Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing

Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing Marc Van Der Hofstadt, Nicolas l’Helgoualch, Juliette Houot-Cernettig, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5772672/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Despite of global efforts, reducing the culling of one-day hatched male chicks in the poultry industry has been a critical priority due to significant socio-economic concerns. Indeed, various molecular assays have been developed to determine the sex of eggs before hatching ( in ovo ) to eliminate male embryos at early developmental stages. Because of their precision-related complexity, needs for advanced infrastructures and time-consuming processes, there is still no widespread commercialization for these assays. In this study, we developed two novel digital readouts assays employing PCR, LAMP and RPA techniques whose sensitivity, specificity and robustness were validated on 82 chick embryos at day 9. Our data demonstrate that, while the two novel PCR based assays correctly and robustly sex the 82 embryos, the LAMP and RPA based assays propose comparable results. Moreover, LAMP and RPA assays propose isothermal amplification associated with naked-eye colorimetric and/or fluorescent detection in a relatively shorter time (20 minutes at 65°C and 30 min at 37°C, respectively). These newly developed assays, not only significantly reduce the complexity of experimental setting but also being faster and more affordable sexing methods, addressing key barriers to in ovo sexing to a future commercialization of a non-invasive in ovo sexing assay. Biological sciences/Biotechnology Biological sciences/Molecular biology PCR LAMP RPA chick embryos in ovo sexing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Large-scale chicken egg production has come under increasing scrutiny in several European and North American countries due to economic and ethical concerns. Efforts to address these challenges have focused on various aspects of the production process, such as extending the laying period of hens to reduce replacement rates or transitioning to organic and non-cage farming systems [ 1 ]. Yet, one-day-hatched culling of male chicks remains a critical issue. To mitigate this concern, two main approaches have been envisioned: (i) dual-propose strains where male chicks are raised for meat production [ 2 , 3 ]; and (ii) egg sexing technologies [ 4 – 6 ] to identify and eliminate male embryos at early developmental stages. However, despite the intensive effort, consumer acceptance of meat from such breeds still remains limited, while existing egg sexing techniques are often complex, time consuming and require advanced infrastructure [ 7 ]. Up to date, only five sexing techniques are being successfully commercialized [ 7 ], leaving the need for novel ones to be developed. Up to date, to establish the sex of the embryo before hatching ( in ovo ), five major technological classifications can be done depending on their biomarker or methodology: 1) volatile organic compounds, 2) morphological inspection, 3) acoustic-based methods, 4) spectral-based methods, and 5) molecular approaches. Among them, molecular-based techniques have drawn significant attention due to their higher accuracy and lower susceptibility to biological variability. Indeed, these assays are PCR-based amplifying sex-specific DNA fragments present either in their mutual chromosome (Z) and/or in the female-specific chromosome (W) of chicks [ 7 ]. The first PCR-based sexing assay, developed in 1998 used the chromobox-helicase-DNA-binding (CHD) gene situated on both sexual chromosome Z (CHD-Z) and W (CHD-W) [ 8 ]. CHD-Z and CHD-W share common sequences at the two extremities of the gene, whilst reserve a female-specific fragment in the middle of the gene. Therefore, CHD sequences have been used to discriminate males consisting of ZZ chromosomes (CHD-Z only, smaller amplicon size) and females consisting of ZW chromosomes (CHD-Z & CHD-W, larger amplicon size) in different avian species with only one mutual primer pair [ 9 – 12 ]. Over time, advancements such as new gene targets [ 13 , 14 ], multiplexing [ 15 ], and high-throughput real-time PCR [ 16 ] have improved both speed and accuracy of molecular based tests. However, analytical success of these tests still depends on amplification techniques requiring sophisticated instrumental facilities (thermocyclers, quantitative thermocyclers, and gel/capillary electrophoresis), limiting their scalability and practical implementation in poultry production. To address these limitations, a myriad of isothermal DNA amplification techniques have emerged in the recent years proposing solution for reducing the need of sophisticated instrumental facilities. Their main attractiveness lays on their constant and lower working temperature (37–65°C) compared to repetitive thermocycles of the conventional PCR. Among these, LAMP has gained prominence in the molecular diagnostic field and start to outcompete the conventional PCR technique [ 17 – 20 ]. In particular, LAMP technique has been endorsed due it its high sensitivity and rapid results (less than 30 minutes) thanks to the employment of multiple primer sets (three sets) and the Bst DNA polymerase with a high strand displacement activity [ 20 ]. More importantly, LAMP is compatible with multiple detection modes ranging from conventional gel electrophoresis to fluorescent or naked eye readout (turbidity or colorimetry). Specifically, naked-eye observation together with the digital response of LAMP reactions (Yes Vs. No readouts) have enabled the establishment of point-of-care diagnostic tools for onsite detection of infectious diseases such as malaria [ 21 ], dengue [ 22 ] or COVID-19 [ 23 , 24 ]. Similarly, RPA technique, works at significantly lower temperature (37–42°C) thanks to the employment of DNA recombinase, proposes fast readout and practical visualization methods for the amplified products [ 25 , 26 ]. Importantly, as RPA reactions operate at low temperatures, which is compatible with egg incubation condition, hence a promising approach for onsite sexing within the poultry production setting. In our previous work [ 9 ], we developed a duplex PCR and a quantitative PCR (qPCR) assays using SWIM/12S and DMRT/Xho-I markers, respectively, to enable specific, rapid and robust sex determination of 9 day-old chicken embryo. In this work, we adopted LAMP and RPA techniques to develop two novel assays based on two newly designed sex-specific DNA markers. First, we identified two new sex-specific DNA fragments, one on the well stablished CHD gene (CHD-W), and a second on the AC182258 locus (fragment AC18), which are both located solely in the female chromosome. Second, we showed that fluorescent LAMP can detect AC18 fragment to discriminate chick sex in under 20 minutes at constant temperature (65ºC). This approach is compatible with naked-eye colorimetric detection and larger-scale screening. Finally, we demonstrate that RPA can be used to determine chick sex at 37ºC in under 30 minutes, reducing significantly both the complexity and the costs of the assay. Additionally, we show that our methods eliminate the need for DNA input normalization, further simplifying the experimental workflow and enhancing feasibility for large-scale implementation in the poultry industry. Results Two novel sexing assays for chicken embryos: PCR validation For the design of the two new sexing assays, we focused on two different genomic locus CHD and AC182258 of the female W chromosome and screened for primers amplifying DNA fragments ranging from 180 to 220 bp (Table S1 ). Of note, for CHD gene, contrary to previous CHD based PCR sexing that focus on size discrimination of the CHD locus between the CHD-Z and CHD-W, we targeted the region of the CHD-W (female) that is not present in the CHD-Z (male). Different combinations of PCR primers were assessed for amplification of solely female specific fragments (here defined as CHD-F and AC18). For the major part of the CHD-F (Figure S1 A) and AC18 (Figure S1 B) primer pairs, a clear single band for females versus males was visualized on agarose gel. Nonetheless, AC18 design was less efficient compared to CHD-F, since several combinations of PCR primers were prone to false positives (Figure S1 B). Following the selection of the best candidate for both assays (F3R1 for CHD-F; F2R1 for AC18, Table S1 ), we tested their performance on 82 samples of 9-day- old chick embryos. To this aim, genomic DNA from dissected brain tissue of chick embryos was extracted and purified [ 9 ]. DNA quantification from 82 ISA Brown eggs revealed a variability in DNA concentrations, ranging from 1 ng/µL up to 226 ng/µL (Figure S2 and Table S2 ). Although we observed a normally distributed sample (p-value 0.08, Kolmogorov-Smirnov test of normality), we noticed that ~ 21% of the samples were significantly low in DNA concentration (< 10 ng/µL). Nevertheless, to assess the robustness of the two assays, no DNA concentration normalization step was performed in the subsequent experiments. For both CHD-F and AC18 assays, in addition to the female specific target, the internal control (12S primers) [ 9 ] was added to the test (CHD-F/12S and AC18/12S, respectively). First, PCR amplification for CHD-F/12S using chosen primer pair (F3R1, table S1 ) was performed. Cappilary electrophoresis (Labchip) analysis of PCR products discriminated two groups by the number of amplicons. The first group (40 samples) contained only the 12S band with an amplicon size of 139.9 ± 0.9 bp (Fig. 1 A&B). The second group (42 samples) presented two bands, the 12S band (142.2 ± 0.2 bp) in addition to a second band of 179.2 ± 0.2 bp, which corresponded to the predicted band size of the CHD-F target (Fig. 1 A&C). At this stage, we could predict that the group 1 classifies male samples while the group 2 are the female ones. For both amplicons 12S (Figure S3A) and CHD-F (Figure S3B), no correlation between concentrations of the extracted DNA and the amplified PCR products measured by Labchip was observed. However, there was a significant difference in the concentration of 12S amplicon between the two groups (p-value < 0.01), in which the predicted female group presents a lower concentrations (Figure S3A). Low concentration amplicon was noticed in one sample (F2) (Figure S3A) for 12S and in three samples (F1, F2 and F3) for CHD-F (Figure S3B). Second, PCR amplification for AC18/12S using the chosen primer pair (F2R1, table S1 ) was performed on the same extracted DNA sample set. Similar to CHD-F assay, analysis of PCR product also revealed two distinct groups, the first group with 40 samples containing one amplicon of size 139.8 ± 1.0 bp (12S amplicon) and a second group of 42 samples containing two amplicons with sizes 142.0 ± 0.2 bp and 229.3 ± 0.2 bp, for 12S amplicon and AC18, respectively (Fig. 1 D, 1 E & 1 F). As expected, sample classification by the AC18 assay was exactly the same as the one performed by the CHD-F assay, demonstrating a 100% concordance (Table S2 ). Again, we observed no correlation between the extracted DNA concentration and the amplified product for both 12S and AC18 amplicons. In the same line, the second group also presented a significantly lower concentration for the 12S amplicon (p-value < 0.01) compared to the first group, in which the sample F2 outstood as significantly low 12S concentration (Figure S4A). For AC18 amplicon, we again noticed the low amplified concentration (up to 43-fold) for the three samples F1, F2 and F3 (Figure S4B). To confirm the sex identity of the two groups detected by the two novel assays, we analyzed this extracted DNA sample set by our previously established SWIM/12S assay [ 9 ]. As expected, PCR product analysis (Fig. 2 ) confirmed the correct sex identification by our two new assays for all the 82 samples consisting of 40 males amplifying only the 12S amplicon (146.7 ± 0.2 bp, Fig. 2 B) and 42 females amplifying both 12S and SWIM amplicons, 148.9 ± 0.3 bp and 231.2 ± 0.3 bp, respectively (Fig. 2 C). Once again, we observed that there was no correlation between the extracted DNA concentration and the amplified concentration of neither 12S nor SWIM amplicons (Figure S5). Differs from 12S/CHD-F and 12S/AC18 assays, SWIM/12S assay is not suffered from low amplification rate of 12S amplicon in the duplex PCR configuration. Indeed, there was no significant difference in concentration of PCR products between the 12S and SWIM (p-value = 0.18). However, we did observe that 12S concentration of F2 sample was 2-fold lower, while, SWIM amplicon was up to 6-fold smaller in the three samples F1, F2 and F3 (Figure S5). Furthermore, the sex validation by SWIM allowed us to discern that female samples had significantly higher extracted DNA concentrations (84.7 ± 8.5 ng/µL) than male samples (57.9 ± 10.2 ng/µL) (Figure S2 ). Loop-Mediated Isothermal Amplification (LAMP) assay for rapid sexing of chick embryos Aiming to make our assays more compatible with field application, we adapted the two novel PCR sexing assays to LAMP whose amplification is isothermal. As such, different LAMP primer combinations (Table S1 ) were designed and validated by endpoint colorimetric LAMP (Figure S6) for their female specific detection. Results showed that designs for both target candidates CHD-F and AC18 allowed sexing by simple naked-eye colorimetry (Assay V for CDH-F; assay D and assay G for AC18). Primer combination for assay D of AC18 was then chosen for large scale analysis on the previously assessed 82 chick embryo samples using fluorescent LAMP. In particular, the obtained data revealed that the AC18 assay is highly robust since it correctly discerned the sex of the 82 samples except for the sample F1 (Table S2 ). In particular, the initiation of the exponential amplification (Ct value) for the female samples was constant (13.5 ± 0.2 min), except for samples F2 and F3 which were significantly higher (19.0 ± 1.1 min) (Fig. 3 A & 3 B). Although we observed heterogeneous fluorescent endpoints and small differences in the Ct values, the concentration of the extracted DNA displayed, once again, no correlation with Ct values (Fig. 3 C). Regarding the negative controls and the male samples, we did not observe any amplification under 40 minute reaction. The AC18 LAMP assay delivers a digital response with either the presence or the absence of the female specific chromosome W. While digital responses simplify data interpretation, the absence of internal controls does not allow technical verification for enzymatic activity of the assay. Since the synthetic plasmid containing the AC18 amplicon displayed a high tau value (Fig. 3 A), we decided to use this plasmid as the internal control for the enzymatic activity of the LAMP reaction. To this aim, we performed a spike-in experiment in which a range of different concentrations of AC18 plasmid was added to a random male (M1) and female (F37) sample prior to amplification. As expected, obtained results (Figure S7) show that increasing the concentration of spiked AC18 plasmid led to increasing detection signal of the non-template negative control and the male sample with the Ct value down to 27.1 at 10 ng/µL. On the contrary, the spike-in plasmid but did not affect the Ct value of the female sample, leaving an elevated ΔCt of 17.3 (Male vs Female) which clearly distinguish between the female and male samples. Recombinase Polymerase Amplification (RPA) assays for in ovo sexing of chick embryos at low temperatures: To further minimize the need for advanced research facilities and costs, we extended the CHD-F and AC18 novel sexing assays to RPA whose reaction occurs at 37°C. Firstly, different RPA primer pairs (Table S1 ) were designed and validated using endpoint RPA assay (Figure S8). We noticed that efficiencies of the best primer pairs to detect the presence of their targets (Pair 5 for CHD-F and Pair 27 for AC18), were equal to their PCR variant assays (Figure S8). These primers were validated by large scale (82 samples) sex identification using fluorescent RPA-exo assay. 12S primers (detected in both male and female) were added as and detected simultaneously with the female specific primers in a duplex configuration (Fig. 4 ). CHD-F/12S fluorescent assay showed exponential amplification of the 12S target in all 82 samples. Moreover, end-point signals for 12S amplification revealed no significant difference (p-value = 0.10) between female and male chicks, nor a correlation to the extracted DNA concentration (Figure S9A). On the other hand, exponential amplification of the CHD-F locus was only observed in female chicks (Fig. 4 B), with the exception of samples F1, F2 and F3 where signal was detected (Fig. 4 C). Again, no correlation was found between the extracted DNA concentration and the end-point signal for the CHD-F locus (Figure S9B). Similarly, AC18/12S assay results were identical to CHD-F/12S assay. 12S locus was detected in all 82 samples, whose signals were dependent from either DNA concentration or sex identity of the analyzed samples (Fig. 4 D and S9C). Likewise, all female samples were detected by the exponential amplification of the AC18 locus whilst all male samples were negative (Fig. 4 E). End-point fluorescence values of AC18 amplification revealed that the average signal of female samples (4.40 ± 0.17 RFU) was 18-fold higher in intensity compared to male samples (0.25 ± 0.01 RFU) enabling the introduction of a sexing cut-off value at 0.56 RFU (even if signals from the three samples F1, F2 and F3 were still significantly lower than the average female sample) (Fig. 4 F). Once again, no correlation was observed between the extracted DNA concentration and the end-point fluorescence of the AC18 locus amplification (Figure S9D). To estimate the impact of the DNA concentration RPA assay, we performed reactions using different volumes of input DNA from highly concentrated samples including M4 (204.7 ng/µL) and F17 (214.3 ng/µL) compared to one female sample (F12) whose DNA concentration was significantly lower (16.5 ng/µL). Fluorescent measurement reveled identical amplification curves for both locus samples M4 and F12 at different volumes of DNA input. However, x significantly impairs in RPA amplification curves for both locus were observed for F17 sample (Figure S10). While this impairment is stronger on the AC18 locus than on the 12S locus, the obtained results indicate that the AC18 assays is specific from at least 0.13 ng/µL up to at least 25.7 ng/µL final DNA concentration. Finally, we assessed the possibility to decrease the temperature down to room temperature (25°C) for the AC18/12S assay (Figure S11). As expected, a longer incubation time (50 minutes) was required to reach the plateau signals for both 12S and AC18 in RPA amplifications. However, at 25°C the AC18/12S assay was not capable to correctly detect samples F1, F2 and F3. Nevertheless, out of these three samples, the other female samples were correctly discriminated (with signals were ~ 3-fold higher than in male samples). These results, hence demonstrate that the AC18 assay is capable of significantly discriminating (P-value < 0.01) the sex of chick samples at 25°C. Discussion Reducing the culling of one-day hatched male chicks is still a major socioeconomical burden for the poultry industry. To reduce the culling, many in ovo molecular sexing procedures have evolved [ 4 – 6 ]. However, their adoption remains limited due to their complexity, reliance on the advance infrastructure, and high temporal and financial costs. We designed two novel digital response assays and adapted them to LAMP isothermal amplification for its rapidity and RPA for its room temperature amplification compatibility. Moreover, these assays do not require DNA concentration normalization, hence simplifying the sexing process. Conventional molecular detection assays often require DNA normalization step to ensure a measurable output [ 9 ]. Our results show that up to a 226-fold difference in concentration of extracted DNA can be obtained (Figure S1 ) from 82 samples, which could be related to different factors such as quantities of input samples, variations in experimental handling or sample conservation. Here, to demonstrate the robustness of our assay, we skipped the normalization step and fix a constant volume of DNA samples added in the assay (and not the concentration of input DNA). Despite of a large concentration range of input DNA (from 3 ng up to 678 ng), the 12S internal control and the two female markers (CHD-D and AC18) were correctly amplified in all samples, demonstrating the robustness of the PCR assays. Furthermore, the fact that the amplicon intensity of all targets are independent of DNA input (Figure S3-S5) indicates that all PCR amplifications had reached their plateau. We previously showed that 1 ng of input DNA was sufficient to achieve PCR saturation in under 30 cycles for female samples and 40 cycles for male samples [ 9 ]. To further decrease the complexity of molecular in ovo sexing assays, we decided to implement digital (yes Vs. no) responses compared to analogue (range of values) responses. This transition is crucial since it reduces the expertise required for the user to interpret the results, as well as the instrumentation requirements. For example, conventional CHD-based PCR sexing assays exploit the size difference between CHD locus present in both the female and male chromosomes (8–12). This size difference can vary between 11 bp up to 165/179 bp, hence would require a corresponding resolution technique and expertise for their downstream analysis. On the contrary, here we demonstrate that the presence or the absence of the amplicon, either CHD-F or AC18 (Fig. 1 C and 1 F), is sufficient to discriminate the sex of the embryo sample (note that the 12S amplicon is only an internal control that is not required for sex discrimination). In particular, we observed that for assays CHD-F/12S and AC18/12S, female samples had lower 12S concentrations than male samples, most likely due to the simultaneous reaction of the duplex PCRs, which might reduce the efficiency of both. For this reason, we predict that in the absence of the 12S internal control, a greater CHD-F and AC18 amplification would occur, further simplifying sex discrimination procedures. Collectively, these findings demonstrate that our two novel PCR assays enable highly robust detection, achieving 100% accurate sex identification without requiring DNA input normalization. This breakthrough significantly enhances the efficiency and scalability for large-scale analyses in industrial applications To streamline in ovo sexing techniques and align with the high yield of egg production, rapid and high throughput approaches are crucial [ 1 ]. To this aim, we implemented LAMP for chick embryo sexing achieving results in less than 20 minutes with binary (digital) readouts. Colorimetric LAMP results (Figure S6) enable sex determination by visual inspection with minimal equipment (only a 65°C incubator) (Table 1 ), hence allowing the development of portable devices [ 28 ]. However, colorimetric assays are limited by their reliance on manual verification, constraining automation for high throughput analysis. To overcome this obstacle, we adapted the LAMP colorimetric to a fluorescence-based version allowing real-time, high-through put measurements with a small reaction volume (12.5 µL). Using AC18 fluorescent LAMP assay, 81 out of 82 samples were accurately sexed based on the presence (female) or the absence (male) of exponential amplifications (Fig. 3 ). Notably, while real-time fluorescence LAMP is generally used for quantitative analysis, its continuous amplification process is highly prone to stochastic variations at low template concentration leading to digital-like rather than quantitative output [ 19 ]. Given that the target locus concentration is independent of the sex phenotype ( i.e. the female chromosome is present or not), fluorescence LAMP assay does not require precise quantitative resolution for sex determination. To meet the stringent requirements of industrial applications, we established that the addition of 0.5 ng/µL of AC18 plasmid serves as an effective internal control, ensuring reliable verification of enzymatic activity in the LAMP reaction. Table 1 Comparative analysis of the three molecular tools used in developed assays for sex identification of chick embryos. Power consumption has been measured at the maximum temperature required by the assay. Items PCR LAMP RPA Detection method Labchip Sybr green /colorimetry Probe Steps required 2 1 1 Time (minutes) 60 + 60 20 25 Machines required Thermocycler & Labchip Incubator (65ºC) & fluorometer Incubator (37ºC) & fluorometer Power consumption (W) 240 (94°C) 150 (65°C) 110 (37°C) Estimated consumable cost per reaction (€) 2.7 2.5 2.1 Accuracy +++ +++ +++ In addition to high accuracy and simple implementation, cost effectiveness is crucial factor for a sexing assay to be industrialized given its significant economic implication. Although we calculated that the consumable cost per reaction not to be different between PCR and LAMP assays, we estimated a 38% reduction for the incubator power consumption for LAMP assay (Table 1 ) (without taking into consideration the surplus energy required for the labchip analysis). Even more cost effective, our RPA sexing assay cost (at 37°C) is reduced by 20% with 58% less in power consumption compared to PCR assays. RPA results revealed that the CHD-F assay clearly assesses sex status with a sensitivity of 96% (79 out of 82 sample) and a 100% specificity. Endpoint AC18 assay displays a 100% sensitivity and specificity with a clear established cut-off (Fig. 4 C). It is worth noting that for RPA assays can also produce a digital readout ( i.e. amplification has occurred or not). However, due to the irregular shape of amplification curves (Fig. 4 A), commercial software struggles to accurately detect the onset of exponential amplification using the second derivative method. As such, a thresholding approach is preferable. We noticed that in female samples, the final fluorescence intensity decreased as the input DNA concentration increased (Figure S10), likely due to an imbalance between the target and primer concentration. However, beyond a certain input threshold, fluorescence level stabilized and remains sufficiently high for reliable sex phenotype identification. This highlights the assay robustness even in the presence of elevated DNA inputs. Lastly, to further explore the potential of onside applications, we tested the AC18/12S RPA-exo assay at room temperature. A slight slot in robustness observed of the assay, as samples F1-F3 failed to achieve exponential amplification within 50 minute. This outcome aligns with the low amplification efficiency already observed at 37°C for these samples, as the reduction in RPA at lower temperature [ 26 ] likely exacerbates this limitation. While other female samples also experience a decrease in performance (with the fold change between female and males samples dropping from 18 to 3-fold), all were still correctly identified, demonstrating the assay’s resilience over temperature-induced impairment. Throughout the study, three samples (F1, F2 and F3) consistently exhibited significantly lower PCR amplification efficiencies for the target locus (Figure S3-S5), with distinct behaviour observed among them. Sample F2, on the one hand, consistently showed the lowest 12S amplification. While it was the female sample with the lowest total extracted DNA concentration, 12 other male samples with even lower total extracted DNA concentrations displayed higher 12S amplicon intensity during simplex amplification, likely due to the absence of competing female targets. This suggest a potential partial impairment of both targets in a duplex amplification configuration. On the other hand, samples F1 and F3 displayed normal 12S amplifications but with a reduced amplification of the female specific targets. The hypothesis that low DNA input might hinder target amplification was ruled out, as F1 (10.3 ng/µL) and F3 (18.3ng/µL) exhibited higher DNA concentrations compared to other female samples (e.g., F39 = 8.1 ng/µL, F12 = 16.5 ng/µL and F10 = 18.1 ng/µL) that achieved robust amplification of female specific targets. These findings suggest variability in the copy number of different targets across samples, significantly impacting PCR results while total extracted DNA concentration is relatively low. Despite these challenges, the PCR assays successfully determined the female phenotype for all these three samples, demonstrating their robustness for chick embryo sexing. Interestingly, while sample F2 showed the lowest amplification in PCR assays, sample F1 was the least efficiently amplified in RPA and failed amplification in LAMP assay. For samples F2 and F3, although lower amplification efficiencies were observed, sex discrimination remained feasible using AC18 LAMP and RPA assays. Conclusions In this study, we have established two innovative molecular assays capable of determining the sex of 9 day-old chicken embryos with straightforward “Yes” or “N o” readout. Initially, we used conventional PCR amplification to validate their efficiency sex identification. Subsequently, we employed LAMP amplification, which significantly reduce experimental time to less than 20 minutes under isothermal incubation at 65°C. Finally, we demonstrated the accurate sexing of all samples by RPA amplification at lower temperature (37°C), offering a significant reduction in both experimental complexity and economic cost. We envision that these two novel assays (CHD-F and AC18) combined with the adapted amplification techniques, present transformative potential for the poultry industry across different production scales. The rapid and high throughput nature of LAMP assays enable efficient and user friendly large-scale in ovo sexing. Meanwhile, the lower temperature requirement for RPA assays coupled with with the possibility of colorimetric readouts pave the way for simple, cost-effective and compatible with an onsite solution for chick sexing [ 29 – 30 ]. Overall, these simple, faster and more affordable novel molecular assays promise to facilitate the automation and commercialization of chick sexing processes. This advancement represents a significant step forward in improving the economic, ethical and environmental sustainability print of the poultry industry. Materials and methods Eggs incubation Fertilized ISA Brown eggs were purchased from a commercial supplier (SFPA, Saint-Marcellin France), stocked at 19˚C for a maximum of 10 days after laying and before incubation. Eggs were incubated in a dedicated incubator (Masson, Soyans France) at 37.5˚C, 55% humidity and tilted every hour. Incubation was arrested at day 9. Sample collection and lysis 10–20 mg of day 9 post-hatching embryo brain tissue from 82 ISA Brown eggs was recovered and treated as previously described [ 9 ]. In brief, collected samples were lysed for 3H at 55°C in 150 µl of lysis buffer containing 10% of chelating beads (Chelex 100 Biorad), 0.2% SDS, 10mM Tris-HCl pH8, 0.2 µg/µl Proteinase K followed bya denaturation step at 95°C for 15 min. Supernatant was recovered by centrifugation for 5 minutes at 13000g at room temperature and submitted to DNA purification using QIAamp DNA mini kit (Qiagen 51306). DNA quantification was performed post-purification using Nanodrop One (Thermo Scientific, Wilmington USA). All samples were stored at -80˚C until used. Primer design Primers for all sex determination assays were designed based on the two female specific sequences located on the female-specific W chromosome: CHD-W (Chromobox-Helicase-DNA-binding, GenBank Accession No GU132944.1) and AC182258 locus (GenBank Accession No AC182258.2). Blasting between CHD-W and CHD-Z was performed to identify specific female fragments (Table S1 ). The un-matched fragment situated from position 151–326 of the CHD gene would allow female identification as it is present only on W chromosome, hence primers were designed based on this identified fragment (Table S1 ). Regarding the locus AC182258, we used a fragment situated on the W chromosome from position 6942–7267 for designing the AC18 primers with various amplicon lengths (Table S1 ). Primers for the internal control were designed based on the 12S ribosomal RNA gene (Accession No MF041985.1). BIP, FIP and loop primers for the LAMP assay were designed using PrimerExplorer V5. CHD-W and AC18 primers had been consciously designed to be compatible with the RPA, following TwistDx guidelines regarding primer and amplicon length. For real time measurement of RPA amplification assisted by exonuclease, corresponding hydrolyse probes for each assay were designed (Table S1 ) following the instruction of TwistDx. Briefly, RPA probes contain a long amplicon specific sequence (about 45 bp) on to which a fluorophore (FAM or TAMRA) and a quencher (BHQ-1) were incorporated. A basic nucleotide analogue (tetrahydrofuran, THF) residue was introduced in between the fluorophore and the quencher as the recognition site for the exonuclease, whose activity will degrade the probe and separate the fluorophore from the quencher. PCR amplification and analysis PCR reaction mix was prepared in 96 well-plate using 5 µL of 2X Master Mix (Invitrogen Platinum Green Hot Start PCR) and 200 nM of primer set SWIM/12S, CHD-W/12S or AC18/12S at a ratio 2:1. 3 µL (constant volume) of purified embryo DNA was added into each well. Amplification was performed on peqSTAR 96X thermocycler (Ozyme, Montigny-le-Bretonneux France) as previously described [ 9 ]. In brief, one initial denaturation at 94˚C for 2 minutes followed by 35 cycles comprising of: 1) 30 sec. at 94˚C for the denaturation, 2) 30 sec at 55˚C for the annealing, and 3) 30 sec at 72˚C for the elongation. A final extension cycle at 72˚C for 5 minutes was performed. Negative Control (Non – template) were performed for each experiment. Sex determination was performed using two reference PCR assays SMIW/12S [ 9 ] and CHD 2250F/2718R [ 27 ]). All samples were analysed by two novel PCR assays AC18 and CHD-W/12S. Negative (non-template) and positive controls (synthetic plasmid containing the target amplicon, purchased from IDT) were added for each isothermal reaction. Real-time qPCR thermal cycling reactions were performed by the LightCycler 480 (Roche, Meylan France) directed by the LightCycler 480 Software (version 1.5.1.62). LAMP sexing assays Reaction mix for fluorescent LAMP assays were prepared on ice using WarmStart LAMP Kit (NEB, E1700) and NEB LAMP fluorescent dye (NEB B1700S). A premix primer solution containing 16 µM of FIP and BIP primers and 2 µM of AC18 primers was prepared prior to the reaction mix preparation. For one reaction, 1.25 µL of the primer mix solution was added to 6.25 µL of 2X NEB LAMP Master Mix, 0.125 µL of 50X NEB LAMP dye and 1.875 of H2O. The reaction mix was distributed on a 348 well-plate (Axygen) followed by the addition of 3 µL extracted DNA sample. LAMP amplification was carried out at 65°C for 40 min using the LightCycler480 (Roch, Meylan, France) with one fluorescent acquisition every 30 second. For each experiment, negative control (water) and positive control (plasmid) were performed. The samples were assessed in triplicate. For colorimetric LAMP assessment, the WarmStart colorimetric LAMP master mix (M1800S) was used, following the recommended protocol (M1800). RPA sexing assays Endpoint RPA amplification reactions were performed as recommended by the manufacture manual (TwistAmp Liquid basic RPA kit, Ref TALQBAS01). In brief, a pre-master mix for each reaction was prepared on ice with 25 µL of 2X buffer, 5 µL 10X E-mix, and 9.2 µL H2O containing 1.8 mM of total dNTPs following by vortexing and a brief spin. 2.5 µL of core reaction mix was then added to the lid of the tube containing the pre-master mix. The 41.7 µL of complete master mix was homogenised by 10 full inversions of the tube, followed by a brief spin. In another tube, on the lid, three reagents were added separately in its different corners including 2.4 µL of 10 µM of each primer, 2.5 µL of MgOAc and 1 µL template DNA. The reaction preparation was finally by the addition of 41.7 µL master mix at the bottom of the tube. RPA reaction was activated by spinning in all the materials from the lid. Amplification was carried out at 37°C for 30 min on a thermoblock (Eppendorf). Real-time RPA amplification reactions were performed at 37°C for 30 min as described in the manufacture’s manual instruction (TwistAmp™ exo Kit, Ref TAEXO02KIT) with a smaller volume adjusted to use the Roche LightCycler 480. Briefly, a pre-reaction mix for each reaction was prepared on ice with 2 µL of the primer mix at 10 µM (ratio 2:1 of sexing target to 12S target), 0.6 µL of 10 µM exo probe mix, 29.5 µL of the Primer Free Rehydration buffer and 9.4 µL H2O. The pre-reaction mix (41.5 µL) was then used to resuspend one lyophilized TwistAmp® exo reagents. The complete reaction mix was homogenised by pipetting. On each well of a 384 well-plate (Axygen), a drop of 3 µL sample (0.5 µL of purified embryo DNA + 2.5 µL H2O unless otherwise stated) was added to the upper part of the well. In the same well, another drop of 1.25 µL MgOAc (280 mM) was added onto the opposing side of the well’s wall followed by the addition of 20.7 µL of the complete reaction mix. Following a brief vortex and centrifugation, real time RPA reactions were performed at 37°C on the LightCycler480 (Roch, Meylan, France for 25 min with one acquisition of fluorescent signal every 30 second or at 25°C on the CFX Opus Real-Time PCR System for 50 min with an acquisition every 42 seconds. For each sexing reaction, water negative controls and positive controls (plasmid consist of target sequence) were added. Endpoint product analysis by agarose gels and capillary electrophoresis Amplification products were loaded on a 2% Gelgreen (Biotium, California USA) stained 1.5% agarose gel in 0.5X TAE buffer and separated by electrophoresis at 100 Volts for 30minutes. 1 µg of DNA ladder (GeneRuler 50 bp or GeneRuler 100 bp, Fisher Scientific, Illkirch France) served as the reference for the migration. Gels were revealed by Blue LED GelPicBox at 430nm (Nippongenetics, Düren Germany). Capillary electrophoresis (Capillary LifeSciences, France) controlled by the Labchip GX version 4.1.1619.0 SP1 software was performed using 10 µl of the amplification products. The time of the capillary electrophoresis results was normalized by a home-made routine code using two internal labchip markers, a lower maker at the beginning of the gel, and an upper maker at 1500 bp and 7000 bp for ladder 1K (for SWIM) and ladder 5k (for CHD and AC18), respectively. Data analysis Home-made python codes (version 3.10.4) were developed to automatize data analysis. Regarding the labchip data, each electropherogram profile had baseline removed prior time normalization using the two extreme markers (Fig. 1 A and 1 D). LAMP and RPA real-time data baseline was removed at the early time points. Scipy version 1.14 was used do statistical analysis, where Mann-Whitney U (scipy.stats.mannwhitneyu) with asymptotic method ( i.e. p-value calculated by comparing to normal distribution and hence correcting for ties) was used. All statistical calculations show the mean values ± standard error of the mean. Abbreviations LAMP: Loop-Mediated Isothermal amplification RPA: Recombinase Polymerase amplification PCR: Polymerase Chain Reaction qPCR: Quantitative Polymerase Chain Reaction CHD: Chromobox Helicase DNA-binding CHD-Z: Chromobox Helicase DNA-binding gene on chromosome Z CHD-W: Chromobox Helicase DNA-binding gene on chromosome W CHD-F: Female specific fragment of Chromobox Helicase DNA-binding gene on chromosome W AC18: DNA fragment situated on AC182258 locus 12S: 12S ribosomal RNA gene Declarations Ethics approval Not applicable. The Institutional Animal Care and Use Committee (IACUC), an Association of New England Medical Center and Tufts, as well as the National Institutes of Health, USA, mandated that a chick embryo that has not reached the 14th day of its gestation period would not experience pain and can therefore be used for experimentation without any ethical restrictions or prior protocol approval, simplifying the planning process. Data availability statement Data is provided within the manuscript and its supporting information files. Competing interests The authors declare that there are no competing interests. Funding The study was a part of SOO project funded by the France Agrimer Institution. Author contributions: Conceptualization: F.M, J.E and TNN.V. Methodology: TNN.V. Investigation: M.VDH, N.L’H, J.HC, P.M, T.M, T.G, J.H, J.E and TNN.V. Data analysis and visualization: M.VDH. Data curation: M.VDH and TNN.V. Validation: M.VDH and TNN.V. Funding acquisition: F.M. Writing – original draft: TNN.V. Writing & editing: M.VDH, J.E and TNN.V. Reviewing the final manuscript: M.VDH, N.L’H, J.HC, P.M, T.M, T.G, J.H, J.E, FM and TNN.V Acknowledgements The authors thank Louis Perrault and Saint Marcellin hatchery for the technical support during the study. We thank the France Agrimer Institution for funding project. The funders had no role in designing or directing the study and editing the manuscript. 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Recombinase Polymerase Amplification Coupled with a Photosensitization Colorimetric Assay for Fast Salmonella Spp. Testing. Anal. Chem. 2021 , 93 (16), 6559–6566. https://doi.org/10.1021/acs.analchem.1c00791. Additional Declarations No competing interests reported. Supplementary Files MarcVanDerHoftadtetalMinimaldataset.xlsx VanderHofstadtetalSupplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Mar, 2025 Reviews received at journal 08 Mar, 2025 Reviewers agreed at journal 14 Feb, 2025 Reviews received at journal 25 Jan, 2025 Reviewers agreed at journal 23 Jan, 2025 Reviewers invited by journal 16 Jan, 2025 Editor assigned by journal 16 Jan, 2025 Editor invited by journal 13 Jan, 2025 Submission checks completed at journal 10 Jan, 2025 First submitted to journal 06 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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09:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5772672/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5772672/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-13013-3","type":"published","date":"2025-07-29T16:38:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73641975,"identity":"52fe3bb7-9d2d-427d-85c0-1bb3684ffb33","added_by":"auto","created_at":"2025-01-13 08:15:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo novel \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein ovo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e PCR assays are capable of sexually discerning 82 chicken samples.\u003c/strong\u003e Capillary electrophoresis results for the CHD-F assay (\u003cstrong\u003eA-C\u003c/strong\u003e) showing \u003cstrong\u003e(A)\u003c/strong\u003e the superposition of the 82 electropherograms, \u003cstrong\u003e(B)\u003c/strong\u003e the samples containing only the 12S amplicon (male samples), or \u003cstrong\u003e(C)\u003c/strong\u003e the samples containing both 12S and CHD-F amplicons. AC18 results (\u003cstrong\u003eD-F\u003c/strong\u003e) done on the same sample set showing \u003cstrong\u003e(D)\u003c/strong\u003e the superposition of the 82 electropherograms, \u003cstrong\u003e(E)\u003c/strong\u003ethe samples containing only the 12S amplicon (male samples), or \u003cstrong\u003e(F)\u003c/strong\u003e the samples containing both 12S and AC18 amplicons. Orange depicts 12S amplicons, while green depicts sex specific target amplicons. In panels B, C, E and F the samples have been position by increasing order of normalized time of the 12S peak (and not by the reference order).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/13539c02e111e56408ea5022.png"},{"id":73641976,"identity":"ba74d383-5bb5-4d67-a65a-941bebf5335d","added_by":"auto","created_at":"2025-01-13 08:15:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of sex identification of the 82 chick embryo samples by the previously established SWIM/12S assay.\u003c/strong\u003eCapillary electrophoresis results for the SWIM/12S assay showing \u003cstrong\u003e(A)\u003c/strong\u003e the superposition of the 82 electropherograms, \u003cstrong\u003e(B)\u003c/strong\u003e the samples only containing the 12S amplicon (males), or \u003cstrong\u003e(C)\u003c/strong\u003e the samples containing both 12S and SWIM amplicons (females). Orange depicts 12S amplicons, while green depicts SWIM amplicons. In panels B and C the samples have been position by increasing order of normalized time of the 12S peak (and not by the reference order).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/d14a9f2b830cb71e44db4323.png"},{"id":73641982,"identity":"e22d4deb-f4f2-4c80-ba9d-efe1f0563186","added_by":"auto","created_at":"2025-01-13 08:15:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapid sex identification of chick embryos by the AC18 LAMP assay. (A)\u003c/strong\u003e Real-time fluorescent curves of AC18 LAMP assay for the 82 samples and the positive control (AC18 plasmid). Ct values (initiation of the exponential amplification) for the curves in panel A depending on the \u003cstrong\u003e(B)\u003c/strong\u003e reference name or \u003cstrong\u003e(C)\u003c/strong\u003e the extracted DNA concentration.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/a678db997088403b121a4868.png"},{"id":73641981,"identity":"abd2b5ee-f41e-43db-8a1a-5d1aba34b442","added_by":"auto","created_at":"2025-01-13 08:15:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":275076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRPA assays for sex identification of chick embryosat 37°C.\u003c/strong\u003e Real-time fluorescence of the CHD-F/12S assay for the simultaneous amplification of the \u003cstrong\u003e(A)\u003c/strong\u003e 12S locus and \u003cstrong\u003e(B)\u003c/strong\u003e CHD-F locus. \u003cstrong\u003e(C)\u003c/strong\u003eEnd-point fluorescence values of panel B. Real-time fluorescence of the AC18/12S assay for the simultaneous amplification of the \u003cstrong\u003e(D)\u003c/strong\u003e 12S locus and \u003cstrong\u003e(E)\u003c/strong\u003eAC18 locus. \u003cstrong\u003e(F)\u003c/strong\u003e End-point fluorescence values of panel E.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/9fdb3ef7a1abdcca3fc13ab6.png"},{"id":88268844,"identity":"23d41510-16fc-4167-8e06-fea2a072a15b","added_by":"auto","created_at":"2025-08-04 16:52:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/04aa74e8-0897-4719-a5bc-95652d5708a0.pdf"},{"id":73641979,"identity":"d3c00654-3b2a-4aef-b0dd-181ae02793f7","added_by":"auto","created_at":"2025-01-13 08:15:14","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":305056,"visible":true,"origin":"","legend":"","description":"","filename":"MarcVanDerHoftadtetalMinimaldataset.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/5803cd82f7e8a461e674adb4.xlsx"},{"id":73642354,"identity":"9ae3be4e-fa1c-4949-8625-0035e252f941","added_by":"auto","created_at":"2025-01-13 08:23:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4272155,"visible":true,"origin":"","legend":"","description":"","filename":"VanderHofstadtetalSupplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5772672/v1/4d9df0b39dd8bf38aa672af7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing","fulltext":[{"header":"Background","content":"\u003cp\u003eLarge-scale chicken egg production has come under increasing scrutiny in several European and North American countries due to economic and ethical concerns. Efforts to address these challenges have focused on various aspects of the production process, such as extending the laying period of hens to reduce replacement rates or transitioning to organic and non-cage farming systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Yet, one-day-hatched culling of male chicks remains a critical issue. To mitigate this concern, two main approaches have been envisioned: (i) dual-propose strains where male chicks are raised for meat production [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; and (ii) egg sexing technologies [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] to identify and eliminate male embryos at early developmental stages. However, despite the intensive effort, consumer acceptance of meat from such breeds still remains limited, while existing egg sexing techniques are often complex, time consuming and require advanced infrastructure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Up to date, only five sexing techniques are being successfully commercialized [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], leaving the need for novel ones to be developed.\u003c/p\u003e \u003cp\u003eUp to date, to establish the sex of the embryo before hatching (\u003cem\u003ein ovo\u003c/em\u003e), five major technological classifications can be done depending on their biomarker or methodology: 1) volatile organic compounds, 2) morphological inspection, 3) acoustic-based methods, 4) spectral-based methods, and 5) molecular approaches. Among them, molecular-based techniques have drawn significant attention due to their higher accuracy and lower susceptibility to biological variability. Indeed, these assays are PCR-based amplifying sex-specific DNA fragments present either in their mutual chromosome (Z) and/or in the female-specific chromosome (W) of chicks [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The first PCR-based sexing assay, developed in 1998 used the chromobox-helicase-DNA-binding (CHD) gene situated on both sexual chromosome Z (CHD-Z) and W (CHD-W) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. CHD-Z and CHD-W share common sequences at the two extremities of the gene, whilst reserve a female-specific fragment in the middle of the gene. Therefore, CHD sequences have been used to discriminate males consisting of ZZ chromosomes (CHD-Z only, smaller amplicon size) and females consisting of ZW chromosomes (CHD-Z \u0026amp; CHD-W, larger amplicon size) in different avian species with only one mutual primer pair [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Over time, advancements such as new gene targets [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], multiplexing [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and high-throughput real-time PCR [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] have improved both speed and accuracy of molecular based tests. However, analytical success of these tests still depends on amplification techniques requiring sophisticated instrumental facilities (thermocyclers, quantitative thermocyclers, and gel/capillary electrophoresis), limiting their scalability and practical implementation in poultry production.\u003c/p\u003e \u003cp\u003eTo address these limitations, a myriad of isothermal DNA amplification techniques have emerged in the recent years proposing solution for reducing the need of sophisticated instrumental facilities. Their main attractiveness lays on their constant and lower working temperature (37\u0026ndash;65\u0026deg;C) compared to repetitive thermocycles of the conventional PCR. Among these, LAMP has gained prominence in the molecular diagnostic field and start to outcompete the conventional PCR technique [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In particular, LAMP technique has been endorsed due it its high sensitivity and rapid results (less than 30 minutes) thanks to the employment of multiple primer sets (three sets) and the Bst DNA polymerase with a high strand displacement activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. More importantly, LAMP is compatible with multiple detection modes ranging from conventional gel electrophoresis to fluorescent or naked eye readout (turbidity or colorimetry). Specifically, naked-eye observation together with the digital response of LAMP reactions (Yes \u003cem\u003eVs.\u003c/em\u003e No readouts) have enabled the establishment of point-of-care diagnostic tools for onsite detection of infectious diseases such as malaria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], dengue [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] or COVID-19 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, RPA technique, works at significantly lower temperature (37\u0026ndash;42\u0026deg;C) thanks to the employment of DNA recombinase, proposes fast readout and practical visualization methods for the amplified products [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Importantly, as RPA reactions operate at low temperatures, which is compatible with egg incubation condition, hence a promising approach for onsite sexing within the poultry production setting.\u003c/p\u003e \u003cp\u003eIn our previous work [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], we developed a duplex PCR and a quantitative PCR (qPCR) assays using SWIM/12S and DMRT/Xho-I markers, respectively, to enable specific, rapid and robust sex determination of 9 day-old chicken embryo. In this work, we adopted LAMP and RPA techniques to develop two novel assays based on two newly designed sex-specific DNA markers. First, we identified two new sex-specific DNA fragments, one on the well stablished CHD gene (CHD-W), and a second on the AC182258 locus (fragment AC18), which are both located solely in the female chromosome. Second, we showed that fluorescent LAMP can detect AC18 fragment to discriminate chick sex in under 20 minutes at constant temperature (65\u0026ordm;C). This approach is compatible with naked-eye colorimetric detection and larger-scale screening. Finally, we demonstrate that RPA can be used to determine chick sex at 37\u0026ordm;C in under 30 minutes, reducing significantly both the complexity and the costs of the assay. Additionally, we show that our methods eliminate the need for DNA input normalization, further simplifying the experimental workflow and enhancing feasibility for large-scale implementation in the poultry industry.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTwo novel sexing assays for chicken embryos: PCR validation\u003c/p\u003e \u003cp\u003eFor the design of the two new sexing assays, we focused on two different genomic locus CHD and AC182258 of the female W chromosome and screened for primers amplifying DNA fragments ranging from 180 to 220 bp (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Of note, for CHD gene, contrary to previous CHD based PCR sexing that focus on size discrimination of the CHD locus between the CHD-Z and CHD-W, we targeted the region of the CHD-W (female) that is not present in the CHD-Z (male). Different combinations of PCR primers were assessed for amplification of solely female specific fragments (here defined as CHD-F and AC18). For the major part of the CHD-F (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and AC18 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) primer pairs, a clear single band for females versus males was visualized on agarose gel. Nonetheless, AC18 design was less efficient compared to CHD-F, since several combinations of PCR primers were prone to false positives (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFollowing the selection of the best candidate for both assays (F3R1 for CHD-F; F2R1 for AC18, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), we tested their performance on 82 samples of 9-day- old chick embryos. To this aim, genomic DNA from dissected brain tissue of chick embryos was extracted and purified [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. DNA quantification from 82 ISA Brown eggs revealed a variability in DNA concentrations, ranging from 1 ng/\u0026micro;L up to 226 ng/\u0026micro;L (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Although we observed a normally distributed sample (p-value 0.08, Kolmogorov-Smirnov test of normality), we noticed that ~\u0026thinsp;21% of the samples were significantly low in DNA concentration (\u0026lt;\u0026thinsp;10 ng/\u0026micro;L). Nevertheless, to assess the robustness of the two assays, no DNA concentration normalization step was performed in the subsequent experiments. For both CHD-F and AC18 assays, in addition to the female specific target, the internal control (12S primers) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] was added to the test (CHD-F/12S and AC18/12S, respectively).\u003c/p\u003e \u003cp\u003eFirst, PCR amplification for CHD-F/12S using chosen primer pair (F3R1, table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was performed. Cappilary electrophoresis (Labchip) analysis of PCR products discriminated two groups by the number of amplicons. The first group (40 samples) contained only the 12S band with an amplicon size of 139.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026amp;B). The second group (42 samples) presented two bands, the 12S band (142.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 bp) in addition to a second band of 179.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 bp, which corresponded to the predicted band size of the CHD-F target (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026amp;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt this stage, we could predict that the group 1 classifies male samples while the group 2 are the female ones. For both amplicons 12S (Figure S3A) and CHD-F (Figure S3B), no correlation between concentrations of the extracted DNA and the amplified PCR products measured by Labchip was observed. However, there was a significant difference in the concentration of 12S amplicon between the two groups (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01), in which the predicted female group presents a lower concentrations (Figure S3A). Low concentration amplicon was noticed in one sample (F2) (Figure S3A) for 12S and in three samples (F1, F2 and F3) for CHD-F (Figure S3B).\u003c/p\u003e \u003cp\u003eSecond, PCR amplification for AC18/12S using the chosen primer pair (F2R1, table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was performed on the same extracted DNA sample set. Similar to CHD-F assay, analysis of PCR product also revealed two distinct groups, the first group with 40 samples containing one amplicon of size 139.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 bp (12S amplicon) and a second group of 42 samples containing two amplicons with sizes 142.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 bp and 229.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 bp, for 12S amplicon and AC18, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). As expected, sample classification by the AC18 assay was exactly the same as the one performed by the CHD-F assay, demonstrating a 100% concordance (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Again, we observed no correlation between the extracted DNA concentration and the amplified product for both 12S and AC18 amplicons. In the same line, the second group also presented a significantly lower concentration for the 12S amplicon (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the first group, in which the sample F2 outstood as significantly low 12S concentration (Figure S4A). For AC18 amplicon, we again noticed the low amplified concentration (up to 43-fold) for the three samples F1, F2 and F3 (Figure S4B).\u003c/p\u003e \u003cp\u003eTo confirm the sex identity of the two groups detected by the two novel assays, we analyzed this extracted DNA sample set by our previously established SWIM/12S assay [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As expected, PCR product analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) confirmed the correct sex identification by our two new assays for all the 82 samples consisting of 40 males amplifying only the 12S amplicon (146.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 bp, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and 42 females amplifying both 12S and SWIM amplicons, 148.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 bp and 231.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 bp, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Once again, we observed that there was no correlation between the extracted DNA concentration and the amplified concentration of neither 12S nor SWIM amplicons (Figure S5). Differs from 12S/CHD-F and 12S/AC18 assays, SWIM/12S assay is not suffered from low amplification rate of 12S amplicon in the duplex PCR configuration. Indeed, there was no significant difference in concentration of PCR products between the 12S and SWIM (p-value\u0026thinsp;=\u0026thinsp;0.18). However, we did observe that 12S concentration of F2 sample was 2-fold lower, while, SWIM amplicon was up to 6-fold smaller in the three samples F1, F2 and F3 (Figure S5). Furthermore, the sex validation by SWIM allowed us to discern that female samples had significantly higher extracted DNA concentrations (84.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 ng/\u0026micro;L) than male samples (57.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 ng/\u0026micro;L) (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLoop-Mediated Isothermal Amplification (LAMP) assay for rapid sexing of chick embryos\u003c/p\u003e \u003cp\u003eAiming to make our assays more compatible with field application, we adapted the two novel PCR sexing assays to LAMP whose amplification is isothermal. As such, different LAMP primer combinations (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed and validated by endpoint colorimetric LAMP (Figure S6) for their female specific detection. Results showed that designs for both target candidates CHD-F and AC18 allowed sexing by simple naked-eye colorimetry (Assay V for CDH-F; assay D and assay G for AC18). Primer combination for assay D of AC18 was then chosen for large scale analysis on the previously assessed 82 chick embryo samples using fluorescent LAMP. In particular, the obtained data revealed that the AC18 assay is highly robust since it correctly discerned the sex of the 82 samples except for the sample F1 (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In particular, the initiation of the exponential amplification (Ct value) for the female samples was constant (13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 min), except for samples F2 and F3 which were significantly higher (19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Although we observed heterogeneous fluorescent endpoints and small differences in the Ct values, the concentration of the extracted DNA displayed, once again, no correlation with Ct values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Regarding the negative controls and the male samples, we did not observe any amplification under 40 minute reaction. The AC18 LAMP assay delivers a digital response with either the presence or the absence of the female specific chromosome W.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile digital responses simplify data interpretation, the absence of internal controls does not allow technical verification for enzymatic activity of the assay. Since the synthetic plasmid containing the AC18 amplicon displayed a high tau value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), we decided to use this plasmid as the internal control for the enzymatic activity of the LAMP reaction. To this aim, we performed a spike-in experiment in which a range of different concentrations of AC18 plasmid was added to a random male (M1) and female (F37) sample prior to amplification. As expected, obtained results (Figure S7) show that increasing the concentration of spiked AC18 plasmid led to increasing detection signal of the non-template negative control and the male sample with the Ct value down to 27.1 at 10 ng/\u0026micro;L. On the contrary, the spike-in plasmid but did not affect the Ct value of the female sample, leaving an elevated ΔCt of 17.3 (Male vs Female) which clearly distinguish between the female and male samples.\u003c/p\u003e \u003cp\u003eRecombinase Polymerase Amplification (RPA) assays for \u003cem\u003ein ovo\u003c/em\u003e sexing of chick embryos at low temperatures:\u003c/p\u003e \u003cp\u003eTo further minimize the need for advanced research facilities and costs, we extended the CHD-F and AC18 novel sexing assays to RPA whose reaction occurs at 37\u0026deg;C. Firstly, different RPA primer pairs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed and validated using endpoint RPA assay (Figure S8). We noticed that efficiencies of the best primer pairs to detect the presence of their targets (Pair 5 for CHD-F and Pair 27 for AC18), were equal to their PCR variant assays (Figure S8). These primers were validated by large scale (82 samples) sex identification using fluorescent RPA-exo assay. 12S primers (detected in both male and female) were added as and detected simultaneously with the female specific primers in a duplex configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCHD-F/12S fluorescent assay showed exponential amplification of the 12S target in all 82 samples. Moreover, end-point signals for 12S amplification revealed no significant difference (p-value\u0026thinsp;=\u0026thinsp;0.10) between female and male chicks, nor a correlation to the extracted DNA concentration (Figure S9A). On the other hand, exponential amplification of the CHD-F locus was only observed in female chicks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), with the exception of samples F1, F2 and F3 where signal was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Again, no correlation was found between the extracted DNA concentration and the end-point signal for the CHD-F locus (Figure S9B). Similarly, AC18/12S assay results were identical to CHD-F/12S assay. 12S locus was detected in all 82 samples, whose signals were dependent from either DNA concentration or sex identity of the analyzed samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and S9C). Likewise, all female samples were detected by the exponential amplification of the AC18 locus whilst all male samples were negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). End-point fluorescence values of AC18 amplification revealed that the average signal of female samples (4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 RFU) was 18-fold higher in intensity compared to male samples (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 RFU) enabling the introduction of a sexing cut-off value at 0.56 RFU (even if signals from the three samples F1, F2 and F3 were still significantly lower than the average female sample) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Once again, no correlation was observed between the extracted DNA concentration and the end-point fluorescence of the AC18 locus amplification (Figure S9D). To estimate the impact of the DNA concentration RPA assay, we performed reactions using different volumes of input DNA from highly concentrated samples including M4 (204.7 ng/\u0026micro;L) and F17 (214.3 ng/\u0026micro;L) compared to one female sample (F12) whose DNA concentration was significantly lower (16.5 ng/\u0026micro;L). Fluorescent measurement reveled identical amplification curves for both locus samples M4 and F12 at different volumes of DNA input. However, x significantly impairs in RPA amplification curves for both locus were observed for F17 sample (Figure S10). While this impairment is stronger on the AC18 locus than on the 12S locus, the obtained results indicate that the AC18 assays is specific from at least 0.13 ng/\u0026micro;L up to at least 25.7 ng/\u0026micro;L final DNA concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we assessed the possibility to decrease the temperature down to room temperature (25\u0026deg;C) for the AC18/12S assay (Figure S11). As expected, a longer incubation time (50 minutes) was required to reach the plateau signals for both 12S and AC18 in RPA amplifications. However, at 25\u0026deg;C the AC18/12S assay was not capable to correctly detect samples F1, F2 and F3. Nevertheless, out of these three samples, the other female samples were correctly discriminated (with signals were ~\u0026thinsp;3-fold higher than in male samples). These results, hence demonstrate that the AC18 assay is capable of significantly discriminating (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) the sex of chick samples at 25\u0026deg;C.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReducing the culling of one-day hatched male chicks is still a major socioeconomical burden for the poultry industry. To reduce the culling, many \u003cem\u003ein ovo\u003c/em\u003e molecular sexing procedures have evolved [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, their adoption remains limited due to their complexity, reliance on the advance infrastructure, and high temporal and financial costs. We designed two novel digital response assays and adapted them to LAMP isothermal amplification for its rapidity and RPA for its room temperature amplification compatibility. Moreover, these assays do not require DNA concentration normalization, hence simplifying the sexing process.\u003c/p\u003e \u003cp\u003eConventional molecular detection assays often require DNA normalization step to ensure a measurable output [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our results show that up to a 226-fold difference in concentration of extracted DNA can be obtained (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) from 82 samples, which could be related to different factors such as quantities of input samples, variations in experimental handling or sample conservation. Here, to demonstrate the robustness of our assay, we skipped the normalization step and fix a constant volume of DNA samples added in the assay (and not the concentration of input DNA). Despite of a large concentration range of input DNA (from 3 ng up to 678 ng), the 12S internal control and the two female markers (CHD-D and AC18) were correctly amplified in all samples, demonstrating the robustness of the PCR assays. Furthermore, the fact that the amplicon intensity of all targets are independent of DNA input (Figure S3-S5) indicates that all PCR amplifications had reached their plateau. We previously showed that 1 ng of input DNA was sufficient to achieve PCR saturation in under 30 cycles for female samples and 40 cycles for male samples [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further decrease the complexity of molecular \u003cem\u003ein ovo\u003c/em\u003e sexing assays, we decided to implement digital (yes \u003cem\u003eVs.\u003c/em\u003e no) responses compared to analogue (range of values) responses. This transition is crucial since it reduces the expertise required for the user to interpret the results, as well as the instrumentation requirements. For example, conventional CHD-based PCR sexing assays exploit the size difference between CHD locus present in both the female and male chromosomes (8\u0026ndash;12). This size difference can vary between 11 bp up to 165/179 bp, hence would require a corresponding resolution technique and expertise for their downstream analysis. On the contrary, here we demonstrate that the presence or the absence of the amplicon, either CHD-F or AC18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), is sufficient to discriminate the sex of the embryo sample (note that the 12S amplicon is only an internal control that is not required for sex discrimination). In particular, we observed that for assays CHD-F/12S and AC18/12S, female samples had lower 12S concentrations than male samples, most likely due to the simultaneous reaction of the duplex PCRs, which might reduce the efficiency of both. For this reason, we predict that in the absence of the 12S internal control, a greater CHD-F and AC18 amplification would occur, further simplifying sex discrimination procedures. Collectively, these findings demonstrate that our two novel PCR assays enable highly robust detection, achieving 100% accurate sex identification without requiring DNA input normalization. This breakthrough significantly enhances the efficiency and scalability for large-scale analyses in industrial applications\u003c/p\u003e \u003cp\u003eTo streamline \u003cem\u003ein ovo\u003c/em\u003e sexing techniques and align with the high yield of egg production, rapid and high throughput approaches are crucial [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To this aim, we implemented LAMP for chick embryo sexing achieving results in less than 20 minutes with binary (digital) readouts. Colorimetric LAMP results (Figure S6) enable sex determination by visual inspection with minimal equipment (only a 65\u0026deg;C incubator) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), hence allowing the development of portable devices [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, colorimetric assays are limited by their reliance on manual verification, constraining automation for high throughput analysis. To overcome this obstacle, we adapted the LAMP colorimetric to a fluorescence-based version allowing real-time, high-through put measurements with a small reaction volume (12.5 \u0026micro;L). Using AC18 fluorescent LAMP assay, 81 out of 82 samples were accurately sexed based on the presence (female) or the absence (male) of exponential amplifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, while real-time fluorescence LAMP is generally used for quantitative analysis, its continuous amplification process is highly prone to stochastic variations at low template concentration leading to digital-like rather than quantitative output [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given that the target locus concentration is independent of the sex phenotype (\u003cem\u003ei.e.\u003c/em\u003e the female chromosome is present or not), fluorescence LAMP assay does not require precise quantitative resolution for sex determination. To meet the stringent requirements of industrial applications, we established that the addition of 0.5 ng/\u0026micro;L of AC18 plasmid serves as an effective internal control, ensuring reliable verification of enzymatic activity in the LAMP reaction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eComparative analysis of the three molecular tools used in developed assays for sex identification of chick embryos.\u003c/b\u003e Power consumption has been measured at the maximum temperature required by the assay.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLAMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRPA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDetection method\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLabchip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSybr green /colorimetry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSteps required\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTime (minutes)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u0026thinsp;+\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMachines required\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermocycler\u003c/p\u003e \u003cp\u003e\u0026amp;\u003c/p\u003e \u003cp\u003eLabchip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncubator (65\u0026ordm;C)\u003c/p\u003e \u003cp\u003e\u0026amp;\u003c/p\u003e \u003cp\u003efluorometer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIncubator (37\u0026ordm;C)\u003c/p\u003e \u003cp\u003e\u0026amp;\u003c/p\u003e \u003cp\u003efluorometer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePower consumption (W)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e240 (94\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150 (65\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e110 (37\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstimated consumable cost per reaction (\u0026euro;)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAccuracy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition to high accuracy and simple implementation, cost effectiveness is crucial factor for a sexing assay to be industrialized given its significant economic implication. Although we calculated that the consumable cost per reaction not to be different between PCR and LAMP assays, we estimated a 38% reduction for the incubator power consumption for LAMP assay (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (without taking into consideration the surplus energy required for the labchip analysis). Even more cost effective, our RPA sexing assay cost (at 37\u0026deg;C) is reduced by 20% with 58% less in power consumption compared to PCR assays. RPA results revealed that the CHD-F assay clearly assesses sex status with a sensitivity of 96% (79 out of 82 sample) and a 100% specificity. Endpoint AC18 assay displays a 100% sensitivity and specificity with a clear established cut-off (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). It is worth noting that for RPA assays can also produce a digital readout (\u003cem\u003ei.e.\u003c/em\u003e amplification has occurred or not). However, due to the irregular shape of amplification curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), commercial software struggles to accurately detect the onset of exponential amplification using the second derivative method. As such, a thresholding approach is preferable. We noticed that in female samples, the final fluorescence intensity decreased as the input DNA concentration increased (Figure S10), likely due to an imbalance between the target and primer concentration. However, beyond a certain input threshold, fluorescence level stabilized and remains sufficiently high for reliable sex phenotype identification. This highlights the assay robustness even in the presence of elevated DNA inputs. Lastly, to further explore the potential of onside applications, we tested the AC18/12S RPA-exo assay at room temperature. A slight slot in robustness observed of the assay, as samples F1-F3 failed to achieve exponential amplification within 50 minute. This outcome aligns with the low amplification efficiency already observed at 37\u0026deg;C for these samples, as the reduction in RPA at lower temperature [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] likely exacerbates this limitation. While other female samples also experience a decrease in performance (with the fold change between female and males samples dropping from 18 to 3-fold), all were still correctly identified, demonstrating the assay\u0026rsquo;s resilience over temperature-induced impairment.\u003c/p\u003e \u003cp\u003eThroughout the study, three samples (F1, F2 and F3) consistently exhibited significantly lower PCR amplification efficiencies for the target locus (Figure S3-S5), with distinct behaviour observed among them. Sample F2, on the one hand, consistently showed the lowest 12S amplification. While it was the female sample with the lowest total extracted DNA concentration, 12 other male samples with even lower total extracted DNA concentrations displayed higher 12S amplicon intensity during simplex amplification, likely due to the absence of competing female targets. This suggest a potential partial impairment of both targets in a duplex amplification configuration. On the other hand, samples F1 and F3 displayed normal 12S amplifications but with a reduced amplification of the female specific targets. The hypothesis that low DNA input might hinder target amplification was ruled out, as F1 (10.3 ng/\u0026micro;L) and F3 (18.3ng/\u0026micro;L) exhibited higher DNA concentrations compared to other female samples (e.g., F39\u0026thinsp;=\u0026thinsp;8.1 ng/\u0026micro;L, F12\u0026thinsp;=\u0026thinsp;16.5 ng/\u0026micro;L and F10\u0026thinsp;=\u0026thinsp;18.1 ng/\u0026micro;L) that achieved robust amplification of female specific targets. These findings suggest variability in the copy number of different targets across samples, significantly impacting PCR results while total extracted DNA concentration is relatively low. Despite these challenges, the PCR assays successfully determined the female phenotype for all these three samples, demonstrating their robustness for chick embryo sexing. Interestingly, while sample F2 showed the lowest amplification in PCR assays, sample F1 was the least efficiently amplified in RPA and failed amplification in LAMP assay. For samples F2 and F3, although lower amplification efficiencies were observed, sex discrimination remained feasible using AC18 LAMP and RPA assays.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we have established two innovative molecular assays capable of determining the sex of 9 day-old chicken embryos with straightforward \u0026ldquo;Yes\u0026rdquo; or \u003cem\u003e\u0026ldquo;N\u003c/em\u003eo\u0026rdquo; readout. Initially, we used conventional PCR amplification to validate their efficiency sex identification. Subsequently, we employed LAMP amplification, which significantly reduce experimental time to less than 20 minutes under isothermal incubation at 65\u0026deg;C. Finally, we demonstrated the accurate sexing of all samples by RPA amplification at lower temperature (37\u0026deg;C), offering a significant reduction in both experimental complexity and economic cost. We envision that these two novel assays (CHD-F and AC18) combined with the adapted amplification techniques, present transformative potential for the poultry industry across different production scales. The rapid and high throughput nature of LAMP assays enable efficient and user friendly large-scale \u003cem\u003ein ovo\u003c/em\u003e sexing. Meanwhile, the lower temperature requirement for RPA assays coupled with with the possibility of colorimetric readouts pave the way for simple, cost-effective and compatible with an onsite solution for chick sexing [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Overall, these simple, faster and more affordable novel molecular assays promise to facilitate the automation and commercialization of chick sexing processes. This advancement represents a significant step forward in improving the economic, ethical and environmental sustainability print of the poultry industry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eEggs incubation\u003c/p\u003e \u003cp\u003eFertilized ISA Brown eggs were purchased from a commercial supplier (SFPA, Saint-Marcellin France), stocked at 19˚C for a maximum of 10 days after laying and before incubation. Eggs were incubated in a dedicated incubator (Masson, Soyans France) at 37.5˚C, 55% humidity and tilted every hour. Incubation was arrested at day 9.\u003c/p\u003e \u003cp\u003eSample collection and lysis\u003c/p\u003e \u003cp\u003e10\u0026ndash;20 mg of day 9 post-hatching embryo brain tissue from 82 ISA Brown eggs was recovered and treated as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In brief, collected samples were lysed for 3H at 55\u0026deg;C in 150 \u0026micro;l of lysis buffer containing 10% of chelating beads (Chelex 100 Biorad), 0.2% SDS, 10mM Tris-HCl pH8, 0.2 \u0026micro;g/\u0026micro;l Proteinase K followed bya denaturation step at 95\u0026deg;C for 15 min. Supernatant was recovered by centrifugation for 5 minutes at 13000g at room temperature and submitted to DNA purification using QIAamp DNA mini kit (Qiagen 51306). DNA quantification was performed post-purification using Nanodrop One (Thermo Scientific, Wilmington USA). All samples were stored at -80˚C until used.\u003c/p\u003e \u003cp\u003ePrimer design\u003c/p\u003e \u003cp\u003ePrimers for all sex determination assays were designed based on the two female specific sequences located on the female-specific W chromosome: CHD-W (Chromobox-Helicase-DNA-binding, GenBank Accession No GU132944.1) and AC182258 locus (GenBank Accession No AC182258.2). Blasting between CHD-W and CHD-Z was performed to identify specific female fragments (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The un-matched fragment situated from position 151\u0026ndash;326 of the CHD gene would allow female identification as it is present only on W chromosome, hence primers were designed based on this identified fragment (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Regarding the locus AC182258, we used a fragment situated on the W chromosome from position 6942\u0026ndash;7267 for designing the AC18 primers with various amplicon lengths (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Primers for the internal control were designed based on the 12S ribosomal RNA gene (Accession No MF041985.1).\u003c/p\u003e \u003cp\u003eBIP, FIP and loop primers for the LAMP assay were designed using PrimerExplorer V5. CHD-W and AC18 primers had been consciously designed to be compatible with the RPA, following TwistDx guidelines regarding primer and amplicon length. For real time measurement of RPA amplification assisted by exonuclease, corresponding hydrolyse probes for each assay were designed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) following the instruction of TwistDx. Briefly, RPA probes contain a long amplicon specific sequence (about 45 bp) on to which a fluorophore (FAM or TAMRA) and a quencher (BHQ-1) were incorporated. A basic nucleotide analogue (tetrahydrofuran, THF) residue was introduced in between the fluorophore and the quencher as the recognition site for the exonuclease, whose activity will degrade the probe and separate the fluorophore from the quencher.\u003c/p\u003e \u003cp\u003ePCR amplification and analysis\u003c/p\u003e \u003cp\u003ePCR reaction mix was prepared in 96 well-plate using 5 \u0026micro;L of 2X Master Mix (Invitrogen Platinum Green Hot Start PCR) and 200 nM of primer set SWIM/12S, CHD-W/12S or AC18/12S at a ratio 2:1. 3 \u0026micro;L (constant volume) of purified embryo DNA was added into each well. Amplification was performed on peqSTAR 96X thermocycler (Ozyme, Montigny-le-Bretonneux France) as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In brief, one initial denaturation at 94˚C for 2 minutes followed by 35 cycles comprising of: 1) 30 sec. at 94˚C for the denaturation, 2) 30 sec at 55˚C for the annealing, and 3) 30 sec at 72˚C for the elongation. A final extension cycle at 72˚C for 5 minutes was performed. Negative Control (Non \u0026ndash; template) were performed for each experiment. Sex determination was performed using two reference PCR assays SMIW/12S [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and CHD 2250F/2718R [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]). All samples were analysed by two novel PCR assays AC18 and CHD-W/12S. Negative (non-template) and positive controls (synthetic plasmid containing the target amplicon, purchased from IDT) were added for each isothermal reaction. Real-time qPCR thermal cycling reactions were performed by the LightCycler 480 (Roche, Meylan France) directed by the LightCycler 480 Software (version 1.5.1.62).\u003c/p\u003e \u003cp\u003eLAMP sexing assays\u003c/p\u003e \u003cp\u003eReaction mix for fluorescent LAMP assays were prepared on ice using WarmStart LAMP Kit (NEB, E1700) and NEB LAMP fluorescent dye (NEB B1700S). A premix primer solution containing 16 \u0026micro;M of FIP and BIP primers and 2 \u0026micro;M of AC18 primers was prepared prior to the reaction mix preparation. For one reaction, 1.25 \u0026micro;L of the primer mix solution was added to 6.25 \u0026micro;L of 2X NEB LAMP Master Mix, 0.125 \u0026micro;L of 50X NEB LAMP dye and 1.875 of H2O. The reaction mix was distributed on a 348 well-plate (Axygen) followed by the addition of 3 \u0026micro;L extracted DNA sample. LAMP amplification was carried out at 65\u0026deg;C for 40 min using the LightCycler480 (Roch, Meylan, France) with one fluorescent acquisition every 30 second. For each experiment, negative control (water) and positive control (plasmid) were performed. The samples were assessed in triplicate. For colorimetric LAMP assessment, the WarmStart colorimetric LAMP master mix (M1800S) was used, following the recommended protocol (M1800).\u003c/p\u003e \u003cp\u003eRPA sexing assays\u003c/p\u003e \u003cp\u003eEndpoint RPA amplification reactions were performed as recommended by the manufacture manual (TwistAmp Liquid basic RPA kit, Ref TALQBAS01). In brief, a pre-master mix for each reaction was prepared on ice with 25 \u0026micro;L of 2X buffer, 5 \u0026micro;L 10X E-mix, and 9.2 \u0026micro;L H2O containing 1.8 mM of total dNTPs following by vortexing and a brief spin. 2.5 \u0026micro;L of core reaction mix was then added to the lid of the tube containing the pre-master mix. The 41.7 \u0026micro;L of complete master mix was homogenised by 10 full inversions of the tube, followed by a brief spin. In another tube, on the lid, three reagents were added separately in its different corners including 2.4 \u0026micro;L of 10 \u0026micro;M of each primer, 2.5 \u0026micro;L of MgOAc and 1 \u0026micro;L template DNA. The reaction preparation was finally by the addition of 41.7 \u0026micro;L master mix at the bottom of the tube. RPA reaction was activated by spinning in all the materials from the lid. Amplification was carried out at 37\u0026deg;C for 30 min on a thermoblock (Eppendorf).\u003c/p\u003e \u003cp\u003eReal-time RPA amplification reactions were performed at 37\u0026deg;C for 30 min as described in the manufacture\u0026rsquo;s manual instruction (TwistAmp\u0026trade; exo Kit, Ref TAEXO02KIT) with a smaller volume adjusted to use the Roche LightCycler 480. Briefly, a pre-reaction mix for each reaction was prepared on ice with 2 \u0026micro;L of the primer mix at 10 \u0026micro;M (ratio 2:1 of sexing target to 12S target), 0.6 \u0026micro;L of 10 \u0026micro;M exo probe mix, 29.5 \u0026micro;L of the Primer Free Rehydration buffer and 9.4 \u0026micro;L H2O. The pre-reaction mix (41.5 \u0026micro;L) was then used to resuspend one lyophilized TwistAmp\u0026reg; exo reagents. The complete reaction mix was homogenised by pipetting. On each well of a 384 well-plate (Axygen), a drop of 3 \u0026micro;L sample (0.5 \u0026micro;L of purified embryo DNA\u0026thinsp;+\u0026thinsp;2.5 \u0026micro;L H2O unless otherwise stated) was added to the upper part of the well. In the same well, another drop of 1.25 \u0026micro;L MgOAc (280 mM) was added onto the opposing side of the well\u0026rsquo;s wall followed by the addition of 20.7 \u0026micro;L of the complete reaction mix. Following a brief vortex and centrifugation, real time RPA reactions were performed at 37\u0026deg;C on the LightCycler480 (Roch, Meylan, France for 25 min with one acquisition of fluorescent signal every 30 second or at 25\u0026deg;C on the CFX Opus Real-Time PCR System for 50 min with an acquisition every 42 seconds. For each sexing reaction, water negative controls and positive controls (plasmid consist of target sequence) were added.\u003c/p\u003e \u003cp\u003eEndpoint product analysis by agarose gels and capillary electrophoresis\u003c/p\u003e \u003cp\u003eAmplification products were loaded on a 2% Gelgreen (Biotium, California USA) stained 1.5% agarose gel in 0.5X TAE buffer and separated by electrophoresis at 100 Volts for 30minutes. 1 \u0026micro;g of DNA ladder (GeneRuler 50 bp or GeneRuler 100 bp, Fisher Scientific, Illkirch France) served as the reference for the migration. Gels were revealed by Blue LED GelPicBox at 430nm (Nippongenetics, D\u0026uuml;ren Germany).\u003c/p\u003e \u003cp\u003eCapillary electrophoresis (Capillary LifeSciences, France) controlled by the Labchip GX version 4.1.1619.0 SP1 software was performed using 10 \u0026micro;l of the amplification products. The time of the capillary electrophoresis results was normalized by a home-made routine code using two internal labchip markers, a lower maker at the beginning of the gel, and an upper maker at 1500 bp and 7000 bp for ladder 1K (for SWIM) and ladder 5k (for CHD and AC18), respectively.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eHome-made python codes (version 3.10.4) were developed to automatize data analysis. Regarding the labchip data, each electropherogram profile had baseline removed prior time normalization using the two extreme markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). LAMP and RPA real-time data baseline was removed at the early time points. Scipy version 1.14 was used do statistical analysis, where Mann-Whitney U (scipy.stats.mannwhitneyu) with asymptotic method (\u003cem\u003ei.e.\u003c/em\u003e p-value calculated by comparing to normal distribution and hence correcting for ties) was used. All statistical calculations show the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLAMP: Loop-Mediated Isothermal amplification\u003c/p\u003e\n\u003cp\u003eRPA: Recombinase Polymerase amplification\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eqPCR: Quantitative Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eCHD: Chromobox Helicase DNA-binding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCHD-Z: Chromobox Helicase DNA-binding gene on chromosome Z\u003c/p\u003e\n\u003cp\u003eCHD-W: Chromobox Helicase DNA-binding gene on chromosome W\u003c/p\u003e\n\u003cp\u003eCHD-F: Female specific fragment of Chromobox Helicase DNA-binding gene on chromosome W\u003c/p\u003e\n\u003cp\u003eAC18: DNA fragment situated on AC182258 locus\u003c/p\u003e\n\u003cp\u003e12S: 12S ribosomal RNA gene\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. The Institutional Animal Care and Use Committee (IACUC), an Association of New England Medical Center and Tufts, as well as the National Institutes of Health, USA, mandated that a chick embryo that has not reached the 14th day of its gestation period would not experience pain and can therefore be used for experimentation without any ethical restrictions or prior protocol approval, simplifying the planning process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript and its supporting information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was a part of SOO project funded by the France Agrimer Institution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: F.M, J.E and TNN.V. Methodology: TNN.V. Investigation: M.VDH, N.L\u0026rsquo;H, J.HC, P.M, T.M, T.G, J.H, J.E and TNN.V. Data analysis and visualization: M.VDH. Data curation: M.VDH and TNN.V. Validation: M.VDH and TNN.V. Funding acquisition: F.M. Writing \u0026ndash; original draft: TNN.V. Writing \u0026amp; editing: M.VDH, J.E and TNN.V. Reviewing the final manuscript: M.VDH, N.L\u0026rsquo;H, J.HC, P.M, T.M, T.G, J.H, J.E, FM and TNN.V\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Louis Perrault and Saint Marcellin hatchery for the technical support during the study. We thank the France Agrimer Institution for funding project. The funders had no role in designing or directing the study and editing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGautron, J.; R\u0026eacute;hault-Godbert, S.; Van De Braak, T. G. H.; Dunn, I. C. 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Recombinase Polymerase Amplification Coupled with a Photosensitization Colorimetric Assay for Fast \u003cem\u003eSalmonella\u003c/em\u003e Spp. Testing. \u003cem\u003eAnal. Chem.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e93\u003c/em\u003e (16), 6559\u0026ndash;6566. https://doi.org/10.1021/acs.analchem.1c00791.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PCR, LAMP, RPA, chick embryos, in ovo sexing","lastPublishedDoi":"10.21203/rs.3.rs-5772672/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5772672/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite of global efforts, reducing the culling of one-day hatched male chicks in the poultry industry has been a critical priority due to significant socio-economic concerns. Indeed, various molecular assays have been developed to determine the sex of eggs before hatching (\u003cem\u003ein ovo\u003c/em\u003e) to eliminate male embryos at early developmental stages. Because of their precision-related complexity, needs for advanced infrastructures and time-consuming processes, there is still no widespread commercialization for these assays. In this study, we developed two novel digital readouts assays employing PCR, LAMP and RPA techniques whose sensitivity, specificity and robustness were validated on 82 chick embryos at day 9. Our data demonstrate that, while the two novel PCR based assays correctly and robustly sex the 82 embryos, the LAMP and RPA based assays propose comparable results. Moreover, LAMP and RPA assays propose isothermal amplification associated with naked-eye colorimetric and/or fluorescent detection in a relatively shorter time (20 minutes at 65\u0026deg;C and 30 min at 37\u0026deg;C, respectively). These newly developed assays, not only significantly reduce the complexity of experimental setting but also being faster and more affordable sexing methods, addressing key barriers to \u003cem\u003ein ovo\u003c/em\u003e sexing to a future commercialization of a non-invasive \u003cem\u003ein ovo\u003c/em\u003e sexing assay.\u003c/p\u003e","manuscriptTitle":"Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 08:15:09","doi":"10.21203/rs.3.rs-5772672/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-10T07:54:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-08T16:38:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48354241399673240031946546285153093655","date":"2025-02-14T15:32:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-25T15:48:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172101324616975004764187717088783543938","date":"2025-01-23T09:24:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-17T02:17:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-17T01:57:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-13T17:58:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-10T12:36:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-06T09:41:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b47abbd5-ca9e-4766-83a9-9f2281295fbe","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42683926,"name":"Biological sciences/Biotechnology"},{"id":42683927,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-08-04T16:49:36+00:00","versionOfRecord":{"articleIdentity":"rs-5772672","link":"https://doi.org/10.1038/s41598-025-13013-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-29 16:38:21","publishedOnDateReadable":"July 29th, 2025"},"versionCreatedAt":"2025-01-13 08:15:09","video":"","vorDoi":"10.1038/s41598-025-13013-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-13013-3","workflowStages":[]},"version":"v1","identity":"rs-5772672","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5772672","identity":"rs-5772672","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}