Secrets in the eyes of Black Oystercatchers: a new sexing technique

ABSTRACT.   Sexing oystercatchers in the field is difficult because males and females have identical plumage and are similar in size. Although Black Oystercatchers ( Haematopus bachmani ) are sexually dimorphic, using morphology to determine sex requires either capturing both pair members for comparison or using discriminant analyses to assign sex probabilistically based on morphometric traits. All adult Black Oystercatchers have bright yellow eyes, but some of them have dark specks, or eye flecks, in their irides. We hypothesized that this easily observable trait was sex-linked and could be used as a novel diagnostic tool for identifying sex. To test this, we compared data for oystercatchers from genetic molecular markers (CHD-W/CHD-Z and HINT-W/HINT-Z), morphometric analyses, and eye-fleck category (full eye flecks, slight eye flecks, and no eye flecks). Compared to molecular markers, we found that discriminant analyses based on morphological characteristics yielded variable results that were confounded by geographical differences in morphology. However, we found that eye flecks were sex-linked. Using an eye-fleck model where all females have full eye flecks and males have either slight eye flecks or no eye flecks, we correctly assigned the sex of 117 of 125 (94%) oystercatchers. Using discriminant analysis based on morphological characteristics, we correctly assigned the sex of 105 of 119 (88%) birds. Using the eye-fleck technique for sexing Black Oystercatchers may be preferable for some investigators because it is as accurate as discriminant analysis based on morphology and does not require capturing the birds.     

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.     

A rapid improved method for sexing embryo of water buffalo

The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.     

Classification and misclassification in sexing the Black femur by discriminant function analysis

Stepwise discriminant function analysis for sex assessment was applied to 130 North American Black femora. The measurements included femoral length and three midshaft dimensions likely to be preserved in archaeologically derived and forensic remains. The method correctly assigned sex for 76.4% of the sample (range 70.8–81.5%). This compares favorably with results achieved with other skeletal parts; it also compares favorably with results using the femur in sexing other racial groups. Among our other conclusions are: (1) a “general size factor” is one of major significance in correct classification and in misclassification of sex, and most misclassified individuals are anomalous for this factor; (2) the inconsistency in the relation between circumference and femoral length, which characterizes the remaining misclassified individuals, suggests that anomalous functional demands of body weight/musculature are at fault, and affect circumference more than length; and (3) discriminant function analysis of the same variables in Whites produced similar results, suggesting that sex overrides race in sex assessment; this was confirmed by cross-validating the predictive accuracy of Black discriminant function coefficients on White data, and vice versa.     

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.     

Morphology versus molecules: sexing Red-winged Blackbird nestlings

ABSTRACT.   Past studies of offspring sex ratios in birds have often relied on sexually size dimorphic species where nestling sex could be determined based on weight at a given age. DNA-based sexing techniques allow us to assess the accuracy of those techniques and to refine them for use when costs or convenience make DNA methods impractical. Using nestling Red-winged Blackbirds ( Agelaius phoeniceus ) whose sex was determined using DNA, we compared sex ratios obtained using different morphological criteria. Conservative criteria from previous studies were completely accurate, but allowed sexing of few nestlings younger than 8 d old, and were more successful for sexing males than females. A new method was developed that allowed accurate sexing of nestlings beginning at day 6 posthatching and was less biased relative to known sex ratios. Using 11 years of data, the conservative method left an average of 55% of nestlings and 36% of fledglings unsexed, compared to 31% and 9% using the new method. Furthermore, the male bias in sex ratio estimates using the conservative method was greater, both absolutely and relative to estimates based on the new method, when the proportion of unsexed nestlings (because they were not weighed when older) was higher. Thus, estimates of population sex ratios will be more accurate as the number of nestlings measured on day 8 or older increases. However, if some nestlings that were not weighed past day 7 fledge, the new method allows more of those individuals to be sexed than the conservative method, and the population sex ratio estimate should be more reliable. Although our approach should apply to other sexually dimorphic species, the criteria used must be developed based on such species-specific attributes as growth patterns and degree of hatching asynchrony.     

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.     

Sex identification by alternative polymerase chain reaction methods in falconiformes

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.     

家禽的性别鉴定方法

现代家禽生产,祖代鸡通过释肛鉴定性别,父母代鸡以羽速（快慢羽）判定公母,而商品代鸡则以羽色自别雌雄。常规性别鉴定方法耗资费力,且慢羽基因（K）与内源病毒基因（ev-21）紧密连锁,导致慢羽鸡群免疫反应降低,成活率和产蛋性能下降。以鉴定W染色体上性连锁DNA序列或基因为基础,从分子水平上鉴定家禽性别的研究有望填补家禽性别鉴定空白。     

用分离精子进行性别控制研究的现状

哺乳动物后代性别控制的方法有两种,即植入前胚胎性别的选择和受精前精子的分离,后者是动物性别控制最有效的途径。目前最有重复性、科学性和有效性的分离精子的方法,是根据精子DNA含量存在差异的原理,利用荧光染料与DNA相结合,并通过流动细胞检索分离仪进行精子的分离。     

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.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

Uncertainty in determination of sex from harvested bobcats

Considerable uncertainty often exists in estimates of demographic parameters based on data collected from harvested furbearer species. We used molecular genetic techniques to estimate rates of error in 2 methods of sex determination of harvested bobcats (Lynx rufus): manual examination of the carcass (field sex) and laboratory-based maximum canine root area (MRA sex). Error rates were high for both sexing techniques, and were associated with age and an age–sex interaction for the field and MRA sexing methods, respectively. These findings do not support the use of the field methods for identifying sex of harvested bobcats. The MRA method may be effective for determining sex of older bobcats but is limited by considerable overlap between sexes in juveniles and yearlings. If critical demographic parameters are estimated from harvest data, efforts should be made to identify and reduce rates of error before data are used to assess population status. © 2011 The Wildlife Society.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

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.     

A biometric analysis of the pelvic acetabulum as an indicator of sex in bovids

Despite its potential importance in the reconstruction of hunting strategies and subsistence patterns, determining sex in zooarchaeological assemblages is a task that has been often neglected because the assemblages consist mainly of fragmentary bones. Only a limited amount of research has been focused on sexing individuals at archaeological sites. Although dimorphic elements in skeletal anatomy (e.g., horns) are the most widely used indicators for sexing, other skeletal parts, such as the pelvic acetabulum provide valuable information to identify sex. The present work builds upon previous research and indicates the most useful indicators in the pelvic acetabulum to distinguish sex in bovids, with the goal of providing an analytical basis to expand interpretations of carcass acquisition strategies by humans.     

Sex identification of owls (family Strigidae) using oligonucleotide microarrays

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.     

Sexing fish by palpation: a simple method for gonadal assessment of fluvial salmonids

This paper presents a non‐destructive sexing method that can be employed in fluvial salmonids and presumably some other taxa of fish. The sex of a fish is identified and assessed by indirectly palpating the gonad.     

Sex determination of bovine embryo blastomeres by fluorogenic probes

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).