A reliable method for sexing unincubated bird eggs for studying primary sex ratio

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.     

Cell number and sex ratio in unfertilized chicken eggs (Gallus gallus domesticus)

In chickens and other birds, females have two different sex chromosomes (ZW), whereas males carry two homologous sex chromosomes (ZZ). The primary sex ratio can thus be determined by genetic analysis of the sex chromosome of the ovum before fertilization. Sex diagnosis is more reliable when there are more cells, i.e. sufficient DNA, for the analysis. In this study, eggs from virgin hens were incubated for 3 days and the number of cells in the germinal discs was counted. A median of 2.5 cells was counted with a range of two to 20 cells. We also counted cells in the germinal discs of unfertilized eggs of inseminated hens and recorded a median of three cells and a range of two to 40 cells. Sex diagnosis based on polymerase chain reaction (PCR) amplification of Z and W chromosomes specific fragments from the CHD1 gene in 30 incubated eggs obtained from 35-week-old virgin hens gave a ratio of 13 Z to 15 W chromosomes with two samples undetermined.The unfertilized eggs of three groups of chickens were subjected to sex diagnosis to supplement the sex ratio data of an incubation experiment (see companion paper). The high proportion of Z chromosomes diagnosed in all three groups by two independent gene products suggests a sex difference on developmental potential and/or a sex chromosome segregation biased toward males in unfertilized eggs especially at the beginning of reproduction.     

鸟类性别鉴定技术研究进展

鸟类性别鉴定曾经通过外科手术对生殖腺直接进行检测,此法不仅繁琐而且有时会导致鸟的不育;细胞学水平上鉴定性别是通过直接观察W、Z性染色体而进行,但由于染色体数目大、分散难度高,很容易引起人为的主观因素导致结果错误。近年来利用鸟类W染色体上的性连锁基因或DNA序列,通过PCR、原位杂交等技术建立起来的．DNA分子水平性别鉴定方法,发展迅猛,准确度高,操作简便。此技术一旦形成体系,对鸟类研究、濒危物种保存等都将具有非常重要的意义。     

Fluorescence in situ hybridization: a new method for determining primary sex ratio in ants

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and/or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Improving the reliability of molecular sexing of birds using a W-specific marker

Molecular techniques for identifying sex of birds utilize length differences between CHD-Z and CHD-W introns, but in some cases these methods can lead to sexing errors. Here we show that an additional W-specific primer can be used in conjunction with a pre-existing sexing primer pair to dramatically improve the reliability of molecular sexing methods. We illustrate the approach with American coots (Fulica americana), a species with CHD-Z polymorphism that could not be accurately sexed using traditional methods. We developed a reverse primer GWR2 designed to sit within the intron of the W chromosome and amplify a distinctively small DNA fragment that serves as a W-specific marker. Analysis of known-sex individuals indicates that this W-specific primer provides an efficient and reliable protocol to identify the sex of F. americana. The development of such sex-specific primers will likely increase the reliability of molecular sexing methods in other birds as well. Comparisons between CHD-Z alleles of coots and common moorhens (Gallinula chloropus) revealed that CHD-Z polymorphism evolved separately in these two closely related species. We discuss the implications of repeated evolution of CHD-Z polymorphisms among birds.     

High‐resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

High‐resolution melting (HRM) analysis is a very attractive and flexible advanced post‐PCR method with high sensitivity/specificity for simple, fast and cost‐effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real‐time PCR systems, along with improved saturating DNA‐binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex‐specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed‐tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real‐time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.     

Retroposon insertions and the chronology of avian sex chromosome evolution

The vast majority of extant birds possess highly differentiated Z and W sex chromosomes. Nucleotide sequence data from gametologs (homologs on opposite sex chromosomes) suggest that this divergence occurred throughout early bird evolution via stepwise cessation of recombination between identical sex chromosomal regions. Here, we investigated avian sex chromosome differentiation from a novel perspective, using retroposon insertions and random insertions/deletions for the reconstruction of gametologous gene trees. Our data confirm that the CHD1Z/CHD1W genes differentiated in the ancestor of the neognaths, whereas the NIPBLZ/NIPBLW genes diverged in the neoavian ancestor and independently within Galloanserae. The divergence of the ATP5A1Z/ATP5A1W genes in galloanserans occurred independently in the chicken, the screamer, and the ancestor of duck-related birds. In Neoaves, this gene pair differentiated in each of the six sampled representatives, respectively. Additionally, three of our investigated loci can be utilized as universal, easy-to-use independent tools for molecular sexing of Neoaves or Neognathae.     

Offspring sex ratios in tree swallows: females in better condition produce more sons

Organisms are expected to adjust the sex ratio of their offspring in relation to the relative fitness benefits of sons and daughters. We used a molecular sexing technique that amplifies an intron of the CHD1 gene in birds to examine the sex ratio at egg-laying in socially monogamous tree swallows (Tachycineta bicolor). We examined all individuals in 40 broods (210 young), including all unhatched eggs and nestlings. Thus, the sex ratio we measured was the same as the sex ratio at laying. Overall, the mean sex ratio per brood (+/- SD) was biased significantly towards males (57 +/- 2% male). Within broods, male-biased sex ratios were associated with females in better body condition, and these females were more likely to produce sons in better condition. Tree swallows have one of the highest known levels of extra-pair paternity in birds (38-76% extra-pair young), and, as a consequence, variance in male reproductive success is greater than that of females. Thus, in tree swallows, investment in sons has the potential for higher fitness returns than investment in daughters, assuming that sons in better condition have greater reproductive success.     

Morphometric sex determination of Milky and Painted Storks in captivity

Logistic regression was applied to develop a morphometric sexing method of two closely related stork species that were previously sexed through amplification of the CHD gene. Tarsus length (TL) and bill length (BL) measurements were recorded from captive populations of adult Milky Stork (Mycteria cinerea) (n = 60) and Painted Stork (Mycteria leucocephala) (n = 58) at Zoo Negara Malaysia. Despite having monomorphic plumages, both stork species exhibited normal sexual size dimorphism in which males were significantly larger than females in the tested variables. Based on logistic regression analysis, BL correctly classified the sex of sampled individuals from Painted and Milky stork with an overall predicted accuracy of 94.8 and 90.0%, respectively. However, TL measurements generated a lower predicted accuracy level of 86.2% and a same accuracy level of 90% on the sex classification of individuals from Painted and Milky stork, respectively. By comparing the measurements of both species, only the average BL measurements of the Milky storks were significantly lower than that of Painted storks (t-test, P80.001). The logistic regression equation in this study may serve as a simple and more practical option for sexing Milky and Painted storks for their breeding and conservation programmes.     

Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification

We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

New and improved molecular sexing methods for museum bird specimens

We present two new avian molecular sexing techniques for nonpasserine and passerine birds (Neognathae), which are more suitable for use with museum specimens than earlier methods. The technique for nonpasserines is based on a new primer (M5) which, in combination with the existing P8 primer, targets a smaller amplicon in the CHD1 sex-linked gene than previously. Primers targeting ATP5A1, an avian sex-linked gene not previously used for sex identification, were developed for passerines. Comprehensive testing across species demonstrated that both primer pairs sex a range of different species within their respective taxonomic groups. Rigorous evaluation of each method within species showed that these permitted sexing of specimens dating from the 1850s. For corn bunting museum specimens, the ATP5A1 method sexed 98% of 63 samples (1857-1966). The M5/P8 CHD1 method was similarly successful, sexing 90% of 384 moorhen specimens from six different museum collections (1855-2001). In contrast, the original P2/P8 CHD1 sexing method only identified the sex of less than half of 111 museum moorhen samples. In addition to dried skin samples, these methods may be useful for other types of material that yield degraded or damaged DNA, and are hence potential new sexing tools for avian conservation genetics, population management and wildlife forensics.     

鸵鸟性别鉴定的分子标记方法

鸵鸟(Struthiocamelus)属于平胸总目鸟类,雌雄鸵鸟在性成熟前外部形态相同,很难通过外观和形态来鉴定性别,给早期分群饲养造成了很大的困难。实验利用鸵鸟羽毛提取基因组DNA,之后利用EE0.6和CHD基因中2个引物组合对3对已知性别和9只未知性别鸵鸟的性别基因片段进行特异性扩增。结果显示,这对引物组合在雄性鸵鸟的DNA中未扩增出片段,在雌性鸵鸟DNA中扩增出1条片段,可以对鸵鸟的性别作出准确鉴定,从而解决幼雏期鸵鸟难以从外貌上区分其性别的问题。     

Non-invasive sex identification of the white-bellied sea eagle (Haliaeetus leucogaster) through genetic analysis of feathers

The white-bellied sea eagle, Haliaeetus leucogaster, displays reversed sexual size dimorphism and is monomorphic for adult plumage coloration. Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters, but since avian W-specific DNA sequences were identified, PCR amplification has become commonly used for molecular sexing. We used a PCR test employing primers that amplify two homologous fragments of both the CHD-W gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with high reproducibility. We conclude that this PCR-based test with feathers as the DNA source is a reliable sexing method for H. leucogaster. This sexing technique is objective and non-invasive and could be used to test sex ratio theories, as well as to help improve conservation and management actions for captive breeding program of this species in Malaysia.     

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.     

Sexing of mid‐incubation avian embryos as a management tool for zoological breeding programs

Skewed sex ratios in zoo breeding programs may require housing single birds of an overrepresented gender, increasing demands on limited resources that could otherwise be diverted to breeding pairs or other important species. The ability to selectively incubate and hatch eggs of a desired sex represents a significant improvement in the long‐term management of avian species. This study describes a successful method for in ovo sexing of embryos from stage 30 through 42 of incubation (Hamburger and Hamilton [1951] J Morphol 88:49–92). A 0.01–1 µl blood sample was collected from either the vitelline vessel (VV) or the blood vessels of the chorio–allantoic membrane (CAM) of embryos at stages 14–18 or 30–42, respectively. DNA was isolated from whole blood using the Chelex method (Walsh et al. [1991] Biotechniques 10:506–513; Jensen et al., [2003] Zoo Biol 22:561–571). Sex was determined by PCR amplification using the previously described P2/P8 (Griffiths et al. [1998] Mol Ecol 7:1071–1075) and 1237L/1272H (Kahn et al. [1998] Auk 115:1074–1078) primers or by commercial vendor. Success rate was calculated as the percent of sampled embryos surviving to hatch. Embryos of the undesired sex were not incubated, thus not included in the calculation. There was a considerable difference in success rate when blood was collected from the stage 14–18 VV (0–25%, average 12%) vs. stage 30–42 CAM (33–100%, average 76%). In conclusion, in ovo sexing of embryos between stages 30 and 42 yields acceptable embryo survival rates while providing enough blood for genetic testing. Zoo Biol 31:694‐704, 2012. © 2011 Wiley Periodicals, Inc.     

No sex difference in yolk steroid concentrations of avian eggs at laying

Yolk steroids of maternal origin have been proposed to influence genetic sex determination in birds, based on sex differences in yolk steroid concentrations of peafowl eggs incubated for 10 days. More recent reports dispute this proposal, as yolk steroids in eggs incubated for 3 days do not show such sex differences. To date, research examining this phenomenon has only analysed incubated eggs, although sex in avian species is determined before incubation begins. This may be a serious methodological flaw because incubation probably affects yolk steroid concentrations. Therefore, we investigated sex differences in yolk steroid concentrations of unincubated avian eggs. We withdrew yolk for steroid analysis from fresh, unincubated Japanese quail (Coturnix japonica) eggs by biopsy, and then incubated those eggs for 10 days, after which we harvested the embryonic material for genetic sexing and the incubated yolk for further steroid analysis. We found no sex differences in fresh Japanese quail eggs; however, sex differences were apparent in yolk steroids by day 10 of incubation, when female eggs had significantly more oestrogen in relation to androgen than male eggs. Concentrations of all yolk androgens decreased dramatically between laying and day 10 of incubation, whereas oestradiol (E2) concentrations increased marginally. Thus, yolk concentrations of androgens and E2 do not appear critical for avian sex determination.     

Heteroduplex molecules cause sexing errors in a standard molecular protocol for avian sexing

Molecular methods are a necessary tool for sexing monomorphic birds. These molecular approaches are usually reliable, but sexing protocols should be evaluated carefully because biochemical interactions may lead to errors. We optimized laboratory protocols for genetic sexing of a monomorphic shorebird, the upland sandpiper (Bartramia longicauda), using two independent sets of primers, P2/P8 and 2550F/2718R, to amplify regions of the sex‐linked CHD‐Z and CHD‐W genes. We discovered polymorphisms in the region of the CHD‐Z intron amplified by the primers P2/P8 which caused four males to be misidentified as females (n = 90 mated pairs). We cloned and sequenced one CHD‐W allele (370 bp) and three CHD‐Z alleles in our population: Z° (335 bp), Z′ (331 bp) and Z″ (330 bp). Normal (Z°Z°) males showed one band in agarose gel analysis and were easily differentiated from females (Z°W), which showed two bands. However, males heterozygous for CHD‐Z alleles (Z′Z″) unexpectedly showed two bands in a pattern similar to females. While the Z′ and Z″ fragments contained only short deletions, they annealed together during the polymerase chain reaction (PCR) process and formed heteroduplex molecules that were similar in size to the W fragment. Errors previously reported for molecular sex‐assignment have usually been due to allelic dropout, causing females to be misidentified as males. Here, we report evidence that events in PCRs can lead to the opposite error, with males misidentified as females. We recommend use of multiple primer sets and large samples of known‐sex birds for validation when designing protocols for molecular sex analysis.