Morphometric dataset of Varanus salvator for non-invasive sex identification using machine learning.

Reliable sex identification in Varanus salvator traditionally relied on invasive methods like genetic analysis or dissection, as less invasive techniques such as hemipenes inversion are unreliable. Given the ecological importance of this species and skewed sex ratios in disturbed habitats, a dataset that allows ecologists or zoologists to study the sex determination of the lizard is crucial. We present a new dataset containing morphometric measurements of V. salvator individuals from the skin trade, with sex confirmed by dissection post- measurement. The dataset consists of a mixture of primary and secondary data such as weight, skull size, tail length, condition etc. and can be used in modelling studies for ecological and conservation research to monitor the sex ratio of this species. Validity was demonstrated by training and testing six machine learning models. This dataset has the potential to streamline sex determination, offering a non-invasive alternative to complement existing methods in V. salvator research, mitigating the need for invasive procedures.

A review of the recent advances for the in ovo sexing of chicken embryos using optical sensing techniques.

The culling of day-old male chicks has caused ethical and economic concerns. Traditional approaches for detecting the in ovo sex of chicken embryos involve opening the eggshell and inner membrane, which are destructive, time-consuming, and inefficient. Therefore, noncontact optical sensing techniques have been examined for the in ovo sexing of chicken embryos. Compared with traditional methods, optical sensing can increase determination throughput and frequency for the rapid sexing of chicken embryos. This paper presented a comprehensive review of the different optical sensing techniques used for the in ovo sexing of chicken embryos, including visible and near-infrared (Vis-NIR) spectroscopy, hyperspectral imaging, Raman spectroscopy, fluorescence spectroscopy, and machine vision, discussing their advantages and disadvantages. In addition, the latest research regarding different detection algorithms and models for the in ovo sexing of chicken embryos was summarized. Therefore, this paper provides updated information regarding the optical sensing techniques that can be used in the poultry industry and related research.

Differential proteomics between unhatched male and female egg yolks reveal the molecular mechanisms of sex-allocation and sex-determination in chicken.

There is a huge demand to identify the sex of unhatched fertilized eggs for laying industry and to understand the differences between male and female eggs as early as possible. Then the molecular mechanisms of sex determination and sex allocation in chicken were revealed. Therefore, TMT proteomic was applied to characterize the variation of molecular matrix between unhatched male and female egg yolks. A total of 411 proteins were identified and 35 differentially expressed proteins (DEPs), including 375332005, 015809562, 763550308 (upregulated, UPs) and 1337178851, 89000557, 89000581 (downregulated, DPs), etc. were confirmed between them. Gene ontology analyses showed that DEPs were mainly involved in response to stimulus, distributed in the extracellular region and participated in binding; KEGG analyses showed that few DPs were participated in cell growth and death, transport and catabolism, signaling molecules, interaction and were enriched in ubiquitin mediated proteolysis, endocytosis, ferroptosis, etc. metabolic pathways. Moreover, most of the DEPs and related metabolic pathways were associated with sex hormones. More importantly, this study supports maternal sex-allocation theory and extends our understanding of the molecular mechanism of sex determination and differentiation in avian. Which also provides a powerful evidence for ovo sexing of unhatched fertilized domestic chicken eggs by nondestructive approach and will be of great significance to eggs processing and production.

Sex identification of the ornamental amazon fish Astronotus ocellatus by videoceloscopy and gonadal biopsy.

This study was conducted to evaluate and validate the efficacy and safety of videoceloscopy and gonadal biopsy as sexing methods for the A. ocellatus. A total of 31 adult individuals were used. Florfenicol (50 mg/kg) and morphine (5 mg/kg) were administered intramuscularly during the pre-surgical period. Animals were maintained in a supine position preceding a ventral midline incision and endoscope optics were then utilized for gonad visualization and sex identification. A gonadal fragment was collected using laparoscopic forceps and conditioned in 10 % formalin. To suture the cavity, polyamide yarn was used in a simple and continuous pattern. At 15 days subsequent to surgery, healing was evaluated, and the stitches were removed. Videoceloscopy accuracy and gonadal biopsy effectiveness were 97 % and 83 %, respectively. Total time devoted in the videoceloscopy, gonadal biopsy and surgery was longer for animals identified as males compared to females The survival rate was 100 %. There were differences regarding food consumption at 24 and 36 h post-surgery when compared to control specimens (pre-surgical) Regarding position in the water column, differences were observed at 24 and 72 h after surgery when compared individually to the control specimens. There were differences for interaction behavior at 24, 36 and 60 h, and regarding search for hiding places at 12 and 24 h after surgery in relation to the control specimens. The applied videoceloscopy and gonadal biopsy surgical techniques are, therefore, effective and safe for A. ocellatus sexing procedures.

Sexing chickens (Gallus gallus domesticus) with high-resolution melting analysis using feather crude DNA.

Identification of sex in broiler chickens allows researchers to reduce the level of variation in an experiment caused by the sex effect. Broiler breeds commonly used in research are no longer feather sexable because of the change in their genetics. Other alternate sexing methods are costly and difficult to apply on a large scale. Therefore, a sexing method is required that is both cost effective and highly sensitive as well as having the ability to offer high throughput genotyping. In this study, high-resolution melting (HRM) analysis was used to detect DNA variations present in the gene chromodomain helicase DNA binding 1 protein (CHD1) on the Z and W chromosomes (CHD1Z and CHD1W, respectively) of chickens. In addition, a simplified DNA extraction protocol, which made use of the basal part of chicken feathers, was developed to speed up the sexing procedure. Three pairs of primers, that is, CHD1UNEHRM1F/R, CHD1UNEHRM2F/R, and CHD1UNEHRM3F/R, flanking the polymorphic regions between CHD1Z and CHD1W were used to differentiate male and female chickens via distinct melting curves, typical of homozygous or heterozygous genotypes. The assay was validated by the HRM-sexing of 1,318 broiler chicks and verified by examining the sex of the birds after dissection. This method allows for the sexing of birds within a couple of days, which makes it applicable for use on a large scale such as in nutritional experiments.

Efficacy of two primer sets used in the sex identification of rufous-winged buzzard (Butastur liventer).

BACKGROUND: The rufous-winged buzzard (Butastur liventer) belongs to the order Accipitriformes, which is monomorphic, resulting in the difficulty to identify the gender. However, sex determination is important for predicting the sex ratio of this buzzard in nature in order to avoid its extinction. AIM: We aimed to develop a primer set that is able to sex the rufous-winged buzzard through polymerase chain reaction (PCR) amplification and compare the efficacy of the two sets of primers by using PCR technique. METHODS: In the following, sensitivity refers to the smallest DNA concentration that allowed us to accurately sex a bird and specificity refers to the ability to clearly distinguish the sex based on the visual appearance of the bands. Blood samples were collected from captive buzzards. The DNA was extracted from them and was diluted to 50, 25, 10, 5, 2.5, 1.67, and 1 ng/µl. Two sets of primers, including P2/NP/MP and 2550F/2718R, were used to amplify the chromo-helicase DNA binding (CHD) gene of known gender buzzards using the PCR process to determine gender and to compare their sensitivity. To measure specificity, both primers were used to amplify CHD gene fragment of other unknown gender birds. RESULTS: The lowest concentration of the DNA template where P2/NP/MP could amplify DNA fragments was 1 ng/µl, and this set of primers could identify the gender of all birds correctly, giving 100% specificity. On the other hand, the 2550F/2718R could amplify the DNA fragments from 5 ng/µl, and it had only 78% specificity. CONCLUSION: The P2/NP/MP primer set was able to correctly identify the gender of rufous-winged buzzard through PCR amplification with high specificity and sensitivity.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

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

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

[Determination of sex steroids in shed skin of the beaded lizard Heloderma suspectum ("Gila Monster")]. / Bestimmung von Sexualsteroiden in abgestoßener Haut der Gila-Krustenechse (Heloderma suspectum).

OBJECTIVE: Measurement of steroid hormones in skin appendages such as mammalian hair or claws and in avian feathers represents a recognized non-invasive method for the determination of these parameters. The aim of this pilot study in the Gila Monster was to investigate whether the measurement of sex steroids in shed skin may be employed for the monitoring of endocrine gonadal function or sex determination in reptiles. MATERIAL AND METHODS: Shed skins were available from 11 female and 7 male adult and sexually mature animals. Large pieces of skin were initially cut into smaller pieces with scissors. The resultant dermal fragments were finely ground under liquid nitrogen and finally extracted with organic solvents. The following parameters were determined radioimmunologically in the dried and re-dissolved extracts: progesterone (P4), estradiol-17ß (E2), testosterone (T), free total estrogens (fGÖ) and free plus conjugated total estrogens (fkGÖ). RESULTS: For P4 (p = 0.0052) and E2 (p = 0.0079) significant sex differences were found with higher concentrations in females compared to males. Unexpectedly, the measured values for T were also significantly higher in females (p = 0.0232) than in males, with the concentrations overall only slightly above the detection limit. Compared to fGÖ, the concentrations of fkGÖ were only slightly higher, with no significant differences between both sexes. CONCLUSION AND CLINICAL RELEVANCE: In this pilot study, the methods employed did not allow for reliable sex determination in individual animals, neither alone nor in combination, due to an overlap between the sexes. In principle, however, the measurement of sex steroids in shed skins could represent a useful method for non-invasive sex determination or endocrine gonadal function assessment in certain reptile species.

Comparison of Cell-Free Fetal DNA Plasma Content Used to Sex Determination Between Three Trimesters of Pregnancy in Torkaman Pregnant Mare.

This investigation aimed to compare the cell-free fetal DNA (cffDNA) plasma present in three trimesters of pregnancy in Torkaman pregnant mare. Peripheral blood samples of 32 pregnant mares in three trimesters of pregnancy were collected in tubes containing ethylenediaminetetraacetic acid at three time points. Circulating cffDNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, a conventional polymerase chain reaction was performed for the sex-determining region Y (SRY) gene present in the Y chromosome. Of the total 32 Torkaman pregnant mares, 24 were carrying male fetuses and eight were carrying female fetuses. In total, the accuracy of the test was 48.75%, 68.75%, and 75% in the first, second, and third trimesters of pregnancy, respectively. The sensitivities were 25%, 58.32%, and 66.66%, respectively, whereas their specificities were 100% in all trimesters. In conclusion, the SRY gene can permit the detection of equine fetal sex with good accuracy through cffDNA analysis in maternal plasma just in the third trimester of pregnancy, although specificity in all duration of pregnancy was 100%.

Quantification of immunoreactive testosterone and estradiol-17ß metabolites to identify the sex of Neotropical otters (Lontra longicaudis annectens) in the field.

Before there was use of ultrasonographic imaging, determination of the ratio of estrogens to androgens in the same individual was a technique used for differentiating the sex of monomorphic animals in captivity, with larger estrogen concentrations in the females. Due to species-specific differences in both concentration and changes throughout the year of these hormones, corroboration of the method is needed in each case. In this study, there was use of a chemo-immuno assay to quantify sex steroids in fecal samples collected from seven (five females and two males) Neotropical otters, Lontra longicaudis. The reproductive season for this species was determined to be between October and March, with increased estradiol in the females and relatively greater concentrations of testosterone in the males as compared with other seasons of the year. Results from utilization of a k-means analysis procedure indicated that the use of steroid ratios in fecal samples to differentiate otter sex is an effective technique when there are evaluations during the breeding season. The estrogen to androgen ratios during this period, however, are the inverse of what was expected, with there being larger testosterone concentrations in the female otters. The ratio of estrogens to androgens in feces of captive otters can be effectively used to determine the sex of otters in the field. We propose this method is reliable for sex determination in wild otter populations during the reproductive season.

Methods for identifying and interpreting sex-linked SNP markers and carrying out sex assignment: application to thornback ray (Raja clavata).

Sex-determining modes remain unknown in numerous species, notably in fishes, in which a variety of modalities have been reported. Additionally, noninvasive individual sexing is problematic for species without external sex attributes or for early life stages, requiring cytogenetic or molecular analyses when sex chromosomes or sex-linked markers have been characterized. Genomics now provide a means to achieve this. Here, we review common sex-determination systems and corresponding statistical methods for identifying sex-linked genetic markers and their use for sex assignment, focusing on single nucleotide polymorphism (SNP) markers derived from reduced representation sequencing methods. We demonstrate the dependence of expected sex assignment error on the number of sex-linked SNPs and minor allele frequency. The application of three methods was made here: (a) identification of heterozygote excess in one sex, (b) FST outlier analysis between the two sexes and (c) neuronal net modelling. These methods were applied to a large SNP data set (4604 SNPs) for 1680 thornback rays (Raja clavata). Using method (a), nineteen putative sex-linked SNPs were identified. Comparison with the reference genome of a related species (Amblyraja radiata) indicated that all 19 SNPs are probably located on the same chromosome. These results suggest that thornback ray has a XX/XY sex-determination system. Method (b) identified eight SNPs probably located on different chromosomes. Method (a) led to the lowest sex assignment error among the three methods (4.2% error for females and 3.7% for males).

[Gender determination in equine fetuses in early pregnancy using two- and three-dimensional ultrasound]. / Geschlechtsbestimmung bei Pferdefeten mittels zwei- und dreidimensionaler Sonografie in der Frühgravidität.

AIM: The aim of this study was to compare transrectal two-dimensional (2D) and three-dimensional (3D) ultrasound examination with regards to required time and accuracy of fetal sex determination in early pregnant mares. MATERIALS AND METHODS: For this purpose 47 mares were examined transrectally once between days 58 and 115 of gestation. Initially, the fetal sex was determined by identifying the location of the genital tubercle (GT) or external genitalia using 2D-ultrasound. Subsequently, the ultrasound machine was switched to 3D-mode to obtain images for later computer-based evaluation. RESULTS: The gestational period between days 58 and 79 of pregnancy was the most appropriate time for sex determination with 77 % (2D and first 3D evaluation) correct diagnoses. The accuracy of the sex determination could be increased by about 16 % by a second evaluation of the 3D-images in a minimum time interval of 2 weeks. For each mare the additional time needed to perform the 3D-examination and to assess the 3D-images was approximately 6-7 minutes. CONCLUSION AND CLINICAL RELEVANCE: This study demonstrates that the accuracy of transrectal fetal gender determination is higher by using 3D-mode compared to the 2D-ultrasound. For experienced examiners however, 3D ultrasound technology does not offer any decisive advantages and is also more time-consuming.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

Technical note: Droplet digital PCR precisely and accurately quantifies sex skew in bovine semen.

Quantifying the relative population of sperm cells bearing the X or Y chromosome in a sexed-semen sample has historically been limited to methods that are either low throughput and sensitive to user subjectivity (e.g., fluorescence in situ hybridization), conterminous (using the same technology to generate and confirm the sex skew), or relatively insensitive (including quantitative PCR with a change detection threshold of 2×). Customers pay a premium for sexed semen and should have access to reliable sex skew data, generated by an accurate, precise test that is orthogonal to the method used to generate sexed semen. Droplet digital PCR (ddPCR) has the capacity to provide an accurate and precise sex skew quantitation by subdividing a pool of template DNA into nanoliter-scale droplets containing either 1 or 0 copies of template DNA. Then PCR amplification is conducted in the droplets, and the number of copies of the amplicon of interest can be counted as the number of fluorescence-positive droplets based on classic quantitative PCR fluorescent reporters. We have optimized and validated a multiplexed ddPCR assay that uses this copy counting method to quantify the sex skew (ratio of X or Y chromosomes) in frozen-thawed bovine sexed semen. The assay interrogates at least 1,000 cells per sample well, quantifying X and Y chromosome copy numbers along with an autosomal gene, GAPDH, used as an internal assay control to confirm total cells counted. The ddPCR sex skew assay achieved a 0.5-percentage-point variance for %X or %Y with a broad linear detection range, from 10 to 95% X, and provided reproducible skew values across a range of 9 to 27 ng of genomic DNA input. This approach overcomes some limitations of other sex skew assays by quantifying absolute X and Y chromosome copy numbers, thus providing a rigorous, independent assessment of sex-skewed semen.

Optimization of timing of insemination of dairy heifers inseminated with sex-sorted semen.

We hypothesized that delaying by approximately 12 h the artificial insemination (AI) of heifers with sex-sorted semen increases pregnancy per AI (P/AI). Holstein heifers (n = 1,207) were fitted with a collar containing an automated estrus-detection device (HR-LDn tags, SCR Engineers Ltd., Netanya, Israel) at 10.7 ± 0.02 mo of age. Once they reached 330 kg, heifers were enrolled in an ovulation synchronization protocol (5-d Cosynch + controlled internal drug release; Zoetis, Parsippany-Troy Hills, NJ). Study personnel recorded the heifers that were in estrus according to the DataFlow II software (SCR Engineers Ltd., Netanya, Israel) twice daily at 0500 and 1500 h from the day of the first PGF2α (Estrumate; Merck Animal Health, Madison, NJ) injection to 72 h later. Heifers enrolled in the conventional (CONV) and early sex-sorted (SSEarly) semen treatments detected in estrus at 0500 and 1500 h were AI at 0600 and 1600 h of the same day, respectively. Heifers enrolled in the late sex-sorted (SSLate) semen treatment detected in estrus at 0500 and 1500 h were AI at 1600 h of the same day and 0600 h of the following day, respectively. All heifers not detected in estrus by 72 h after the first PGF2α injection received a GnRH (Fertagyl; Merck Animal Health, Madison, NJ) injection at 0500 h. Heifers in the CONV and SSEarly treatments were AI at fixed time at 0600 h, whereas heifers in the SSLate treatment were AI at fixed time at 1600 h. Among heifers detected in estrus, the ranges of the interval from the onset of estrus to AI were 3.6 to 28.5 h, 0.0 to 25.5 h, and 9.4 to 36.8 h for the CONV, SSEarly, and SSLate treatments, respectively. Among heifers AI at fixed time, the ranges of the interval from the GnRH injection to AI were 0 h for heifers in the CONV and SSEarly treatments and 8.5 to 11.7 h for heifers in the SSLate treatment. The P/AI at 62 ± 1 d after the first AI was greater for CONV (63.1 ± 2.6%) compared with SSEarly (43.3 ± 2.6%) and SSLate (44.8 ± 2.7%). A greater percentage of heifers enrolled in the SSEarly (65.8 ± 2.5%) and SSLate (70.0 ± 2.5%) treatments produced a live female calf compared with the CONV treatment (40.5 ± 2.7%). When the values of 1-d-old female and male calves were USD $0 and the cost of replacement heifers was $750, the cost of raising heifers from enrollment to calving was lesser for the CONV treatment than the SSEarly treatment, but SSLate treatment did not differ from CONV and SSEarly treatments. When the values of a 1-d-old female calf ≥$130 and 1-d-old male calf ≥$30 and the cost of replacement ≥$1,000, no differences were observed among treatments in the cost from enrollment to calving. We conclude from this experiment that the P/AI with sex-sorted semen is not improved when insemination is delayed by approximately 12 h.

Biomolecular analyses reveal the age, sex and species identity of a near-intact Pleistocene bird carcass.

Ancient remains found in permafrost represent a rare opportunity to study past ecosystems. Here, we present an exceptionally well-preserved ancient bird carcass found in the Siberian permafrost, along with a radiocarbon date and a reconstruction of its complete mitochondrial genome. The carcass was radiocarbon dated to approximately 44-49 ka BP, and was genetically identified as a female horned lark. This is a species that usually inhabits open habitat, such as the steppe environment that existed in Siberia at the time. This near-intact carcass highlights the potential of permafrost remains for evolutionary studies that combine both morphology and ancient nucleic acids.

Endoscopy gender determination and reproductive hormone profiles of Painted Terrapins (Batagur borneoensis) subjected to ex situ incubation.

Chelonian exhibit temperature dependent sex determination, and ex situ incubation of eggs in conservation hatcheries may render a gender bias. The gender of juvenile Painted terrapins (Batagur borneoensis) produced at a conservation hatchery in Malaysia was determined by endoscopy of the gonads. Circulating reproductive hormones (testosterone, progesterone and estradiol) were profiled for 31 juveniles and nine captive-reared non-breeding adult terrapins. Endoscopy revealed a gender bias of 96.8% (30/31) females. Testosterone levels in the juvenile females (2.49 ± 1.29) were significantly lower than that of the adult females (12.20 ± 4.29), and lower than values in the juvenile male (9.36) and adult males (27.60, 35.62). The progesterone levels in the juvenile females (107.12 ± 68.68) were significantly higher than that of the adult females (51.13 ± 24.67), but lower than values in the juvenile male (33.27) and adult males (3.43, 8.51). Estrogen levels were significantly lower in the juvenile females (1.57 ± 1.35) compared to the adult females (77.46 ± 53.45). Negative correlations were observed between levels of progesterone and testosterone, and progesterone and estrogen. A positive correlation was noted between estrogen and testosterone. The present study constitutes the first attempt to determine the gender and reproductive hormone profiles of juvenile Painted terrapins produced by ex situ incubation, and captive non-breeding adults. Endoscopy of the gonads is a useful techniques for gender determination among juvenile turtles, while the use of testosterone as a gender biomarker warrants further investigation.

Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants.

The genetic sexing of animals having long gestation periods offers significant benefits in regard to breeding management among their populations living in captivity. In our study, a new increased-sensitivity PCR method for fetal sexing was developed and tested successfully on elephants, from only a small volume of maternal plasma. Suitable sensitivity was obtained by using short, reduced amplicon lengths with fluorescent labelling for capillary electrophoresis detection. The fundamental principle for this technique was based on the detection of two Y-specific markers (AmelY and SRY), the presence of which indicates the mother is carrying a male fetus and the absence of these markers designates a female fetus. As a reaction control, the X-chromosomal marker (PlpX) was used. To the best of our knowledge, this is the first report on this topic, confirming the presence of fetal cell-free DNA from the plasma of a pregnant captive elephant, and demonstrating a new opportunity for non-invasive assessment in fetal sex determination.