 132 pregnant women were 100% in agreement with the ultrasound. Sensitivity and specificity for detection of Y chromosome sequences was possible using Real-Time Polymerase Chain Reaction since the 4th week of gestation. This non-invasive early determination could be employed in fetal gender and also to be extended to detection of genetic diseases in the shortest possible time avoiding invasive methods that puts the fetus at risk.

Dental pulp fibroblast and sex determination in controlled burial conditions / Fibroblastos de la pulpa dental y la determinación del sexo en condiciones controladas de enterramiento

The need to identify bodies that are found as a result of disappearances with a diversity of causes, illegal burials and massive disasters, represent a wide percentage of dentistry practice on forensic research. The following study determined the performance of Barr Body Test, in fibroblasts of healthy teeth, under different conditions of burial (in vitro) with variations in pH, humidity and salinity in terms of general accuracy and sensitivity for men and women. Analyzed sample considered 47 dental pulps, taken from teeth under burial conditions during a period of a month. From dental pulps samples, 265 histological cuts valid for this study, were obtained, which were observed with an optical microscope under conventional H/E staining. Results showed a 98.9% of well-diagnosed cases, which correspond to the overall accuracy of the method. Sensitivity for men was 97.5% and 100% for women, over the analyzed sample. In low humidity conditions, 3 samples of badly diagnosed cases in men were observed, with a group accuracy of a 90%, with a sensitivity of 25% for men and 100% for women. The present study establishes that based on these results, the performance of Barr Body Test in fibroblasts, proposed for healthy pulp teeth, is not affected by burial conditions in terms of pH (acid-alkaline), salinity (high-low) and high humidity.

La necesidad de identificar cuerpos que resultan como consecuencia de desapariciones de causas variadas, inhumaciones ilegales y desastres masivos representa un porcentaje amplio en el quehacer odontológico en un escenario de investigación forense. El presente estudio determinó el rendimiento de la prueba diagnóstica de observación del cuerpo de Barr en células de la pulpa de dientes sanos, sometidos a distintas condiciones de enterramiento (in vitro) con variación de pH, humedad y salinidad en términos de exactitud general y sensibilidad para hombres y mujeres. La muestra analizada consideró 47 pulpas dentales, extraídas de dientes sometidos a condiciones de enterramiento durante un mes. De las pulpas dentarias se obtuvieron 265 cortes histológicos válidos para el estudio, los cuales mediante la tinción convencional H/E, fueron observados al microscopio óptico. Los resultados arrojaron un 98,9% de casos bien diagnosticados, que correspondió a la exactitud general del método. La sensibilidad para hombres fue de 97,5% y para mujeres de 100% sobre el total de la muestra analizada. Las condiciones de pH (ácido y alcalino), salinidad (alta y baja) y alta humedad presentaron una exactitud de grupo de 100%, con una sensibilidad para hombres y mujeres de 100%. En la condición de baja humedad se observaron 3 muestras de hombres mal diagnosticadas con una exactitud de grupo de 90% y sensibilidad para hombres de 25% y para mujeres de 100%. A la luz de los resultados, el presente estudio establece que el rendimiento de la prueba diagnóstica de observación del cuerpo de Barr en fibroblastos, propuesto para pulpas de dientes sanos, no se afecta con las condiciones de enterramiento propuestas bajo pH ácido ­ alcalino, salinidad alta ­ baja y humedad alta.

Sex verification tests: ethical, legal and social aspects.

Lay people often wonder at all the fuss about identifying the biological sex of an individual. They may recall a granny or midwife, immediately after a delivery, even in the dim light, declaring with ease that it’s a girl, or a boy, to the rejoicing crowd waiting eagerly outside the delivery room. When it is so simple, why are doctors, sports administrators and investigators making such a hue and cry about this? How do you identify the biological sex of an individual? What is this fuss about sex verification tests? Are they the same as gender verification tests? What are the ethical, legal and social aspects of these sex verification tests? I will try to answer some of these questions.

Sex determination in Turdus amaurochalinus (Passeriformes: Muscicapidae): morphometrical analysis supported by CHD gene

Sex determination is important for conservation and population studies, particularly for reproduction programs of threatened species and behavioural ecology. Turdus amaurochalinus, Creamy-bellied Thrush, only exhibits sexual dimorphism during the breeding season, when males are considered to show intense yellow bills, and females and immature males show dark brown bills. The objectives of this study were: 1) to determine the sex of individuals using genetic techniques, and 2) to test the hypothesis that sex dimorphism can be detected by morphometry. This study was carried out at Parque Nacional da Restinga de Jurubatiba, a preserved area located on the North coast of Rio de Janeiro State. The birds were captured using ornithological nets, singly marked with metal rings, weighed, measured and had blood samples collected before being released. The sex of 42 T. amaurochalinus individuals was determined using the CHD gene marker. A total of 20 males and 22 females were identified from June to August, with peak capture frequency in June. Turdus amaurochalinus females and males differed significantly in morphometrical measures. The most important traits to distinguish males from females were wing length (Student t-test=4.34, df=40, p=0.0001) and weight (Student t-test=2.08,df=40, p=0.044): females were heavier and had significantly shorter wing length than males. Females and males were correctly classified in 86% and 75% of cases, respectively, using Discriminant Analysis. The molecular analysis was the most secure method for sex determination in the studied species. Rev. Biol. Trop. 59 (2): 789- 794. Epub 2011 June 01.

La determinación del sexo es importante para la conservación y los estudios poblacionales. Turdus amaurochalinus no presenta aparente dimorfismo sexual. El objetivo de este estudio fue determinar el sexo a través de una técnica genética, mediante el uso del marcador del gen CHD y se puso a prueba la hipótesis de que el dimorfismo sexual puede ser detectado por morfometría. Este estudio se llevó a cabo en el Parque Nacional da Restinga de Jurubatiba, una zona protegida situada en la costa norte de Río de Janeiro. Las aves fueron capturadas con redes de niebla, los individuos se marcaron con anillos de metal, se pesaron, medieron y se les tomó una muestra de sangre antes de ser liberados. Un total de 20 machos y 22 hembras fueron identificados en el área de estudio desde junio hasta agosto, con la frecuencia máxima de captura en junio. La prueba de t-student fue usada para evaluar si hembras y machos se diferencian considerablemente en relación a medidas morfométricas. Los rasgos más importantes para distinguir machos de hembras fueron la longitud del ala y el peso: las hembras eran más pesadas y tenían longitud de ala considerablemente más corta que los machos. Hembras y machos fueron correctamente clasificados en un 86% y 75% de casos respectivamente, donde se usó un análisis discriminante. El análisis molecular es el método más seguro para la determinación sexual en la especie estudiada.

Sex determination by observation of barr body in teeth subjected to high temperatures / Determinación del sexo mediante la observación de la cromatina de barr en piezas sometidas a altas temperaturas

Sex determination is one of the keys in the identification process. A useful histological method for sex determination is the observation of Barr chromatin or Barr body. This study determines the effect of high temperatures on the diagnostic performance of the Barr chromatin observation on teeth. Were used 50 healthy teeth from 25 male and 25 female individuals aged between 14 and 44 years. The teeth were divided into 5 groups (each group with 5 female and 5 male teeth) and were exposed to controlled temperatures of 200, 400, 600, 800, and 1000 degrees C for 5 minutes. The coronal pulp was obtained and the tissue was processed and stained with hematoxylin-eosin. Four histological slides of male and 4 of female individuals were randomly selected, for each temperature level, which were observed by conventional microscopy at 100X magnification, each showing 50 cells per plate. The presence of 1 cell with visible sex chromatin was considered positive for females. It was only possible to evaluate the samples from groups subjected to 200 and 400 degrees C. In the groups analyzed, the test showed 100 percent accuracy. The average number of cells found to be positive Barr chromatin was 15 (SD 3.9) at 200 degrees C and 11 (SD 2.8) at 400 degrees C. Hence, it was possible to detect the sex at these temperatures by observing chromatin of the Barr body in dental pulp.

La determinación del sexo es uno de los pilares del proceso de identificación, Un método histológico útil para la determinación del sexo es la observación de cromatina de Barr. El objetivo de este estudio fue determinar el efecto de altas temperaturas sobre el rendimiento diagnóstico de observación de cromatina de Barr en piezas dentarias. Se utilizaron 50 piezas dentarias sanas, 25 de individuos de sexo masculino y 25 de sexo femenino con edades comprendidas entre los 14 y 44 años. Las piezas dentarias fueron divididas en 5 grupos, (cada grupo con 5 piezas de sexo femenino y 5 de sexo masculino) y fueron expuestas a temperaturas controladas de 200°C, 400°C, 600°C, 800°C y 1000°C por 5 minutos. La pulpa coronaria fue obtenida y el tejido fue procesado para H-E. Se seleccionaron aleatoriamente 4 placas histológicas de individuos de sexo masculino y 4 de sexo femenino, por cada nivel de temperatura, las cuales fueron observadas por microscopía convencional a un aumento de 100X, observándose 50 células por placa. La presencia de 1 célula con cromatina sexual visible se consideró positiva para el sexo femenino. Sólo fue posible evaluar las muestras provenientes de los grupos sometidos a 200°C y 400°C. En los grupos analizados la prueba presentó un 100 por ciento de exactitud. El número medio de células Barr positivas encontradas fue de 15 (DS 3,9) a 200°C y 11 (DS 2,8) a 400°C, por lo que a estas temperaturas fue posible el diagnóstico del sexo mediante la observación de la cromatina sexual en pulpa dental.

Costicartilage analysis inspection technology in the application of forensic medicine / 法医学杂志

The traditional costicartilage analysis inspection is limited to morphological inspection. In recent years, with the development of forensic radiology and molecular genetics, the costicartilage analysis inspection technology has been further enriched and developed. At present, the costicartilage analysis inspection technology have been able to be used in the practice of forensic medicine. This paper reviews the research advances about the costicartilage analysis inspection technology in the identification of human gender, age and so on in order to provide the references for forensic appraisers.

Sex chromatin in dental pulp: performance of diagnosis test and gold atandard generation / Cromatina sexual en pulpa dental: rendimiento de la prueba diagnóstica y generación del gold standard

The sex chromatin or Barr body is a condensation of chromatin present at the nucleus of cells in female individuals. Their observation is possible in different cell types and is used for the rapid diagnosis of biological sex. The observation of this condensation in cells of the pulp tissue can be useful in the forensic diagnosis of sex, primarily because of the protection it give dental hard tissues, but its diagnostic value has not been tested. The purpose of this study is to analyze the diagnostic value of the observation of sex chromatin in cells of the pulp tissue. We used 40 premolars and third molars 20 from men with a mean age of 20.6 years (SD 8.8) and 20 from women with a mean age of 20.4 years (SD 7.4). The pulp was obtained from teeth extracted and localized 50 cells per plate. The presence of a cell with visible Barr body was considered positive for women. With these results, we evaluated the diagnostic performance of the test. The performance of the test was 100 percent; of the 50 cells observed per plate, mean Barr bodies positive cells was 20.4 (SD 0.44) in female samples. There was no positive cell in preparations of male subjects. The diagnostic test observation of sex chromatin in dental pulp is a reliable test for sex determination. This study provides a gold standard for performance assessment in which the teeth have suffered extreme physical conditions, such as incineration and dumping.

La cromatina sexual o de Barr es una condensación de cromatina presente a nivel del núcleo de las células de individuos de sexo femenino. Su observación es posible en diversos tipos celulares y es utilizado para el diagnóstico rápido del sexo biológico. La observación de esta condensación en células del tejido pulpar puede ser útil en el diagnóstico forense del sexo, principalmente debido a la protección que los tejidos duros dentarios le otorgan, sin embargo su valor diagnóstico no ha sido analizado. El propósito de este estudio es analizar el valor diagnóstico de la observación de la cromatina sexual en células del tejido pulpar. Se utilizaron 40 premolares y terceros molares maxilares, 20 de hombres con una media de edad de 20,6 años (DS 8,8) y 20 de mujeres con una edad media de 20,4 años (DS 7,4). La pulpa fue obtenida a partir de piezas extraídas, se realizó la extirpación pulpar y el tejido fue procesado para H-E. Se seleccionaron aleatoriamente 4 placas histológicas, las cuales fueron observadas de manera sistemática utilizando una microscopia Olimpus CX21 con aumento 100X, localizándose 50 células por placa. La presencia de 1 célula con cromatina sexual visible se consideró positivo para sexo femenino. Con estos resultados se evaluó el rendimiento diagnóstico de la prueba. El rendimiento de la prueba fue del 100 por ciento, de las 50 células observadas por placa, la media de células cromatina sexual positiva fue de 20,4 (DS 0,44) en muestras de sexo femenino. No se observó ninguna célula positiva en preparaciones de sujetos de sexo masculino. La prueba diagnóstica observación de la cromatina sexual en pulpa dentaria es una prueba confiable para la determinación del sexo. Este estudio establece un gold estándar para la evaluación del rendimiento en los que las piezas dentarias han sufrido de condiciones físicas extremas como incineración, inmersión, etc.

Diagnóstico prenatal no invasivo: Ácidos nucleicos de origen fetal en sangre materna / Non invasive prenatal diagnosis: Fetal nucleic acid analysis in maternal blood

Las técnicas actuales de diagnóstico prenatal de enfermedades génicas y cromosómicas incluyen procedimientos invasivos que conllevan un pequeño, pero significativo, riesgo. Por muchos años se ha estudiado la posibilidad de utilizar células fetales en circulación materna; sin embargo, ha fracasado su implementación clínica debido a su escasez y persistencia luego del parto. Desde hace más de una década se detectó ADN fetal libre en sangre de embarazadas. Este sería de origen placentario e indetectable después del parto, y fuente de material fetal para el desarrollo de técnicas diagnósticas utilizando sangre materna. No obstante, la mayoría del ADN libre en circulación materna es de origen materno con una contribución fetal del 3% al 6% aumentando a lo largo de la gestación. Dado que los métodos actuales no permiten separar el ADN libre fetal del materno, las aplicaciones se focalizan en el análisis de genes no presentes en la madre, tales como secuencias del cromosoma Y, o gen RHD en madres Rh negativas, o mutaciones paternas o de novo. Asimismo, la detección de ARN fetal libre en sangre de embarazadas abrió la posibilidad de obtener información acerca de patrones de expresión génica de tejidos embrionarios y, utilizando genes que se expresan sólo en la unidad feto-placentaria, se podría establecer un control de presencia de material fetal, independiente del material genético de la madre. El presente trabajo describe las evidencias acerca del pasaje de ácidos nucleicos fetales a circulación materna, su aplicación actual en el diagnóstico prenatal y posibles usos futuros.

Current prenatal diagnosis of monogeneic and chromosomal diseases, includes invasive procedures which carry a small but significant risk. For many years, analysis of fetal cells in maternal circulation has been studied, however it has failed its clinical use due to the scarcity of these cells and their persistance after delivery. For more than a decade, the presence of cell-free fetal DNA in maternal blood has been identified. These fetal DNA fragments would derive from the placenta and are not detected after delivery, making them a source of fetal material for carrying out diagnosis techniques using maternal blood. However, the vast majority of cell free DNA in maternal circulation is of maternal origin, with the fetal component contributing from 3% to 6% and rising towards term. Available methodologies do not allow separation of fetal from maternal cell free DNA, so current applications have been focused on the analysis of genes not present in the mother, such as Y chromosome sequences, or RHD gene in RhD-negative women, or paternal or de novo mutations. Also, the detection of cell-free fetal RNA in maternal blood offers the possibility of obtaining information regarding genetic expression profiles of embrionic tissues, and using genes expressed only at the feto-placental unit, controls for the presence of fetal material could be established, regardless of maternal genetic tissue. The present article describes the evidences regarding the passage of fetal nucleic acids to maternal circulation, its current prenatal diagnosis application and possible future perspectives.

Iris colour as an indicator of age feature in female Brazilian tanagers (Passeriformes: Emberizidae) confirmed by a molecular sexing technique

The Brazilian tanager, Ramphocelus bresilius is an endemic species from Brazil that is sexually dimorphic in adult plumage. Young males are similar to adult and young females until their second year. Adults and young females are not distinguishable in plumage. We tested whether iris colour can be used to separate adult females from immature females. We used for the first time the molecular sexing technique based on CHD-genes to confirm the sex of the individuals classified as "female plumage with red iris", and to identify the sex of individuals classified as "female plumage and brown iris". The adult males were used as a positive control. DNA samples from 190 individuals were analysed. The sizes of the PCR products were identified as 350 base pairs (bp) for CHD-Z and 388 bp for CHD-W. We confirmed that adult females have a red iris and the young females a brown iris. We could also separate young males and females which present the same iris colour and plumage. Although there are indications that the iris colour can be used by birds to identify the adults in co-operative breeding species such as the Brazilian tanager, more behavioural data are required to understand the role of iris coloration in this species. Rev. Biol. Trop. 56 (4): 1629-1633. Epub 2008 December 12.

El ave Ramphocelus bresilius es una especie endémica de Brasil con dimorfismo sexual en el plumaje del adulto. Los machos jóvenes son similares a las hembras adultas y jóvenes hasta el segundo año de vida. Adultos y hembras jóvenes son indistinguibles por el plumaje. Evaluamos si el color del iris puede ser utilizado para distinguir hembras adultas de hembras inmaduras. Utilizamos por primera vez la técnica molecular de identificación de sexos basada en los genes CHD para confirmar el género de individuos clasificados como plumaje femenino con iris rojo, y para identificar el sexo de los individuos clasificados como plumaje femenino e iris marrón. Usamos machos adultos como control. Analizamos muestras de DNA de 190 individuos. Los tamaños de los productos del PCR fueron identificados como 350 pares de bases (pb) para CHD-Z y 388 pb para CHD-W. Pudimos confirmar que las hembras adultas presentan iris rojo y las hembras jóvenes iris marrón. También pudimos distinguir machos jóvenes de hembras, que presentan el mismo color de iris y plumaje.

Prenatal diagnosis of fetal sex by molecular analysis of maternal plasma after 8 weeks of gestation

Prenatal diagnosis of fetal sex is usually performed by invasive methods such as sampling through amniocentesis or chorionic villus sampling. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. The objective of our study was to investigate the feasibility of using fetal DNA in maternal plasma for prenatal diagnosis of fetal sex. In this experimental study, a nested polymerase chain reaction [PCR] techniques was developed for fetal SRY gene identification using cell-free fetal DNA in maternal plasma. Peripheral blood samples were obtained from 32 pregnant women at the gestational period from 8 to 13 weeks and cell-free DNA was extracted by the phenol/chloroform method from plasma. The nested PCR was carried out to amplify the fragment of SRY gene by two sets of PCR primer pairs. Analysis was then performed on the PCR product. Specifically, the presence of Y-chromosome sequences in maternal blood plasma indicated that the fetus is male, whereas lack of signal will indicate that the fetus is female. Among the 32 pregnant women, SRY sequences were detected in 14 plasma samples after nested PCR amplification, while the 18 women bearing female fetuses had the negative results. The sensitivity of this technique was 87.5%. The phenol/chloroform extraction of fetal DNA in maternal plasma is an effective and simple method, and the nested PCR amplification of SRY sequence is a convenient and low-cost approach for the non-invasive early prenatal diagnosis of fetal sex

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis.

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.

Homologous amelogenin gene test of archaeological samples / 法医学杂志

OBJECTIVE@#Based on the sequence differences of Amelogenin homologous gene in the X and Y chromosomes, a pair of specific primers was designed to identify the sex of archaeological samples.@*METHODS@#Ancient DNA fragments were extracted from the bones and teeth of sacrificial slaves with an improved method that combines phenol-chloroform extraction, silicon dioxide adsorption with ultrafiltration concentration. The polyacrylamide gel electrophoresis (PAGE) was used to detect PCR products.@*RESULTS@#Seven in sixteen samples from eight graves showed positive results and the targeted segments were visible: a male with two bands of 106bp (Amel-X) and 112 bp (Amel-Y), while a female with only one band of 106 bp (Amel-X). Ancient DNA analyzing results from tooth samples are more marked than that from bones.@*CONCLUSION@#The improved extraction method is more effective for ancient DNA extraction, which reduced the PCR inhibitors and lowered experimental costs. The sex determination technology based on Amelogenin homologous gene is an important and feasible method in the molecular archaeological research.

Sexing of sacrum by sacral index and kimura's base-wing index.

A study for sexing of sacra was carried on 64 sacra (32 male & 32 female sacra) by two methods.One method used was sacral index and the other method was Kimura's base-wing index. The measuring instrument used was sliding vernier caliper. The method of sacral index showed high success rate as compared with Kimura's base-wing index method.

Estimation of age and sex from radiological examination of hyoid bones in Egyptians

Although cranial and pelvic bones are the preferred skeletal materials used by forensic anthropologists to assign unknown individuals to their most age and sex together with long bones. In the present study the hyoid bone was investigated as another tool for estimating age and sex especially when these skeletal remains are unavailable. There is considerable age variation in fusion of the greater cornua to the hyoid body. Although the proportion of people with bilateral fusion steadily increases with increasing age, many elderly individuals have either unilateral or bilateral nonfusion. Hyoid bones radiographs [n=130] of both sexes were examined. Their ages ranged from 15-75 years for males with mean value of 42.3 +/- 15.40 and from 16-60 for females with mean value of 39.85 +/- 12.41. The radiographs converted into digital images using a high resolution scanner and an image analysis system was used to take a series of certain measurements on each bone. In addition, the degree of fusion of the greater cornua to the hyoid bodies was recorded. The study revealed that non-fusion is more common in men than in women and that no bilateral fusion was recorded before the age of 35 years in either sex. The measurements of the right variables taken from hyoid bones are the measurements of greatest sex difference as they correctly classify sex by 81.5%

Reliable sex determination of mouse preimplantation embryos by PCR amplification of male-specific genes in single blastomeres.

OBJECTIVE: To assess the reliability of sex determination in mouse preimplantation embryos using the two-step polymerase chain reaction method. SETTING: Division of Immunology, Department of Microbiology and Division of Reproductive Medicine, Department of OB/GYN. METHODS: The Sry and Zfy genes, known to be present in the sex-determining region of mouse Y chromosome, were selected for Y-specific target sequences and DXNds 3 locus located on mouse X chromosome was served as the internal control sequence. DNAs extracted from heart blood of male and female mice were used to test the correctness and specificity of the selected primers using the two-step PCR method. The same experimental conditions were then used to amplify the single copy genes in single mouse blastomeres with two pairs of primers for each of the target sequences. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. RESULTS: All male and female blood DNA sample results confirmed the correct sex identification of the origin (100%). Nineteen of 20 single blastomeres showed the accurate diagnosis when compared with theirs 7/8 embryos. The sex of 36 of 37 mouse pups born from biopsied male and female embryos agreed with the predicted sex. CONCLUSION: The reliable genetic analysis of sex chromosome- specific sequences in single cell is possible by the two-step PCR method and could be applied for diagnosis of defective genes of human preimplantation embryos derived from the in vitro fertilization program.

Sonographic determination of foetal sex

In order to determine foetal sex antenatally, 300 patients were examined sonographically over a period of two years [June 1993 - May 1995] using a 3.5 MHz sector probe. Gestational age ranged between 26 - 30 weeks. Foetal scrotum, penis and labia majora were identified to establish the foetal gender. Of 258 ladies who reported back after confinement, diagnosis was correct in 214 [82.9%]. There were 111 [43%] males and 103 [40%] females. In 44 [17%] of our cases sex could not be ascertained. Main factors contributing to unsuccessful outcome were breech presentation [38%], multiple gestation [23.8%], oligohdramnios [11.9%], and non-optimal foetal position [26.2%]. There were only two false positive results. Foetal hydrocele was noticed in three cases