Sex Sorting Research Articles

Ultrasonography for non-invasive sex identification and reproductive assessment in Nile tilapia (Oreochromis niloticus).

Sex identification and the selection of monosex male tilapia are crucial for tilapia aquaculture. In this study, we evaluated the application of ultrasonography as an alternative procedure for sex identification and reproductive assessment in Nile tilapia (Oreochromis niloticus). Ultrasonography was performed on 23 fish weighing 232-1,281 g to capture longitudinal and transverse images of the ovaries and testes. Female tilapia were identified by the presence of numerous small egg granules and gray or light gray ovarian tissue and male tilapia by the homogeneous echogenicity and uniform gray tubular appearance of the testes. These distinct echogenic patterns allowed for accurate differentiation between the female and male fish. Ultrasonography achieved a 95% accuracy in sex identification, with minimal stress to the fish during the procedure, compared to manual sex sorting, which demonstrated an accuracy of 87%. Furthermore, the method facilitated the assessment of the reproductive status of the fish, including the maturation stages and fecundity potential. The use of ultrasonography offers significant advantages over traditional methods, such as animal welfare enhancements and improved breeding program efficiency. Our findings support the integration of ultrasonography into aquaculture practices and will promote sustainable and humane fish farming while optimizing reproductive management and productivity.

Gene Drive and Symbiont Technologies for Control of Mosquito-Borne Diseases.

Mosquito-borne diseases, such as dengue and malaria, pose a significant burden to global health. Current control strategies with insecticides are only moderately effective. Scalable solutions are needed to reduce the transmission risk of these diseases. Symbionts and genome engineering-based mosquito control strategies have been proposed to address these problems. Bacterial, fungal, and viral symbionts affect mosquito reproduction, reduce mosquito lifespan, and block pathogen transmission. Field tests of endosymbiont Wolbachia-based methods have yielded promising results, but there are hurdles to overcome due to the large-scale rearing and accurate sex sorting required for Wolbachia-based suppression approaches and the ecological impediments to Wolbachia invasion in replacement approaches. Genome engineering-based methods, in which mosquitoes are genetically altered for the modification or suppression of wild populations, offer an additional approach for control of mosquito-borne diseases. In particular, the use of gene drive alleles that bias inheritance in their favor is a potentially powerful approach. Several drives are frequency dependent, potentially giving them broadly similar population dynamics to Wolbachia. However, public acceptance and the behavior of released drives in natural mosquito populations remain challenges. We summarize the latest developments and discuss the knowledge gaps in both symbiont- and gene drive-based methods.

Impact of sperm sex sorting on sperm quality and in vitro embryo production in bovine

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen – CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.

Gamma-Irradiated Female Aedes aegypti Mosquitoes Exhibit Greater Susceptibility to Mayaro Virus.

Mayaro virus (MAYV) is an alphavirus endemic in many parts of Central and South America transmitted to humans by Aedes aegypti. Currently, there is no vaccine or treatment of Mayaro infection, and therefore it is essential to control transmission by reducing populations of Ae. aegypti. Unfortunately, Ae. aegypti are extremely difficult to control with traditional integrated vector management (IVM) because of factors such as growing resistance to a dwindling list of registered insecticides and cryptic immature and adult habitats. The sterile insect technique (SIT) by irradiation is gaining traction as a novel supplemental tool to IVM. The SIT is being used operationally to release large numbers of sterilized colony-reared male mosquitoes in an intervention area to overwhelm females in the natural population, eventually causing population decline because of high frequencies of unfertilized eggs. However, little is known about the effect of irradiation on vector competence for mosquito-borne viruses such as MAYV in females that may be accidentally reared, irradiated, and released alongside males. In this investigation, we exposed female Ae. aegypti pupae to radiation and evaluated vector competence after inoculation with MAYV. Infection and dissemination rates of irradiated (10 and 40 Gy) Ae. aegypti were higher than those of non-irradiated cohorts at 7 and 14 days after infection. Although these results indicate a need to maintain effective sex sorting prior to irradiation and release of Ae. aegypti, our results are consistent with several previous observations that vectorial capacity and vector competence are likely lower in irradiated than in nonirradiated females.

Establishing a Male-Positive Genetic Sexing Strain in the Asian Malaria Vector Anopheles stephensi.

Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit human pathogens. This reduces the risk of spreading pathogens while enabling effective population control. Robust sex sorting methods to enable early larval sorting in mosquitoes need to be developed to allow for scalable sex sorting for genetic biocontrol interventions. This study applies the SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) system, previously developed for Aedes aegypti, to the Asian malaria vector Anopheles stephensi. We hypothesized that the intron from the doublesex gene in Anopheles gambiae would function in An. stephensi due to evolutionary conservation. Our results confirm that the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species. This system enables reliable positive male selection from first instar larval to pupal stages. RT-PCR analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. The SEPARATOR system's independence from sex-chromosome linkage confers resistance to meiotic recombination and chromosomal rearrangements. This approach may facilitate the mass release of males, and the cross-species portability of SEPARATOR establishes it as a valuable tool for genetic biocontrol interventions across various pest species.

Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male–female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

Effect of 2 sex-sorting time schedules on SIT facility management.

Improvements are needed in mosquito mass-rearing to effectively implement the sterile insect technique (SIT). However, managing this technique is challenging and resource intensive. SIT relies on mass rearing, sterilization, and release of adult males to reduce field populations. Maintaining an acceptable level of female presence, who can transmit viruses through biting, is crucial. Females are also essential for facility sustainability. Sex sorting plays a vital role in the production process, and our current mechanical sorting approach aims to obtain a high number of adult males with minimal female contamination within 24 h of pupation. Utilizing protandry helps control female contamination. While the 24-h sorting period achieves desired contamination levels, it may not yield enough females to sustain breeding lines, leading to increased labor costs that impact project sustainability. By delaying the sorting procedure to 48 h, we obtained sufficient females to sustain breeding lines, achieving a balance between male production and female contamination using the automatic version of the Fay-Morlan device as the sorting tool.

The effect of sex-sorting on stallion semen quality

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.

Radiation dose-fractionation in adult Aedes aegypti mosquitoes

Balancing process efficiency and adult sterile male biological quality is one of the challenges in the success of the sterile insect technique (SIT) against insect pest populations. For the SIT against mosquitoes, many stress factors need to be taken into consideration when producing sterile males that require high biological quality to remain competitive once released in the field. Pressures of mass rearing, sex sorting, irradiation treatments, packing, transport and release including handling procedures for each step, add to the overall stress budget of the sterile male post-release. Optimizing the irradiation step to achieve maximum sterility while keeping off-target somatic damage to a minimum can significantly improve male mating competitiveness. It is therefore worth examining various protocols that have been found to be effective in other insect species, such as dose fractionation. A fully sterilizing dose of 70Gy was administered to Aedes aegypti males as one acute dose or fractionated into either two equal doses of 35Gy, or one low dose of 10Gy followed by a second dose of 60Gy. The two doses were separated by either 1- or 2-day intervals. Longevity, flight ability, and mating competitiveness tests were performed to identify beneficial effects of the various treatments. Positive effects of fractionating dose were seen in terms of male longevity and mating competitiveness. Although applying split doses generally improved male quality parameters, the benefits may not outweigh the added labor in SIT programmes for the management of mosquito vectors.

Near-infrared imaging for automated tsetse pupae sex sorting in support of the sterile insect technique.

Tsetse flies are the cyclical vectors of African trypanosomes and one of several methods to manage this vector is the sterile insect technique (SIT). The ability to determine the sex of tsetse pupae with the objective to separate the sexes before adult emergence has been a major goal for decades for tsetse management programmes with an SIT component. Tsetse females develop faster and pharate females inside the pupae melanise 1-2 days before males. This earlier melanisation can be detected by infrared cameras through the pupal shell, and the newly developed Near InfraRed Pupae Sex Sorter (NIRPSS) takes advantage of this. The melanisation process is not homogeneous for all fly organs and the pupa needs to be examined ventrally, dorsally and laterally to ensure accurate classification by an image analysis algorithm. When the pupae are maturing at a constant temperature of 24°C and sorted at the appropriate age, 24 days post-larviposition for Glossina palpalis gambiensis, the sorting machine can efficiently separate the sexes. The recovered male pupae can then be sterilised for field releases of males, while the rest of the pupae can be used to maintain the laboratory colony. The sorting process with the new NIRPSS had no negative impact on adult emergence and flight ability. A mean male recovery of 62.82±3.61% was enough to provide sterile males to an operational SIT programme, while mean contamination with females (4.69±3.02%) was low enough to have no impact on the maintenance of a laboratory colony.

Developing the radiation-based sterile insect technique (SIT) for controlling Aedes aegypti: identification of a sterilizing dose.

The sterile insect technique (SIT) is emerging as a tool to supplement traditional pesticide-based control of Aedes aegypti, a prominent mosquito vector of microbes that has increased the global burden of human morbidity and mortality over the past 50 years. SIT relies on rearing, sterilizing and releasing large numbers of male mosquitoes that will mate with fertile wild females, thus reducing production of offspring from the target population. In this study, we investigated the effects of ionizing radiation (gamma) on male and female survival, longevity, mating behavior, and sterility of Ae.aegypti in a dose-response design. This work is a first step towards developing an operational SIT field suppression program against Ae.aegypti in St. Augustine, Florida, USA. Exposing late-stage pupae to 50 Gy of radiation yielded 99% male sterility while maintaining similar survival of pupae to adult emergence, adult longevity and male mating competitiveness compared to unirradiated males. Females were completely sterilized at 30 Gy, and when females were dosed with 50 Gy, they had a lower incidence of blood-feeding than unirradiated females. Our work suggests that an ionizing radiation dose of 50 Gy should be used for future development of operational SIT in our program area because at this dose males are 99% sterile while maintaining mating competitiveness against unirradiated males. Furthermore, females that might be accidentally released with sterile males as a result of errors in sex sorting also are sterile and less likely to blood-feed than unirradiated females at our 50 Gy dose. © 2022 Society of Chemical Industry.

New self-sexing Aedes aegypti strain eliminates barriers to scalable and sustainable vector control for governments and communities in dengue-prone environments.

For more than 60years, efforts to develop mating-based mosquito control technologies have largely failed to produce solutions that are both effective and scalable, keeping them out of reach of most governments and communities in disease-impacted regions globally. High pest suppression levels in trials have yet to fully translate into broad and effective Aedes aegypti control solutions. Two primary challenges to date-the need for complex sex-sorting to prevent female releases, and cumbersome processes for rearing and releasing male adult mosquitoes-present significant barriers for existing methods. As the host range of Aedes aegypti continues to advance into new geographies due to increasing globalisation and climate change, traditional chemical-based approaches are under mounting pressure from both more stringent regulatory processes and the ongoing development of insecticide resistance. It is no exaggeration to state that new tools, which are equal parts effective and scalable, are needed now more than ever. This paper describes the development and field evaluation of a new self-sexing strain of Aedes aegypti that has been designed to combine targeted vector suppression, operational simplicity, and cost-effectiveness for use in disease-prone regions. This conditional, self-limiting trait uses the sex-determination gene doublesex linked to the tetracycline-off genetic switch to cause complete female lethality in early larval development. With no female progeny survival, sex sorting is no longer required, eliminating the need for large-scale mosquito production facilities or physical sex-separation. In deployment operations, this translates to the ability to generate multiple generations of suppression for each mosquito released, while being entirely self-limiting. To evaluate these potential benefits, a field trial was carried out in densely-populated urban, dengue-prone neighbourhoods in Brazil, wherein the strain was able to suppress wild mosquito populations by up to 96%, demonstrating the utility of this self-sexing approach for biological vector control. In doing so, it has shown that such strains offer the critical components necessary to make these tools highly accessible, and thus they harbour the potential to transition mating-based approaches to effective and sustainable vector control tools that are within reach of governments and at-risk communities who may have only limited resources.

Automatic exposure correction algorithm for online silkworm pupae (Bombyx mori) sex classification

In the process of online sex identification and sorting of silkworm pupae by machine vision technology, the collected images of silkworm pupae gonad suffer underexposure or overexposure due to the biological characteristics of silkworm pupae and the mechanism movement. As a result, the partial texture characteristics of silkworm pupae gonad are not obvious and thus lead to a large error for the subsequent identification of male and female pupae. In view of the overexposed or underexposed problem existed in online image collecting of silkworm pupae gonad, an algorithm model is proposed in this paper. It can automatically determine whether the silkworm pupae gonad image has exposure problem according to the distribution law of gray values. Moreover, an extra correction model based on the combination of the Retinex (retina and cortex) model and the weighted least squares method is put forward to correcting the exposure of gonadal images. The experiment results demonstrate that the proposed algorithm not only can find the silkworm pupae gonad image with exposure problem completely and accurately, but also make the restored gonadal texture clearer. Furthermore, this algorithm has a good effect on the exposure correction of other objects. After the original data sets are processed by the algorithm, the correct sex identification rate in the classification network based on Resnet50 training reaches 90.52%. It is 7.08% higher than the original silkworm pupae gonad images, which can verify the effectiveness of the algorithm. And the recovery time of a single image is within 0.3 s, which can meet the needs of online automatic identification. The proposed algorithm is of great significance for the online male and female identification of silkworm pupae based on machine vision.

Transgenic expression of Nix converts genetic females into males and allows automated sex sorting in Aedes albopictus

Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we establish Ae. albopictus transgenic lines ectopically expressing Nix. Several are composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness is marginally impaired, while their flight performance is similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

Constraints Affecting Fertility of Sex Sorted Semen: An Overview

In most mammals, the male to female sex ratio of offspring is about 50% because half of the sperm contains either the Y chromosome or the X chromosome. Desired sex either male or female which is produced from semen having X or Y bearing sperm is known as sexed semen. Flow cytometry is the only proven method for semen sexing to be commercially viable with more than 90% accuracy to produce calves of desirable sex. The sexed semen technology has several advantages, the production of more female calves and more milk, and lowered risk of dystocia. Increased efficiency of progeny testing, embryo transfer and in-vitro fertilization program has proposed a demand of feasible technology for sex sorting of spermatozoa. In India, this revolutionary technology is in its budding stage. This paper aimed to review the constraints affecting the fertility of sex sorted semen.

Temperature-Inducible Precision-Guided Sterile Insect Technique.

Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.

P–121 Selection and separation of X- and Y- chromosome-bearing mammalian sperm using SPERMAN technology

Abstract Study question How can we select and separate X and Y-chromosome bearing sperm cells without causing cellular damage and reduced fertility? Summary answer AI powered SPERMAN technology can sort X and Y-chromosome bearing sperm populations without using harmful fluorescent dyes and lasers. What is known already Most common and reliable method to sex sorting (separation of X and Y- chromosome bearing sperm cells) is by using fluorescence activated cell sorting ( FACS) which takes advantage of the difference of amount of DNA in X and Y-chromosomes. Unfortunately this method causes reduced fertility and cellular damages due to the lasers and fluorescent dyes that are used. There are new and experimental developments in sex-sorting such as using immunological approaches to separate sperm cells. Current sorting method damages sperm cells. However, there is no automated and easy to use sperm sorting technology available. Study design, size, duration In this study, we have compared the quality parameters (motility, viability, AR status, DNA packaging) of sorted sperm cells to unsorted sperm cells. We have used epididymal sperm samples from 10 C57BL/6 mice (Charles River) and frozen semen samples from 6 Holstein bulls (Sexing Technologies) and frozen semen samples from 8 human sperm donors (Fairfax Cryobank). Samples were divided into two groups: sex-sorted and unsorted sperm. Participants/materials, setting, methods Sperm samples from 8-week-old C57BL/6 mice were collected from epididymal region, frozen bull semen samples and human semen samples were thawed at 37ºC then separated into two groups as sorted and unsorted sperm. Sorted samples in modified SP-TALP medium were loaded on the microfluidic chip of Sperman device, after the 30min long sorting process ended, all samples were centrifuged gently at 100g for 5 min, quality parameters were measured by using CASA and FACS. Main results and the role of chance Sperman device sorted X-bearing sperm cells at 81% purity and Y-bearing cells at 73% in mouse samples. Sperman device was able to sort X-bearing sperm cells at 78% purity and Y-bearing cells at 70% in bull samples.S perman device sorted X-bearing sperm cells at 85% purity and Y-bearing cells at 76% in human samples. Our study shows that in mouse, bull and human sperm samples, sperm DNA quality, sperm concentration, progressive motility and AR status results from the sorting with SPERMAN device are comparable with the unsorted samples. For sperm DNA quality, both the Spearman rank correlation coefficient and concordance correlation coefficient are above 0.97, indicating a high agreement between the unsorted samples and SPERMAN sorted samples. Limitations, reasons for caution In this study we only compared sperm quality parameters between unsorted sperm cells and Sperman-sorted sperm cells. Ideally a follow up study would be to look in detail at fertilization success rates and embryo growth. Also, genomic and proteomic profiles of unsorted and sorted sperm cells could be different. Wider implications of the findings: In livestock, sperm sex sorting has high value in terms of economic impact on the livestock management. Unfortunately, there is one expensive single technology dominates the sex-sorting market causing high fees poor outcomes. We believe that Sperman technology provides farmers more cheaper and better sex-sorting technology. Trial registration number Not applicable

Sterile Insect Technique in an Integrated Vector Management Program against Tiger Mosquito Aedes albopictus in the Valencia Region (Spain): Operating Procedures and Quality Control Parameters.

Aedes albopictus and Aedes aegypti are the main vectors of arboviral diseases such as dengue, Zika and chikungunya viruses. About a third of the world population is currently at risk of contracting Aedes-borne epidemics. In recent years, A. albopictus has drastically increased its distribution in many countries. In the absence of efficient mosquito vector control methods, the sterile insect technique (SIT) is presented as a very promising and environment-friendly control tool. The Agriculture Department of the Valencian Region is promoting an ongoing pilot project to evaluate the efficacy of an integrated vector management program (IVM) based on the use of the SIT as the main method of control. The laboratory studies for evaluating the entomological efficacy of SIT through the phased conditional testing process recommended by World Health Organization and the International Atomic Energy Agency (WHO-IAEA) are addressed. This study describes the routine operating procedures and quality control parameters for the medium-scale rearing of sterile male A. albopictus. More than 15 million sterile males have been produced and released in an area of 80 ha between 2018 and 2020. Of the initial L1 larvae, we recovered 17.2% of male pupae after sex sorting to be sterilized and released on the field, while the rest of the pupae remained available to maintain the rearing colony. The residual percentage of females after sex sorting was on average 0.17%. The obtained values in terms of production and quality control as well as the proposed rearing methodology can be useful for designing a medium-scale mosquito-rearing pipeline.

OPTIMIZATION OF FACS SORTING FOR THE IMPROVEMENT OF LIVESTOCK SEMEN QUALITY

Fluorescence-activated cell sorting (FACS) of spermatozoa was mainly used for sex sorting. Recently, FACS has been used to eliminate DNA-damaged human spermatozoa using YO-PRO-1 staining. Fragmentation of sperm DNA is considered as one of the reasons of male infertility. Moreover, YO-PRO-1 can effectively mark apoptotic as well as dead spermatozoa. Till now, only two FACS instruments were used for the spermatozoa sorting. However, both of them are not more commercially available from different reasons. In this study, we used novel FACSMelody Cell Sorter for the elimination of apoptotic and dead cells from the rabbit and ram semen samples in order to improve their overall quality. Briefly, semen samples were stained using YO-PRO-1 dye (apoptotic and dead cells) and/or propidium iodide (PI; only dead cells). Three different sorting experiments were performed: E1 – YO-PRO-1 and PI stained rabbit sperm cells were sorted into the tubes containing 1 ml of PBS; E2 – PI stained rabbit sperm cells were sorted into tubes that were washed with FBS prior adding PBS; and E3 – YO-PRO-1 and PI stained ram sperm cells were sorted into tubes washed with FBS prior adding PBS. As a sheath fluid sterile PBS was used. All samples, control (before sorting), negatively and positively sorted fractions, were analysed using CASA for motility assessment. Moreover, all sorted samples were re-stained with PI for viability assessment. In conclusion, elimination of dead (PI+) sperm from rabbit samples might improve their quality, since their progressive motility increased significantly (P&lt;0.001) after sorting from 40 to 65%. However, ram spermatozoa seem to be sensitive to sorting procedure thus further optimisation of this procedure is required.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.