Utilization of a Gender-Based Sorting Machine for Crustacean Selection in Bioconcentration Studies with the Freshwater Amphipod Hyalella azteca.

Bioconcentration factors (BCFs) are determined by fish flow-through tests performed according to Organisation for Economic Co-operation and Development test guideline 305. These are time-consuming and expensive and use a large number of animals. An alternative test design using the freshwater amphipod Hyalella azteca for bioconcentration studies has been recently developed and demonstrated a high potential. For bioconcentration studies using H. azteca, male amphipods are preferred compared with female organisms. Manual sexing of male adult amphipods is, however, time-consuming and requires care and skill. A new fully automatic sorting and dispensing machine for H. azteca based on image analysis has recently been developed by the company Life Science Methods. Nevertheless, an anesthesia step is necessary prior to the automatic selection. In the present study, we show that a single-pulse of 90 min of tricaine at the concentration of 1 g/L can be used and is recommended to select H. azteca males manually or automatically using the sorting machine. In the second part, we demonstrate that the machine has the ability to select, sort, and disperse the males of a culture batch of H. azteca as efficiently as manual procedures. In the last part of the study, BCFs of two organic substances were evaluated using the H. azteca bioconcentration test (HYBIT) protocol, with an anesthetizing step and robotic selection compared with manual selection without an anesthetizing step. The different BCF values obtained were in accordance with those indicated in the literature and showed that an anesthetizing step had no effect on the BCF values. Therefore, these data validated the interest in this sorting machine for selecting males to perform bioconcentrations studies with H. azteca. Environ Toxicol Chem 2023;42:1075-1084. © 2023 SETAC.

A novel method for sexing day-old chicks using endoscope system.

Sexing day-old chicks is important for layer and broiler production. A novel method for sexing day-old chicks was developed using an endoscope system. The probe of the endoscope was inserted from the cloaca into the intestine of a chick, and the presence of testes or ovary was observed through the wall of the intestine. The picture image was displayed on the monitor. Sexing was performed in White Leghorn (WL) and Rhode Island Red (RIR) chicks using this new system. The accuracy of sexing was 91.1% in WL chicks and 88.3% in RIR chicks, confirmed by observing gonads after laparotomy or appearances at 80 d of age. Accuracy of sexing male chicks (95.0%) was higher than that of female chicks (86.0%). The overall accuracy of sexing was 90.2% in the present study, and the accuracy would be improved by continuous training in the handling of the endoscope. The endoscope system devised in this study requires no specific skills and anyone can perform sexing of chicks after short-term training.

Evaluation of dried blood spot collection paper blotters for avian sexing by direct PCR.

Abstract 1. Avian sexing by polymerase chain reaction (PCR) plays an important role in sexual identification of avian species with similar phenotypes. Dried blood spots (DBSs) on paper blotters can help reduce the cost and problem of sample transportation and processing. 2. In the first experiment, several kinds of papers were evaluated for collecting DBS for chicken sexing by direct PCR with different processing methods. The most practical method with cost optimality was the utilisation of Whatman grade 1 filter paper with the combination of methanol fixation and boiling. 3. A second experiment was performed to determine whether cross-contamination could occur among samples cut with the same scissors. No-clean scissors were compared with ones cleaned with bleach-ethanol combination or 0.3N HCl. The PCR results showed that all three methods provided correct amplicon sizes without any false-positives regardless of the utilisation of cleaning intervention. 4. In conclusion, a technique that is suitable for DBS collection for avian sexing by direct PCR with cost efficacy was developed, and it was also shown that the utilisation of the same pair of scissors for several DBS samples did not affect the PCR results.

Optical penetration-based silkworm pupa gender sensor structure.

This paper proposes and experimentally demonstrates for what is believed to be the first time a highly sought-after optical structure for highly-accurate identification of the silkworm pupa gender. The key idea is to exploit a long wavelength optical beam in the red or near infrared spectrum that can effectively and safely penetrate the body of a silkworm pupa. Later on, simple image processing operations via image thresholding, blob filtering, and image inversion processes are applied in order to eliminate the unwanted image noises and at the same time highlight the gender gland. Experimental proof of concept using three 636 nm wavelength light emitting diodes, a two-dimensional web camera, an 8 bit microcontroller board, and a notebook computer shows a very high 95.6% total accuracy in identifying the gender of 45 silkworm pupae with a measured fast identification time of 96.6 ms. Other key features include low cost, low component counts, and ease of implementation and control.

A primer set to determine sex in the small Indian mongoose, Herpestes auropunctatus.

To enable the accurate sexing of individuals of introduced populations of the small Indian mongoose, Herpestes auropunctatus, we designed a primer set for the amplification of the sex-specific fragments EIF2S3Y and EIF2S3X. Using this primer set, the expected amplification products were obtained for all samples of genomic DNA tested: males yielded two bands and females a single band. Sequencing of each PCR product confirmed that the 769-bp fragment amplified from DNA samples of both sexes was derived from EIF2S3X, whereas the 546-bp fragment amplified only from male DNA samples was derived from EIF2S3Y. The results indicated that this primer set is useful for sex identification in this species.

A simple and rapid PCR-based method for ostrich sexing using micro amounts of DNA.

The aim of the present study is to identify ostrich sex by using polymerase chain reaction (PCR) on micro amounts of DNA from blood, bloodstain and feathers. Sixteen male and 18 female ostriches were used as test individuals. Genomic DNA as a template was extracted by the Chelex method. Ostrsex-P1 and P2 primers were designed to perform PCR amplification on the template. PCR products were checked using agarose gel electrophoresis with ethidium bromide staining and ostrich sex was determined directly by the bands shown on the gel. The results demonstrate that ostrich sex can be determined by the extraction of DNA from as little as 0.0125 microl blood using Chelex, whereby the use of large amounts of organic solvents such as phenol and chloroform are unnecessary. In addition, it is possible to identify ostrich sex using micro amounts of DNA extracted from bloodstains and/or feathers. The use of feathers particularly avoids unwanted sampling problems such as the difficulty of collecting ostrich blood, the stress to the ostrich caused by bleeding, and the demand for a lot of manpower for ostrich restraint.

Misuse of prenatal diagnostic technology for sex-selected abortions and its consequences in India.

During 1800, the British Government found that there were no daughters in a village in the Eastern Uttar Pradesh region of India. According to the 2001 Census, there were less than 93 women for every 100 men in the Indian population. The prevailing concept that the birth of a female child can signal the beginning of financial ruin and extreme hardship for a poor Indian family is understandable. What is surprising is that even high-income families do not want a female child. The Government of India in its 10th Plan recognized the rights of the female child to equal opportunity, to be free from hunger, illiteracy, ignorance and exploitation. In the National Policy for the Empowerment of Women 2001, a policy framework was laid down for the elimination of discrimination against, and violation of, the rights of the female child. However, the situation continues to worsen, and studies have revealed that sex-selected abortions are practised among all communities despite enactment of laws prohibiting prenatal sex determination. In this paper, we examine the functioning and consequences of the misuse of this technology.

Sex separation of tsetse fly pupae using near-infrared spectroscopy.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis.

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.

Current status of sexing mammalian spermatozoa.

Thousands of offspring have now been produced via artificial insemination with spermatozoa sexed by flow cytometry and cell sorting. We are unaware of any other practical approach to sexing spermatozoa that maintains fertility. Accuracy of sexing usually is 85-95% in most species, but somewhat lower with human spermatozoa. Spermatozoa are sexed in series, one at a time, at routine rates of about 3000 live spermatozoa of each sex per second for most species, and nearly twice that rate under optimal conditions for some species. Owing to various constraints and statistical considerations, there appears to be an upper theoretical limit to sexing spermatozoa of about 10,000 live spermatozoa of each sex per second with current methodology. About a quarter of the spermatozoa processed are sexed; the rest are discarded in the process or lost due to logistical constraints. Spermatozoa undergo some damage during sorting, although much less in terms of viability than with routine cryopreservation; fertility is lower with sexed than control spermatozoa. Offspring from sexed spermatozoa appear to have no more abnormalities than do controls, and both groups grow and thrive similarly. Despite high costs and complex procedures, sexing spermatozoa, usually followed by cryopreservation, is being used commercially for cattle and horse production in several countries, and is used to produce girls to avoid X-chromosome-linked genetic diseases.

The "fetoscope"--a new clinical tool for prenatal genetic diagnosis.

Our experience in 65 patients using a "fetoscope" and local anesthesia prior to saline abortion is described. Visualization of the intrauterine contents improved with experience, was optimum at 16 to 18 weeks' gestation, and was superior to the quality of photographic records. Four fetal skin biopsies and 6 amnion biopsies were done. Six blood samples were taken from the fetal surface of the placenta. The limitations of this instrument are described. No significant complications have occurred. Current and probable future indications for fetoscopy are reviewed. Until both the safety of the procedure is proved and problems of visualization due to limitation of visual field are solved, extreme caution is urged in the employment of this instrument in genetic high-risk pregnancy.