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Sex determination in monomorphic birds is a precondition for captive breeding programs and management and conservation strategies for threatened species. Most species of the order Psittaciformes often present complications since these birds lack external sexual phenotypic traits, making it impossible to differentiate males and females. In the present study, we used molecular techniques to determine the sex of 31 individuals belonging to nine species of the order Psittaciformes kept under human care at the Akumal Monkey Sanctuary & Rescued Animals in Quintana Roo, Mexico. This is a useful and low-cost methodology based on the analysis of the conserved region of the CHD1 gene, which was amplified by PCR with two sets of primers: P8/P2 and 2550F/2718 R. All individuals were successfully sexed with the first set of primers, while only 28 out of 31 samples (90%) could be amplified with the second set. Out of the 31 individuals analyzed, fifteen are female, and seventeen are male. This information represents a handy tool for adequately managing birds under human care, resulting in their reproduction and eventual reintegration into their natural habitat. [ABSTRACT FROM AUTHOR]

Psittacine beak and feather disease virus (PBFDV) is a significant viral pathogen affecting the development of beak and feather cells, leading to feather abnormalities in avian species. This study aimed to investigate the molecular detection rate of PBFDV among Psittaciformes in Iran and to evaluate the influence of factors such as sex, species, season, and bird origin on PBFDV prevalence. Feather samples were collected from 1335 Psittaciformes, including Agapornis roseicollis, Pyrrhura molinae, Myiopsitta monachus, Aratinga solstitialis, Nymphicus hollandicus, Psittacula krameri, Psittacus erithacus, Poicephalus senegalus, Eos bornea, and Agapornis fischeri, and subjected to polymerase chain reaction (PCR) for PBFDV detection. The overall PBFDV detection rate across all species was 56.2% (751/1335, 95% CI: 53.6%-58.9%). The results showed that Agapornis roseicollis had a 2.23-fold higher likelihood of PBFDV infection compared to the reference species, Myiopsitta monachus (95% CI: 1.36-3.66). Furthermore, a significant interaction effect between season and species was observed. During autumn, Aratinga solstitialis and Pyrrhura molinae were 10.00 (95% CI: 5.10-20.59) and 3.72 (95% CI: 2.28-6.08) times more likely to be infected with PBFDV compared to spring, respectively. These findings underscore a high prevalence of PBFDV in Iran's psittacine population. The study emphasizes the importance of such investigations and the exploration of various risk factors for disease control and prevention strategies. It also underscores the necessity of virological assessments prior to bird export and import. [ABSTRACT FROM AUTHOR]

Chub mackerel Scomber japonicus and blue mackerel S. australasicus have become important aquaculture target species in Japan. For an efficient and stable aquaculture production of mackerels, it is desirable to work with a female-biased broodstock and thus to be able to manage precisely their sex ratio. As these two mackerel species do not have any external sexual dimorphism, direct biopsy of the gonads or measurement of sex hormone plasma levels is used in research to determine their phenotypic sex. However, these methods are associated with handling stress that results in gonadal atresia. Besides, it is difficult to identify sex using these methods in immature individuals. Therefore, genetic sexing would be desirable in these species to overcome these problems. In this study, we developed molecular sex identification tools for both chub and blue mackerels using sex-specific single-nucleotide variations (SNVs) identified by pool-sequencing (pool-seq) analyses. Genomic DNA was isolated from 30 males and females, in each species, and sequenced as pooled samples. Results of these pool-seq analyses identified a clear XX/XY male heterogametic sex-determination system in blue mackerel and a ZZ/ZW female heterogametic system in chub mackerel. Using this pool-seq information, we designed primer sets for genetic sex identification in both species. Results demonstrated that these primer sets are robust and accurate in assessing the genotypic sex of both blue and chub mackerels (accuracy > 99.5% in the chub, and > 94.5% in the blue mackerel) in various geographical samples of different ages. This method can now be applied for the efficient management of sex ratios in both mackerel aquaculture populations and wild-caught animals for fisheries management. [ABSTRACT FROM AUTHOR]

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Abstract Birds have a ZZ and ZW sex chromosome system (male and female, respectively). On the short arm of the Z chromosome of Gallus gallus domesticus there is a repetitive region called MHM region, which is absent on the W chromosome, that causes a natural copy number difference of the MHM region between the sexes, making possible the development of a quantitative PCR (qPCR) sexing assay, based on the Z chromosome double dose. Twenty-seven samples of tissues from eight adult Gallus gallus domesticus (four males and four females) were used to establish the parameters of the MHM copy-number sexing assay. We blinded sexed 20 chicks using 140 samples from different tissues (heart, brain, gonad, kidney, lung, muscle, and blood). The success rate of the assay was 100% (140/140). It required small amounts of DNA (0.39 ng), opening the perspective to the use of the assay in studies in which the amount of available DNA is limited. Highlights • A new chicken molecular sexing assay based on the dose of the Z chromosome. • Chicken sexing assay using qPCR and repetitive MHM region. • Chicken molecular sexing assay using small amount of DNA. [ABSTRACT FROM AUTHOR]

Genetic markers are a useful tool for bird population monitoring, especially when combined with ringing data, and particularly so in vulnerable species. This study compared the effectiveness of two DNA extraction methods: a standard protocol and a commercially available kit. The molecular sex-typing was performed using PCR-based methods with a 2550F/2718R set of primers in three species of herons: Great Egret, Purple Heron, and Grey Heron. Genomic DNA was isolated from feathers, eggshells, and eggshell swabs from 26 individuals. Overall, better DNA yields and purity were obtained by using the standard protocol isolation method. The highest DNA yield was obtained from the pin feathers compared to the contour feathers and eggshells, both of which had lower yields. Eggshell swabs indicated possible contamination with parental/sibling DNA. Our evaluation demonstrates that the optimization of laboratory procedures is beneficial, particularly when different types of noninvasive tissue samples are available. [ABSTRACT FROM AUTHOR]

This study presents the development of a PCR-based method for sex determination of caprine embryos by Sry and Aml-X gene detection. DNA sequencing of the PCR products amplified from the target loci confirmed that these amplicons were indeed amplified from portions of the caprine Sry and Aml-X genes. Furthermore, the primer sets were found to be specific for sexing of caprine of various breeds. An alternative PCR-sexing protocol was also developed to verify embryo sexing results and it was a pit-stop using Sry and DVEPC041 primer sets. For the pitstop PCR protocol, the Sry primer was best added after the first 6 cycles to obtain bands of equal intensity at 116 bp and 160 bp when identifying the male sex. However, application of the same pit-stop PCR protocol using genomic DNA of various caprine breeds also produced acceptable and accurate sex identification despite showing slight diversion of the expected result. [ABSTRACT FROM AUTHOR]

Gibbons of the genus Nomascus have a strong sexual dimorphism and dichromatism. As they mature, both sexes develop sex-specific pelage colour. In combination with physical similarities in the genitalia with both sexes, there are problems with determining the sex of young individuals compared to other genus of gibbons. This is a pilot study applying a multiplex polymerase chain reactions based on a non-invasive method for sex determination of gibbons. The study was conducted on 22 faecal samples from gibbons of the genus Nomascus. The animals were monitored by staff so that the samples were identified correctly and each sample was collected immediately after the defecation. Results confirmed the sex in all adult and juvenile animals with known sex; and 2 females and 5 males in juveniles were determined with unknown sex. The results of direct examination completely corresponded with the PCR results. The PCR reaction with template DNA isolated from faecal material required BSA usage, however, we observed the occurrence of nonspecific fragments. This did not affect the reliability of our results and we confirmed the usability of this method for this genus. [ABSTRACT FROM AUTHOR]

Twenty bitches were seen in consultation at the Department of Reproduction at ONIRIS (College of Veterinary Medicine, Food Science and Engineering, Loire Atlantique, Nantes, France) between 25 and 50 days of gestation for early sex determination of the canine foetus using ultrasound. The genital tubercle is not visible before 26 days; between 26 and 30 days, it is visible between the pelvic limbs; between 33 and 50 days, the position of the genital tubercle enables sex determination as it migrates caudally in the female and cranially in the male. Good statistical concordance between sexing via ultrasound and sexing at birth has been established (kappa coefficient of 0.8). Macroscopic, microscopic, and histological examinations of the external genital organs were also performed on 10 foetuses at 35 days of gestation; a cartilaginous structure was visualized in the genital apparatus of the male but also in half of the females. Finally, the development of a PCR technique on the SRY gene using formaldehyde-preserved tissues has been described for the first time in this study. It served as a reference for sexing canine foetuses. [ABSTRACT FROM AUTHOR]

The molecular methods for sex identification in birds are important resources commonly used in population studies, management and conservation plans, breeding programs and commercial poultry/farms. Recently, advanced polymerase chain reaction (PCR) methods based on melting curve analysis have been reported for Neognathae (non-ratite) birds. These genotyping strategies are simple, rapid, highly sensitive/specific and cost-effective, allowing high-throughput applicability. The evolutionary divergence and ancestral state of the sex chromosomes in the Palaeognathae (ratite) birds have hindered the utilisation of the sex-linked markers developed for non-ratite species. Therefore, the current PCR-based protocols for ratite species are restricted to agarose and polyacrylamide gels. The screening of new candidate sex-linked markers is mandatory to implement advanced PCR-based approaches in the routine molecular sexing of ratites. In this study, the nucleotide sequences of the Ostrich Z and W gametologous genes were used for the identification of new potential sex-linked markers, considering the optimisation of a molecular sexing protocol using the high-resolution melting (HRM) analysis. Four candidate markers (NTRK2, RASEF, TMEM2 and DAPK1) were characterised for four ratite species (Ostrich, Greater Rhea, Emu and Southern Cassowary). The male and female genotypes identified were accurately differentiated based on specific melting curve profiles generated. The discussion of the polymorphic patterns obtained and their influence on the reliability of molecular sexing are complemented with an overview of the classical PCR-based methods for ratite birds. This study highlights the potential and usefulness of the recently available genomic data for ratite sex chromosomes to identify new candidate sex-linked markers with simple in silico approaches. [ABSTRACT FROM AUTHOR]

Embryonic mice have been used widely to study organ development. Days 14-16 are critical for sex organ development and differentiation in mice. Current methods for sex identification are limited. Even the simplest polymerase chain reaction method may injure the embryo. We determined that morphologic analysis of embryonic mammary anlagen could be used for rapid atraumatic sex identification of day 14-16 mice. The accuracy of our method was verified by molecular and anatomical approaches. [ABSTRACT FROM AUTHOR]

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing. [ABSTRACT FROM AUTHOR]

Captive breeding of endangered species is often difficult, and may be hampered by many factors. Sexual monomorphism, in which males and females are not easily distinguishable, is one such factor and is a common problem in captive breeding of many avian and reptile species. Species-specific nuclear DNA markers, recently developed to identify portions of sex chromosomes, were employed in this study for sex determination of Komodo dragons (Varanus Komodoensis). Each animal was uniquely tagged using a passive integrated micro-transponder (TROVAN 100A type transponders of 13 mm in length and 2 mm in diameter). The sex of a total of 81 individual Komodo dragons (44 samples from Ragunan zoo, 26 samples from Surabaya zoo, and 11 samples from Gembira Loka zoo) were determined using primers Ksex 1for and Ksex 3rev. A series of preliminary PCR amplifications were conducted using DNA from individuals of known sex. During these preliminary tests, researchers varied the annealing temperatures, number of cycles, and concentrations of reagents, in order to identify the best protocol for sex determination using our sample set. We thus developed our own PCR protocol for this study, which resulted in the amplification of band A in females and band C in males. Results from band B, however, turned out to be non-determinative in our study because, for females, band B was not always visible, and for males sometimes a similar, but lighter band was also amplified, making interpretation difficult. In this study, sex determination was based mainly on the difference in size between the female-specific 812 bp fragment and the homologous, longer fragment amplified for males. [ABSTRACT FROM AUTHOR]

Abstract: Sex identification of birds is of great interest in ecological studies, however this can be very difficult in many species because their external features are almost monomorphic between the sexes. Molecular methodology has simplified this process but limitations still occur with widely accepted methods using polymerase chain reaction and gel electrophoresis, especially when applied to degraded DNA. Real-time polymerase chain reaction assays are emerging as a more efficient, sensitive, and higher throughput means of identification, but there are very few techniques validated using fecal samples and small target sizes. We present a real-time melt curve analysis assay targeting a small region of the CHD-1 gene allowing for high-throughput, sensitive, specific, and easy-to-interpret sexing results for a variety of Southern Ocean seabirds using fecal and tissue samples. [Copyright &y& Elsevier]

Flow cytometry is a widely used application for validating the accuracy of sperm sexing. However, this method is relatively expensive and requires considerable technical support. An alternative method employing simpler technology at low cost could be suitable for the evaluation of bovine semen in laboratories with low budgets. We used a SYBR Green Real-Time PCR assay to determinate sex ratio in bovine semen. The PLP and SRY genes were amplified to isolate the specific fragments of X- and Y-chromosome sequences, respectively. Two certified standard curves were obtained using two plasmids containing PLP and SRY amplicons. Our results show no significant difference in semen sex ratio in unsorted semen (54.7±0.52% X and 47.6±0.60% Y). However, significant difference was observed in X/Y-sorted semen (93.3±0.08% X and 91.4±0.06% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data. The evolution of X-chromosome bearing sperm content in unsorted samples showed an average of 52.6 for ejaculates and 51.8 for the commercial semen. In order to confirm our results, the accuracy, repeatability and reproducibility of the method were tested resulting in 98.2% accuracy, repeatability of CV=5.59% and reproducibility of CV=5.40%. Thus, this method is demonstrated to be a reliable and inexpensive way to test sexual chromosome content in semen samples. [ABSTRACT FROM AUTHOR]

Abstract: Sex determination of domestic animal's meat is of potential value in meat authentication and quality control studies. The job becomes easier if the same assay could serve the purpose for all three species i.e. cattle, sheep and goat. The objective of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to cattle, sheep and goat meat. Based on the amelogenin gene located on the X and Y chromosomes, a pair of primers was designed and the system of PCR was optimized. Upon PCR amplification, male tissue showed 2 bands, while female tissue resulted in only one band. The accuracy and specificity of the primers was assessed using genomic DNA extracted from meat samples of known sex. The protocol was subjected to a blind test and showed 100% concordance, proving its accuracy and reliability. The technique also claims its utility in resolving legal cases related to sex origin of these animals. [Copyright &y& Elsevier]

The applicability of simple PCR-based approaches for sex discrimination in the three European Phalacrocoracidae species was tested, using 93 individuals of known sex and two sets of primers (1237L/1272R and 2550F/2718R) for the amplification of the avian sex-specific chromo-helicase-DNA-binding protein gene. We evaluated the accuracy of each set of primers in providing the correct sex for each individual. The first primer set did not produce reliable results. The second provided a band pattern for each sex, easily distinguishable with agarose gel electrophoresis, which correctly identified all the individuals, even in samples of low DNA yield. The amplification products were sequenced and aligned revealing important nucleotide diversity among Phalacrocoracidae species. Compared with morphometric discriminant analysis and DNA-fingerprinting techniques previously applied, the PCR-based sexing with the 2550F/2718R primers is more accurate, less invasive and widely applicable to both adults and chicks, using a variety of DNA sources such as blood, tissue, feathers, egg shells and others. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35–48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle. [Copyright &y& Elsevier]

Analysis of the sex of cattle meat by fast and reliable molecular techniques is an important measure to ensure correct allocation of export refunds, particularly in European countries and also in countries such as India where female cattle (cow) slaughter is banned legally in many areas because of religious beliefs. Based on the male-specific SRY gene, X and Y chromosome specific amelogenin gene 2 pair of primers was designed and the system of PCR was optimized. Upon PCR amplification, male tissue showed 3 bands, while female tissue resulted in only 1 band. The accuracy and specificity of the primers were assessed using DNA template extracted from cattle meat of known sex. The protocol was subjected to a blind test and showed 100% concordance, proving its accuracy and reliability. [ABSTRACT FROM PUBLISHER]

Some studies showed analysis of fetal DNA in maternal plasma had been introduced as a new method for non-invasive prenatal diagnosis. Fetal sexing is possible at 8th week of pregnancy, using maternal blood sample testing. The aim was providing a rapid, reliable and non-invasive method for sexing of bovine fetuses. Maternal blood samples were collected from 38 pregnant cows during the 8th-38th w of gestation. Plasma was obtained by centrifugation and DNA was extracted by phenol-chloroform method from 350 μL maternal plasma. Two primer pairs for bovine amelogenin, Y-encoded, and testis-specific protein gene were used to amplification three fragments; 260 bp (Y-encoded, testis-specific protein gene), 341 and 467 bp (Y and X chromosome amelogenin gene). The polymerase chain reaction has been optimized for fragments amplification. The 467 bp fragment was detected in all samples. The 341 and 260 bp fragments were detected in 24 of 38 plasma samples of cows with male fetuses. The sensitivity and specificity of test was 100% with no false negative and positive results. The results showed that phenol-chloroform method was a simple and sensational to isolation fetal DNA in maternal plasma. The polymerase chain reaction is a favorable noninvasive method for bovine fetal sexing. [ABSTRACT FROM AUTHOR]

Contents The objective of this study was to test the suitability of a duplex PCR assay for sex and scrapie resistance genotype determination in fresh embryos. Duplex PCR amplified a repetitive and specific fragment of Y chromosome, used for sex diagnosis, and a PrnP fragment. PrnP codons 134 and 156, and codon 171 were genotyped by restriction fragment length polymorphisms and allele-specific PCR, respectively, after re-amplification of PrnP fragment. The specificity of the method was first assessed by testing 359 blood samples from Rasa Aragonesa sheep breed (161 males and 198 females). No amplification failures and total agreement between genotypic and phenotypic sex were found. In the same way, PrnP genotype determination by duplex PCR assay was in agreement with the PrnP animal's genotype established by sequencing. Finally, 73 samples of 1-10 cells from compact morulae were aspirated through the zona pellucida and genotyped for sex and PrnP. The efficiency was 96% when three or more cells were sampled. These results confirm that the duplex PCR assay reported in this work can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (96%) when three or more cells are sampled, allowing sexed fresh embryos of known PrnP genotype to be transferred in multiple ovulation and embryo transfer programmes. [ABSTRACT FROM AUTHOR]

The article presents a study on sex determination of sika deer using the feces collected in the field. The study was conducted by a group of researchers who used a nested polymerase chain reaction (nested PCR) for the amplification of the partial sequences of the sex determination region of the Y chromosome (SRY) gene and X zinc finger protein (ZFX) gene. The researchers yielded successful sex determination as they matched the sequences of target amplicons between the muscle and feces from the rectum.

We report the development of a reliable and efficient method for molecular sexing of all extant elephant taxa. We developed primers that amplify two short Y-specific fragments ( SRY1 and AMELY2) and one longer X-specific fragment ( PLP1), developed from elephant sequences in one multiplex PCR. All fragments were designed to be short (<200 basepairs) for use with degraded DNA and to be 50 basepairs apart to optimize visualization on agarose gels or as electropherograms. The multiplex PCR method matched sexes for at least 97.9% of the noninvasive savannah elephant samples and produced the expected female/male banding patterns for 14 African forest and 11 Asian elephant samples. We found this method to be more robust, efficient and less prone to contamination than previously developed sexing methods for elephants. [ABSTRACT FROM AUTHOR]

The article measures the sperm development in adult bulls through quantitative real-time polymerase chain reaction. The study was performed on semen extracted from bulls and cow embryos, which were classified based on being applied by either Percoll or OptiPrep density gradient. Results prove that sexing sperm through either Percoll or OptiPrep gradients can enhance the birth process of cattle.

Purpose: We analyzed the sex chromosome-encoding ZFX-ZFY genes and tested molecular sexing using the amplification patterns of intron 9 of ZFX-ZFY in the horse. Methods and results: The amplification of the ZFX-ZFY produced two distinct patterns, reflecting sexual dimorphism based on a length difference between the X and Y chromosomes. The amplification products from foals showed two distinct bands: one was common to all foals and mares, indicating that this band was amplified from ZFX, while the other was specific to some foals, indicating that it was from ZFY. The result based on the PCR assay was identical to the results of amplification of the Y chromosome-specific SRY gene and those of investigations of the phenotypic gender in three different horse populations. Conclusion: We suggest that this PCR strategy for determining sexes by comparing the amplification patterns of ZFX-ZFY genes is a convenient and precise method for discriminating sexes in horses. [ABSTRACT FROM AUTHOR]

Sex pre-selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR-specific primers are derived from the few single-copy Y-chromosome-specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male-specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male-specific markers (OPC16 and OPF10) by means of RAPD. These markers were successfully converted into SCARs (OPC16 and OPF10) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16 marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16 is also highly similar to a bubaline Y-chromosome-specific sequence. The primers derived from the two Y-chromosome-specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing. [ABSTRACT FROM AUTHOR]

Abstract: Consumer awareness has increased concerning castration of piglets without analgesia or anaesthesia. On the other hand the occurrence of boar taint is not tolerated by consumers. Currently no reliable methods exist for the on-line detection of boar taint in the slaughterhouse or for genetic sexing of pigs. Therefore, as an alternative the detection of male pork meat was sought. Based on detection of a length polymorphism of the sex chromosomal amelogenin gene a reliable, specific and highly sensitive PCR method for qualitative and semi-quantitative determination of male pork tissue in meat and meat products was determined. A set of 25 male and 25 female meat samples could be correctly identified and mixtures with as little as 0.1% male meat content could be detected. Therefore the method can be used for production and control of specific meat products containing low amounts of male pork meat and thus avoiding boar taint. [Copyright &y& Elsevier]

We determined a polymerase chain reaction (PCR)-based method to identify the sex of greater roadrunners (Geococcyx californianus). We found that previously reported primer combinations used in other bird species did not work reliably in roadrunners. However, a novel primer combination (P2-1237L) allowed for consistent and reliable sex identification of roadrunners. Conducting several PCR replicates per sample helped to identify occasional preferential amplification of the Z band that could occur in females (the heterogametic sex; i.e., WZ). Identifying the sex of individuals will allow for examination of sex-associated differences in home range size and habitat use, population demographics, and parenting behavior, as well as for a better understanding of the social system of roadrunners. [ABSTRACT FROM AUTHOR]

Abstract: Yak meat is of great economic importance to the people living in the cold high altitude area. In the present work, a multiplex PCR-based method was proposed for the sexing of yak meat by amplifying the target sequence of male-specific SRY gene. In a single reaction set, two DNA fragments of 121- and 290-bp were amplified from male meat DNA using two pairs of primers, but only one DNA fragment of 290-bp was amplified from female meat DNA. The assay proposed herein was applied to raw and heat-treated yak meat samples, and the identification results obtained were in perfect agreement with the anatomical sex of the yak meat samples. The method is fast, reliable and cheap. It is potentially suitable for the sexing of yak meat in routine analyses. It can be considered a helpful tool in the quality control of yak meat and meat products. [Copyright &y& Elsevier]

Abstract: A previously described canine-specific 9-STR multiplex, now including two markers for sex determination, was tested for the genotyping of 23 wolves from Northern and Central Portugal. The samples were collected at necropsies and presented varying states of preservation. Complete profiles were obtained in 74% of the samples, partial profiles in 22% and one completely null profile. This survey revealed 15 alleles not previously described in dogs, distributed among 6 STR loci. It is shown that this genotyping system, previously tested in domestic dogs, can be reliably used for obtaining complete genetic profiles in wolves with a matching probability of 2.45×10−9 and compatible sex identification, even in sub-optimal samples. Moreover, a population structure analysis using the observed genotypes revealed that this multiplexed 11-loci panel may potentially be used for discriminating between wolves and dogs. [Copyright &y& Elsevier]

Abstract: The aim of this study was to use polymerase chain reaction (PCR) by amplifying DNA from bovine (Bos taurus) fetal cells recovered through uterine puncture and subsequent amniotic fluid aspiration and to compare the effectiveness of the PCR method with amniotic dihydrotestosterone (DHT) levels in gender determination. Amniotic DHT levels between sexes were significantly higher in males than in females in all periods except the period 91 to 120 d. The differences among the amniotic DHT levels at different gestation periods (61 to 90, 91 to 120, 121 to 150, 151 to 180, 181 to 210 d) were not significant in females but were significant in males in the period 61 to 90 d compared with three other periods. Sensitivity was equal to 97.8% (95% CI=88.2% to 99.6%), and specificity was equal to 85.4% (95% CI=80.0% to 97.6%). These two values correspond with a cutoff of DHT in amniotic fluid. Distributions of the two sex groups were classified according to the 192.1 pg/mL cutoff value. A total of 93 amniotic fluid samples were examined by PCR analysis. The sex determination of 91 samples by PCR and electrophoresis was in agreement with the visual sexes of the fetuses. In two amniotic fluid samples, DNA was not isolated, and thus no sex determination was made. Fetal gender was correctly identified by PCR in 44 of 45 males and in 47 of 48 females. In PCR, one band (at the length of 102bp) and two bands (at the lengths of 102 and 226bp) were observed respectively for female and male fetuses. It may be concluded that the levels of amniotic DHT and PCR might be used for embryo sexing in pregnant cows. [Copyright &y& Elsevier]

The article focuses on the effectiveness of a deoxyribonucleic acid (DNA) test in sex determination of birds developed by the authors. The test uses a novel primer (P0) specific for chromohelicase-DNA- (CHD)-W gene, a multiplex polymerase chain reaction (PCR) and simple agarose-gel electrophoresis. The PCR was able to identify males and females in 14 families due to varying bands amplified by primers P0, P2 and P8. Sequence alignment and direct sequencing were able to detect partial CHD-W gene amplified by P2 and P8 in additional band in males.

Gender identification in bird is important for both conservation program and forensic investigation. Since many bird species exhibit sexual monomorphism, this could lead to fraudulent cases in related communities. PCR-based techniques are the current standards for avian gender identification. Most assays target the specific introns of the chromo-helicase DNA binding gene (CHD) on sex chromosomes, which generate different fragment sizes on agarose gel electrophoresis. In this study, we validated four primer sets previously reported for avian sex determination (P2/P8, CHD1F/CHD1R, 2550F/2718R, and 1237L/1272H) using 1405 samples from 70 avian species commonly found in Thailand. The result showed that all four primer sets can be used for gender investigation of these avian species. However, the most efficient primer set vary depending on the species being investigated. Primer sets providing 100% identification accuracy for each bird species are reported. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis. [ABSTRACT FROM AUTHOR]

For molecular sexing of the naked mole-rat ( Heterocephalus glaber), we designed a PCR primer set to amplify part of the Y-linked DBY gene. When this primer set was applied to the samples of known sex with the 16S rRNA gene (16S rDNA) primers as control, PCR products were successfully obtained as two DNA bands in males, a male-specific 163 bp DBY band and a 446 bp band of 16S rDNA shared with females, whereas females showed only the common band. This result shows that this multiplex PCR assay is useful for sex identification of H. glaber. [ABSTRACT FROM AUTHOR]

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192-196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245-1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211-1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4). [ABSTRACT FROM AUTHOR]