An approach to rapidly identify the gender of the pigeon by using cross-priming ampli�cation with immune-chromatographic strip

Most of birds are monomorphic species and breeds, which makes it di�cult to determine their gender by appearances, especially the pigeon is a farm animal with an economic interest in meat production, ornamentals, sports, and experimental animals. Until now, only endoscopy, laparoscopy, karyotyping, and DNA testing are available, but these methods are expensive, invasive, or time-consuming for the determination gender of the pigeon. Therefore, an easy, accurate, sensitive, on-site, affordable, and applicable rapid identi�cation of the gender of the pigeon is widely needed for the owner of the pigeon. The purpose of this study was to develop and evaluate the e�cacy of Cross-priming ampli�cation (CPA) combined with an immune-chromatographic strip (CPA-strip) for gender identi�cation of the pigeon. The methodology was optimized through various experimental trials. Subsequently, ten samples collected from pigeons were subjected to analysis using the optimized CPA-strip assay, and the results indicated that all female samples were accurately detected. In contrast, the blood samples collected from chickens and ducks were negative when tested with the CPA-strip assay. In conclusion, our study demonstrates the successful establishment of an immune-chromatographic CPA-strip assay for the on-site gender determination of pigeons with high accuracy.


Introduction
Pigeons or doves are the most common birds worldwide, comprise the bird family Columbidae, and are usually considered a symbol of peace [1].
The domestic pigeon (Columba livia domestica) serves a multifaceted role in society.It is utilized for meat production, as an ornamental animal and a pet, and for racing which holds signi cant economic value [2] [3].Furthermore, it has been acknowledged as a valuable experimental animal model, often selected for medical research and studies on genetic variation and behavior [4].
The di culty in accurately determining the sex of pigeons base on external appearance is attributed to the lake of pronounced sexual dimorphism [5].Traditionally, sex identi cation in pigeons according to behavioral observations [6], or through invasive procedures such as endoscopy, laparoscopy, and laparotomy [7].These methods have been widely criticized for being highly invasive, stressful and challenging, particularly when the size of birds is taken into consideration.With the advent of molecular diagnostics, new methods of sex identi cation, the polymerase chain reaction (PCR) or based on the PCR method within genetic differences between the male and female have been developed[8] [9][10].
The development of PCR-based techniques for the determination of gender in pigeons has garnered signi cant attention in recent years.Although PCR-based methods have proven to be effective, they can be time-consuming and require laboratory facilities.The PCR method is based on the ampli cation of CHD-W and CHD-Z genes, with the ampli ed CHD-Z and CHD-W products exhibiting a 20bp size difference.To differentiate these amplicons, high-resolution acrylamide gel electrophoresis is required[8] [9].
The application of isothermal ampli cation techniques in the eld of genetic diagnosis represents a novel approach [11].One such technique, loop-mediated isothermal ampli cation (LAMP), has been speci cally developed for the sex identi cation of Columbidae birds [12].Another novel isothermal ampli cation technique, cross-priming ampli cation (CPA), has gained signi cant attention in recent years [13].The CPA-based method has been successfully utilized in a wide range of diagnostic applications, including the detection of Mycobacterium tuberculosis, African swine fever virus, pseudorabies virus infection, and for meat species authentication [14][15][16] [17].
In our study, we developed a novel Cross-priming Ampli cation (CPA) assay combined with an immunochromatographic strip (CPA-strip) for the on-site identi cation of the sex of pigeons.The assay targeted the female-speci c gene of pigeons and was optimized by designing the appropriate primers and probes.The products generated by the CPA were labeled with biotin and FITC, and the recognition of these labels by anti-biotin and anti-FITC monoclonal antibodies was demonstrated on the test line of the strip, which was xed with gold nanoparticles.

Sample collection, storage, and DNA extraction
Blood samples were collected from pairs male and female Columba livia (C.livia) individuals.The gender of each bird was determined through anatomical inspection.All the samples were stored at -20℃ until further analysis.DNAs were extracted from the blood sample using DNA kit (protocol for animal blood: CWbiotech, universal genomic DNA kit).

Primers and probes design
In accordance with the NCBI database, a female-speci c sequence of Columba livia (GenBank accession No. AY944219) was utilized as a reference.Based on sequence alignment, multiple primers and probes were designed and synthesized by Sangon Biotech (Shanghai, China), as shown in Table 1.

preparation of the immunochromatographic strip
The preparation of the immunochromatographic strip was conducted according to a previously published procedure [18].In brief, gold nanoparticles were synthesized through the sodium citrate reduction method.
The anti-FITC monoclonal antibody (McAb) was mixed with the AuNPs to form a colloidal solution, which was then added to the conjugate pad.The anti-biotin McAb and anti-mouse IgG were immobilized on the Millipore membrane and added to the test line and control line, respectively, as depicted in Fig. 1.The immunochromatographic strips were manufactured by Hangzhou Ustar company.
The reaction volume was set at 25 µL, consisting of the following components: 2.5 µL of ThermoPol buffer (10 ×), 1.5 µL of the cross primer (0.6 µM), 0.5 µL of the upstream primer (0.2 µM), 0.5 µL of the outer primer (0.2 µM), 1.0 µL of the probe (0.4 µM), 2.5 µL of dNTPs (10 mM), 1.5 µL of Mg 2+ (6.0 mM), 1.0 µL of Bst DNA polymerase (8 U/µL), 2.0 µL of betaine (8 U/µL), 2.0 µL of extracted DNA from the pigeon sample, and the volume was completed with nuclease-free water.The reaction was ampli ed at 57-63℃ for 1 hour.The ampli cation was veri ed by electrophoresing the CPA products in 1.5% agarose gel.Subsequently, 8 µL of the products was mixed with 92 µL of PBS and added to the sample pad.After a 15-minute incubation, the appearance of the test and control lines were observed visually with the naked eye.

Sensitivity, reproducibility, and speci city tests of the CPA-strip assay
In order to obtain promising results, blood samples from chickens, ducks, and other avian species were selected to evaluate the speci city of the CPA assay.The extracted DNA was serially diluted from 1 ng to 100 ng in order to assess the sensitivity of the assay.

Optimization of the CPA assay
Based on the published female-speci c sequence alignment of Columba livia in the NCBI database, three sets of primers and probes were designed.Among them, set 3 demonstrated ampli ed bands (as depicted in Fig. 2), making it the chosen set for subsequent testing.
To enhance the ampli cation e ciency of the CPA products, the temperature was varied from 57 to 63℃.No ampli cation was observed at 57 and 58℃.However, results from gel electrophoresis indicated no difference in ampli ed bands at temperatures above 60℃ (as depicted in Fig. 3)

Sensitivity, reproducibility, speci city, and of CPA-strip assay
To decide the detection range of the CPA reaction, the concentration of DNA was set as follows: 1, 5, 10, and 100ng; CPA-strip assay results showed the assay's detection limit was 10ng extracted DNA (Fig. 4).The color intensity among the test lines were similar when the extracted DNA was more than 10ng in the CPA assay.But the 5ng extracted DNA for the ampli cation, and the test line was faint.
The ten pigeons' blood samples were tested by the CPA-strip assay, ve females (positive) and ve males (negative) (Fig. 5), and the result is identical to the anatomic result.
While other birds, including chickens and ducks, showed negative results (Fig. 6).Only the female pigeon showed a positive outcome.
In order to determine the detection range of the CPA reaction, the concentration of extracted DNA was varied at 1, 5, 10, and 100 ng.Results from the CPA-strip assay indicated that the detection limit was 10 ng of extracted DNA (as depicted in Fig. 4).No signi cant differences in color intensity were observed among the test lines when the extracted DNA was greater than 10 ng.However, when the concentration of extracted DNA was 5 ng, the ampli ed test line appeared faint.
Ten blood samples from pigeons were evaluated using the CPA-strip assay, with ve samples from females (positive results) and ve samples from males (negative results) (as depicted in Fig. 5), which were consistent with the results of anatomical inspection.
In contrast, other avian species, including chickens and ducks, resulted in negative outcomes (as depicted in Fig. 6).The assay only produced a positive result for the female pigeon.

Discussion
Bird sex identi cation is not only relevant to veterinary, also relevant to ecological sciences and helpful in enforcing legislation to protect endangered bird species and resolving paternity disputes [19].An investigation into the patterns of distribution and dispersal of commensal pigeons has the potential to inform the formulation of policies and urban design strategies that mitigate the impact of humaninduced eco-evolutionary alterations and enhance ecological and societal outcomes.[20] [21].
In the eld of veterinary medicine, the accurate and e cient identi cation of the sex of an individual pigeon is a crucial task.This is particularly relevant for pigeon owners in the meat production industry, as well as for breeding centers and zoological gardens where large numbers of pigeons are bred and traded.
In these contexts, on-site and cost-effective sex identi cation is imperative.[10].
More than fty percent of bird species are monomorphic [22], and identifying their sex based on external morphology is always impossible.Accurate identi cation of the sex of individuals is crucial in numerous bird protection programs aimed at preserving various species through intensive bird breeding [23].The accurate assessment of bird population size in a given area is crucial for monitoring population stability and variations [24] as well as migration patterns in response to climate change [25].Our methods of CPA assay can provide insight for identifying the gender of other birds.
There are several methods for sex identi cation of pigeons, such as by using the PCR for sex identi cation of pigeon, the PCR and electrophoretic apparatus, gel image system are required, however, compared with it, CPA-strip assay has several advantages, the ampli ed products can be directly observed by naked eyes.This method does not need technicians to perform it, and it also does not need PCR apparatus.
The results of the CPA-strip assay for the sex identi cation of pigeons demonstrated 100% agreement when tested with ten blood samples, showcasing its high sensitivity and speci city.The high concordance between the CPA-strip assay and the anatomic results indicates the feasibility and practicality of using this novel assay for the accurate sex identi cation of pigeons.
In summary, the study has demonstrated the utility of the CPA-strip assay as a reliable, on-site, costeffective, high speci city and sensitivity and convenient tool for sex identi cation in pigeons.

Conclusion
In conclusion, sex identi cation of pigeons is essential for avian research and breeding programs.The PCR-based method has been the most commonly used method in the past.However, it requires specialized laboratory facilities and is time-consuming.The CPA method, combined with an immunochromatographic strip, offers a novel, on-site, and user-friendly diagnostic tool for the sex identi cation of pigeons.This method has several advantages, such as simplicity, high speci city, and sensitivity, and requires only basic laboratory facilities.Thus, the CPA-strip assay developed in this study has signi cant potential as a rapid and cost-effective alternative method for sex identi cation of pigeons.

Figures
Figures

Figure 1 description
Figure 1