Sex Determination In Birds Research Articles

Evaluation of primers targeting chromo helicase DNA-binding gene (CHD) for molecular sexing identification in four bird families

<p><span class="fontstyle0">Objective: </span></p><p><span class="fontstyle2">Sex determination of birds is crucial role in breeding and conservation purposes. Genomic approaches by using basis of bird sex polymorphism are accepted to generate an accurate sexing procedure. The PCR method is commonly used and known to provide accurate test results in determining the sex of birds, but its successes is strongly influenced by the compatibility of the primers used with the DNA template. This study aimed to determine the potential use of CHD1LF/CHD1LR targeting CHD gene fragment in 4 species from Phasianidae, Psittacidae, Estrildidae, and Passeridae families.</span></p><p><span class="fontstyle2"><br /></span><span class="fontstyle0">Method: </span></p><p><span class="fontstyle2">Blood samples (n= 30) from representatives of species in 4 families above were collected for DNA isolation, then PCR test was performed using CHD1LF/CHD1LR primers. The PCR results obtained were compared with positive controls, necropsy examination and information from bird sellers.</span></p><p><span class="fontstyle2"><br /></span><span class="fontstyle0">Result: </span></p><p><span class="fontstyle2">The results showed that the primer CHD1LF/CHD1LR could be used in Phasianidae,<br />Psittacidae, Estrildidae, and Passeridae families. The PCR product was confirmed to be same as well as the positive control and necropsy results. It is demonstrated that male birds showed a single band (474 bp), while double bands (474 and 319 bp) were observed in female birds. Different accuracies were observed during this study between molecular approach and manual sexing by bird seller i.e:<br />sparrows (46%) and finches (50%), while the accuracy of parakeets was 75%, and quails 80%.</span></p><p><span class="fontstyle2"><br /></span><span class="fontstyle0">Conclusions: </span></p><p><span class="fontstyle2">Primer CHD1LF/CHD1LR can be used to determine the sex of birds from the families<br />Phasianidae, Psittacidae, Estrildidae, and Passeridae. Our study showed that molecular-based method is valuable and able to reduce error rate to 100% in bird sexing procedures.</span> <br /><br /></p><p> </p>

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).

Short Communication: A new primer set in CHD1 gene for bird sex identification

Abstract. Vera F, Wajjwalku W, Yuda P, Daryono BS. 2021. Short Communication: A new primer set in CHD1 gene for bird sex identification. Biodiversitas 22: 4977-4982. Determine sex is difficult for many bird species that are sexually monomorphic or only dimorphic in the adult stage. Many molecular markers have been developed for DNA sexing, which were mostly based on identifying Z and W chromosomes of avians. The sex determination of birds was mostly applied universal primer set such as P2/P8 or 2550F/2718R. However, those universal primers that were designed sometimes could not good result consistency in non-ratite birds or ratite birds. Therefore, we improved a specific primer design with deletion site in W chromosome to amplify the female-specific segments on Chromodomain-helicase DNA binding protein-1 (CHD1) gene from alignment sequences CHD1-Z and CHD1-W of Macrocephalon maleo S. Müller, 1846. This study aims to design a new pair of primer targeting a section of the CHD1 gene that can be amplified in non-ratite birds, the name of a new primer is In-Sex F/In-Sex R. A new primer set amplified DNA fragments in around 650 or 550 base pairs of CHD1-Z and also, 350 base pairs of CHD1-W. The result has successfully amplified the sex of multiple species on orders Galliformes, Passeriformes, Accipitriformes, and Strigiformes. This analysis can be helpful to the effort of in-situ or ex-situ management and conservation programs e.g the mating system in birds. And also, the analysis can be helpful for sexing data in the case of captive birds before releasing in natural habitat or reintroduction programs.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

DIFERENCIACIÓN DEL SEXO DEL GORRIÓN DE BAIRD (Centronyx bairdii) POR MEDIO DE REDES NEURONALES ARTIFICIALES Y DATOS MORFOMÉTRICOS

Las estimaciones indican que 148 especies de aves en Norteamérica enfrentan amenaza alta o severa, pues sus poblaciones están disminuyendo. El gorrión de Baird (Centronyx bairdii) es una de las especies con declive poblacional, presenta plumaje monomórfico y su sexo no se puede determinar a simple vista. La determinación del sexo de las aves permite conocer su comportamiento social y proporción en la dinámica de sus poblaciones. Un método de clasificación es el de redes neuronales artificiales y se ha utilizado en diversos campos, como la clasificación de plantas y semillas y la diferenciación de especies productoras de madera. En el ámbito de la ornitología se ha utilizado poco. Por lo tanto, el objetivo de este estudio fue desarrollar una red neuronal artificial para diferenciar el sexo de los gorriones de la especie Centronyx bairdii, con datos zoométricos obtenidos en campo. La hipótesis fue que las redes neuronales artificiales pueden predecir el sexo de las aves de la especie Centronyx bairdii. La red neuronal construida a partir de los datos de peso, cuerda alar, longitud de cola, culmen, ancho del pico y profundidad del pico de 111 aves, permitió diferenciar el sexo de la especie con grado de certeza de 92.3%.

DETERMINACJA PŁCI I ROZWÓJ GONAD KURY DOMOWEJ

Poznanie procesu determinacji płci i rozwoju płciowego ptaków zawdzięczamy głównie badaniom kury domowej. U gatunku tego, jak u reszty ptaków, płcią heterogametyczną jest płeć żeńska (ZW), a homogametyczną jest płeć męska (ZZ). Jak dotąd nieznany jest nadrzędny gen determinujący płeć ptaków. Za kluczowy gen uznaje się DMRT1, który znajduje się na chromosomie Z i ulega ekspresji w rozwijających się gonadach męskich. Natomiast w rozwoju gonad żeńskich kluczową rolę odgrywa aromataza, enzym odpowiedzialny za syntezę estrogenów. Geny potencjalnie zaangażowane w determinację płci ulegają ekspresji w rozwijających się gonadach. U ptaków jajnik i jajowód rozwijają się jedynie po stronie lewej, natomiast prawa gonada ulega znacznej redukcji. Okazuje się, że estrogeny odgrywają istotną rolę w rozwoju płciowym ptaków. Modulując aktywność aromatazy można uzyskiwać osobniki o odwróconej płci. Rola testosteronu w rozwoju gonad kur jest niejasna, natomiast opisano jego rolę w rozwoju drugorzędowych cech płciowych. Zestawienie dotychczasowych badań nad determinacją płci u ptaków wskazuje, że dalsze badania są konieczne dla pełnego poznania tego procesu.

Hormonal Sex Inversion and Some Aspects of Its Genetic Determination in Chicken

Different mechanisms of genetic sex determination in birds are considered. Special emphasis is placed on the influence of external factors (reduction of estrogen synthesis, inactivation of DNA methylases) on expression of key sex determining genes. The possibility of sex inversion in domestic chicken by aromatase inhibition, genomic DNA demethylation, and the effect of estrogen analogs on estrogen receptors was demonstrated. The results of these effects were analyzed and their effect on the development of gonads in one-day embryos was demonstrated. A suggestion on earlier aromatase activity in chicken embryos, weak effect of DNA demethylation with 5-azacytidine, and different mechanisms of the effect of estrogens and their analogs (tamoxifen) on the gonad development in chicken was made.

Validation of loop-mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds.

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR-based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP-based primer set for sex determination across the modern birds (NEO-W) based on a fragment of the gene chromo-helicase-DNA-binding protein located on the female-specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO-W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.

Percutaneous Endoscopic Retrieval of Gizzard Foreign Body in a Goose Using an Operative Telescope

Background: Foreign bodies are most commonly diagnosed in stomach (proventriculus and gizzard) of galliformes birds and waterfowl. Endoscopyis routinely used to assess birds’ respiratory, gastrointestinal and urogenital tracts. Endosurgeryis also used for sex determination and intra-coelomaticorgan biopsy. Benefits of endoscopic approaches are widely reported in birds. Conventional surgical approaches are far more invasive and risky for those patients. Thus, the purpose of this study was to describe a successful case of gizzard foreign body removal in a greylag goose (Anseranser), using a rigid endoscopic approach through a minimally invasive percutaneous access. Case: A 2-year-old female greylag goose, weighting 3,116 g, was referred to a Veterinary Teaching Hospital presenting apathy and anorexia for at least 24 h. The results on hematologic assessment were within normal range for the species. Radiographic examination indicated presence of radiopaque content, resembling gizzard sediment. Thus, the patient undergone to endoscopy for examination and aspiration of the content. The goose was fasted for 6 h. Anesthesia was induced by face mask and maintained by endotracheal tube, using isoflurane vaporized in 100% oxygen. The patient was positionedin the lateral recumbence. The patient’s neck was longer than the working length of the rigid endoscope. Thus, the telescope was inserted into the esophagus following a small distal esophagotomy, carried out on the caudal third of the neck. A 0º 10-mm operative rigid telescope, with a 6-mm working channel, and a 5-mm diameter and 42-cm in length laparoscopic Babcock forceps were used. The endoscope was inserted through the esophagotomy up to the ventricle level. Sand sediments and stones were viewed. The stones were grasped and retrieved, and the sediments were rinsed with normal saline solution and aspirated with a suction cannula through the working channel of the telescope. Inspection following sediment and foreign body retrieval revealed moderate inflammation of the gizzard mucosa. Total procedure time was 24 min. The patient recovered uneventfully and was discharged following 72 h. Discussion: Rigid endoscopy provided accurate visualization of the thoracic esophagus, proventriculus and gizzard. In general, endosurgery is usually employed for sex determination in birds. It is also useful to access digestive tract using rigid endoscopy, in order to remove foreign body. Such minimally approach reduces postoperative morbidity, which is usually seen following conventional surgical approaches. This technique has been used for removal of foreign body in the digestive tract of several species. Foreign bodies found within crop should require ingluviotomy. However, could be easily removed by endoscopy. If the foreign body is within the proventriculus or gizzard, rigid endoscopy may be useful, as celiotomy could cause morbidity. The small esophagotomy access at the base of the neck was essential for the procedure. In short-necked birds, a 30º Trendelenburg positioning provides more caudal access to the digestive tract. In this case, such positioning was unnecessary. In conclusion, the use of an operative telescope provided accurate retrieval of foreign bodies and drainage of sediments within the digestive tract. Moreover, it can be used as a minimally invasive approach to foreign bodies in the esophagus, crop or stomach (esophagus and gizzard) of geese.

Experimental Sex Inversion of Chicken Embryos at Aromatase Inhibition, Estrogen Receptor Modulation, DNA Demethylation and Progesterone Treatment

The work was carried out by microinjections of an aromatase inhibitor letrozole, an estrogen receptor modulator tamoxifen in incubated eggs Gallus g. domesticus, the first day of incubation. It also used 5-azacytidine (5-AC) at the same time. Injection of progesterone was carried out before the onset of meiosis prophase 1 on 16 h of incubation. Morphologically and histologically and by PCR, sex of the 17-day embryos was controlled. According to information received microinjection of letrozole caused almost a 100% inversion of genetic males to females, which is manifested in the morphology of the gonads. In other experiments, sex reversal is not revealed. The results are obtained in this study, and the data suggest that the presence of gonadogenesis in female chickens makes earlier emergence of aromatase in the beginning of incubation, than that according to the classical scheme of sex determination in birds. Presumably this kind of synthesis is triggered by some W-chromosomal factors. It failed to detect the phenomenon of gender inversion—the transformation of males into females after exposure to demethylating agent 5-AC that casts doubt on the participation of male hypermethylated (MHM)-RNA-segment in regulating the activity of sex determining genes.

CHD genes: a reliable marker for bird populations and phylogenetic analysis?Case study of the superfamily Sylvioidea (Aves: Passeriformes)

The chromo-helicase-DNA binding protein (CHD) genes are widely used markers for sex determination in birds and provide a rapid and low-cost method with applicability to a large number of taxa. A good phylogenetic marker displays highly conserved domains and a slow evolution rate, proprieties that CHD genes seem to have. This gives rise to the following question: is the CHD gene a reliable marker for phylogenetic and bird population analysis? The aim of this study is to investigate whether the CHD gene is a reliable tool for molecular phylogeny and for population analysis within the superfamily Sylvioidea. Our results reveal that CHD genes are good markers for these two analyses, even better than myoglobin.

Historical overview of methods for sex determination in birds

Determining the sex in birds is very difficult, primarily because over 50% of species is monomorphic (no morphological differences between the sexes). Before the application of molecular genetic methods, there were used numerous methods all of which were un?reliable. Because of the importance of the analyses, they have to be reliable, economical, safe and prompt. Highly conserved CHD gene is defined in 1995. on W chromosome in birds, while on Z chromosome it was defined two years later. The difference in the length of the intronic sequences of CHD gene of Z and W chromosomes enables the distinguishing of the sexes after amplification of specific fragments by the application of specific primers. Molecular genetic methods have the supremacy over all the other methods because, ex?cept for being safe (both for birds and people), they provide reliable results, and also can be applied in all bird species.

Spatiotemporal expression patterns of doublesex and mab-3 related transcription factor 1 in the chicken developing gonads and Müllerian ducts

Sex of birds is genetically determined by the inheritance of sex chromosomes (ZZ for male and ZW for female), and the Z-linked gene named doublesex and mab-3 related transcription factor 1 (DMRT1) is a candidate sex-determining gene in avian species. However, the mechanisms underlying sex determination in birds are not yet understood, and the expression patterns of the DMRT1 protein in urogenital tissues have not been identified. In the current study, we used immunohistochemistry to investigate the detailed expression patterns of the DMRT1 protein in the urogenital systems (including Müllerian ducts) in male and female chicken embryos throughout embryonic development. Gonadal somatic cells in the male indifferent gonads showed stronger expressions of DMRT1 compared with those in the female indifferent gonads well before the presumptive period of the sex determination, and Sertoli cells forming testicular cords expressed DMRT1 in the testes after sex determination. Germ cells expressed DMRT1 equally in males and females after sex determination. The expression was continuous in males, but in females it gradually disappeared from the germ cells in the central part of the cortex of the left ovary toward both edges. The DMRT1 was also detected in the tubal ridge, which is a precursor of the Müllerian duct, and at the mesenchyme and outermost coelomic epithelium of the Müllerian duct in both sexes. Strong expression was observed in the males, but it was restricted to coelomic epithelium after the regression of the duct started. Thus, we observed the detailed spatiotemporal expression patterns of DMRT1 in the developing chicken urogenital systems throughout embryonic development, suggesting its various roles in the development of urogenital tissues in the chicken embryo.

Differential expression of the MHM region and of sex-determining-related genes during gonadal development in chicken embryos

The chicken (Gallus gallus) embryo has been used as a classic model system for developmental studies because of its easy accessibility for surgical manipulation during embryonic development. Sex determination in birds is chromosomally based (ZZ for males and ZW for females); however, the basic mechanism of sex determination is still unknown. Here, the dynamics of expression of candidate genes implicated in vertebrate sex determination and differentiation were studied during embryonic chicken gonadal development. Gene expression profiles were obtained before, during, and after gonadal sex differentiation in females and males for DMRT1, SOX3, SOX9, DAX1, SCII, HINTZ, HINTW, and the male hypermethylated (MHM) region. Transcripts for the HINTZ, DMRT1, DAX1, SCII, and SOX9 genes were observed in both sexes, but expression was higher in male gonads and may be correlated with testicular differentiation. The expression patterns of HINTW, SOX3, and MHM suggest that they may act in ovary development and may be involved in meiosis entry. MHM was upregulated and DMRT1 was downregulated in females at the same developmental stage. This may indicate a regulation of DMRT1 by MHM ncRNA. Similar dynamics were observed between HINTW and HINTZ. This study reports on the MHM expression profile during gonadal development and its correlation with the expression of genes involved in vertebrate sex determination.

Yolk concentrations of hormones and glucose and egg weight and egg dimensions in unincubated chicken eggs, in relation to egg sex and hen body weight

Birds can manipulate offspring sex ratio under natural and experimental conditions and maternal hormones have been shown to be involved in this process. Studies also provided evidence for the presence of sex specific concentrations of yolk hormones in avian eggs. These findings led to the suggestion that yolk hormones could influence genetic sex determination in birds. However, in previous studies, yolk hormone concentrations and egg sex were studied in incubated eggs, although incubation of the eggs and embryonic development can alter yolk hormone concentrations and measured sex ratio. This study is the first to determine a wide array of egg components and hen body weight in relation to the sex of the egg in unincubated eggs. Egg parameters studied were yolk concentrations of testosterone, estradiol, androstenedione, progesterone, dihydrotestosterone, and glucose, and egg weight and dimensions. In addition, we studied the associations among all measured parameters. Associations were found between a number of yolk hormones (progesterone associated with testosterone, estradiol and androstenedione; androstenedione with testosterone; dihydrotestosterone with estradiol and androstenedione) as well as between yolk testosterone and egg length and egg weight. There were no significant overall differences between male and female chicken eggs in any of the measured egg parameters. However, there were a few interactions such as the interaction of egg sex with dihydrotestosterone and with hen body weight which predicted estradiol levels and an interaction of estradiol levels with egg width for predicting sex of egg. Their biological relevance need, however, further study.

Just how conserved is vertebrate sex determination?

Sex determination in vertebrate embryos has long been equated with gonadal differentiation into testes or ovaries. This view has been challenged over the years by reports of somatic sexual dimorphisms pre-dating gonadal sex differentiation. The recent finding that sex determination in birds is likely to be partly cell autonomous has again called for a broader definition of sex determination. Inherent sexual differentiation in each and every cell may apply widely among vertebrates, and may involve more than one "master sex gene" on a sex chromosome. At the gonadal level, key genes required for proper sexual differentiation are conserved among vertebrates, but their relative positions in the ovarian and testicular cascades differ. We illustrate these differences by comparing key sex genes in fishes versus birds and mammals, with emphasis on DM domain genes and the SOX9-AMH pathway in the testis and the FOXL2-Aromatase pathway in the ovary. Such comparisons facilitate the identification of ancient versus derived genes involved in gonadal sex determination. The data indicate that vertebrate sex-determining cascades are not as conserved as once thought.

RNA sequencing reveals sexually dimorphic gene expression before gonadal differentiation in chicken and allows comprehensive annotation of the W-chromosome

BackgroundBirds have a ZZ male: ZW female sex chromosome system and while the Z-linked DMRT1 gene is necessary for testis development, the exact mechanism of sex determination in birds remains unsolved. This is partly due to the poor annotation of the W chromosome, which is speculated to carry a female determinant. Few genes have been mapped to the W and little is known of their expression.ResultsWe used RNA-seq to produce a comprehensive profile of gene expression in chicken blastoderms and embryonic gonads prior to sexual differentiation. We found robust sexually dimorphic gene expression in both tissues pre-dating gonadogenesis, including sex-linked and autosomal genes. This supports the hypothesis that sexual differentiation at the molecular level is at least partly cell autonomous in birds. Different sets of genes were sexually dimorphic in the two tissues, indicating that molecular sexual differentiation is tissue specific. Further analyses allowed the assembly of full-length transcripts for 26 W chromosome genes, providing a view of the W transcriptome in embryonic tissues. This is the first extensive analysis of W-linked genes and their expression profiles in early avian embryos.ConclusionSexual differentiation at the molecular level is established in chicken early in embryogenesis, before gonadal sex differentiation. We find that the W chromosome is more transcriptionally active than previously thought, expand the number of known genes to 26 and present complete coding sequences for these W genes. This includes two novel W-linked sequences and three small RNAs reassigned to the W from the Un_Random chromosome.

Triploid plover female provides support for a role of the W chromosome in avian sex determination

Two models, Z Dosage and Dominant W, have been proposed to explain sex determination in birds, in which males are characterized by the presence of two Z chromosomes, and females are hemizygous with a Z and a W chromosome. According to the Z Dosage model, high dosage of a Z-linked gene triggers male development, whereas the Dominant W model postulates that a still unknown W-linked gene triggers female development. Using 33 polymorphic microsatellite markers, we describe a female triploid Kentish plover Charadrius alexandrinus identified by characteristic triallelic genotypes at 14 autosomal markers that produced viable diploid offspring. Chromatogram analysis showed that the sex chromosome composition of this female was ZZW. Together with two previously described ZZW female birds, our results suggest a prominent role for a female determining gene on the W chromosome. These results imply that avian sex determination is more dynamic and complex than currently envisioned.

Assessment of CHD-specific primers for gender determination in Red-billed Oxpeckers Buphagus erythrorhynchus

Red-billed Oxpeckers Buphagus erythrorhynchus are morphologically similar and do not display distinctive phenotypic difference between males and females. The development of DNA-based gender determination techniques constituted a breakthrough in reliable sex determination in birds. Two DNA-based methods of gender determination were evaluated to determine the preferred method for the Red-billed Oxpeckers. DNA-based gender determination of the Red-billed Oxpeckers was conducted so that specific sexes could be relocated to new release sites within South Africa. The two primer sets used were 2550F/2718R and P2/P8. When comparing the results of the two primer sets, it was determined that 17% (n = 25) of individuals that were identified as having one sex by the 2550F/2718R primer set changed their DNA gender determination when the P2/P8 primer set was used. Based on molecular evidence and the pathology results for three recorded mortalities at the National Zoological Gardens of South Africa, it was determined that the P2/P8 primer set would be preferable to the 2550F/2718R primer set for DNA gender determination of Red-billed Oxpeckers.

Morphological sexing of passerines: not valid over larger geographical scales

Sex determination of birds is important for many ecological studies but is often difficult in species with monomorphic plumage. Morphology often provides a possibility for sex determination, but the characters need to be verified. We tested whether five passerine species can be sexed according to standard morphological measurements applying a forward logistic regression with sex determined by molecular analysis as the dependent variable. Furthermore, we tested whether the results can be used on a larger geographic scale by applying morphological sexing methods gained by similar studies from other regions to our data set. Of the five species of this study only Garden Warblers Sylvia borin could not be sexed morphologically. In the Robin Erithacus rubecula, 87.2% of all individuals were sexed correctly. For Reed Warblers Acrocephalus scirpaceus, Willow Warblers Phylloscopus trochilus and Reed Buntings Emberiza schoeniclus, the respective values were 77.6, 89.4 and 86.4%. When the logistic regression functions from similar studies on Robins and Reed Buntings in Denmark and Scotland were applied to the birds from south-western Germany, they performed less well compared to the original dataset of these studies and compared to the logistic regression function of our own study. The same was the case for Willow Warblers when a wing length criterion used in Great Britain was applied to the birds of our study. These discrepancies may have several explanations: (1) the models are optimised for the dataset from which they were extracted, (2) inter-ringer variation in measurements, (3) the use of different age cohorts, (4) different morphology due to different habitat availability around the study site, or, most likely, (5) different morphology due to different migratory behaviour. We recommend that morphological sex differentiation methods similar to this study (1) be only used population specific, (2) only with one age cohort and (3) to adjust the extracted equations from time to time.