Chicken Sex Determination using SWIM Gene Revealed its Location in Both Autosomes and Sex Chromosome

In contrast to mammals, birds have heterogametic females with two distinct sex chromosomes (ZW) and homogametic males with one sex chromosome (ZZ). The W chromosome is transferred through the ovum of female birds to determine the sex of the progeny (Stevens, 1997; Bachtrog et al., 2011). Males have two Z sex chromosomes in this arrangement while females have Z and W sex chromosomes. The W chromosome only contains a small number of genes, but the Z chromosome is bigger and carries many genes (Graves, 2014). During the embryonic stage, the chick’s sex can be determined using gene sequences specific to the W chromosome. Gender in chickens and other bird species is determined manually via cloacal or vent sexing, as well as feather sexing based on plumage colour. Although manual gender determination approaches are less expensive, they require highly skilled workers and are more susceptible to errors (Seemann, 2003). However, sexing of the chick at embryonic stage can also be performed using polymerase chain reaction (PCR) with genes located on both Z and W chromosomes. The PCR of chromo helicase DNA binding 1 (CHD-1) which is present in both Z (CHD-1Z) and W chromosome (CHD1-W) are used for sexing in non-ratite birds which had produced two bands in females and one band in males on agarose gel electrophoresis (Fridolfsson and Ellegren, 1999). Similarly, PCR based assay on Xhol repeats on the W chromosome was detected using gene specific primers where a band of size 415 bp appeared in females and no band in males (Clinton et al., 2001). While the amplification of ECoRI repeats on W chromosome will help in sexing of the carinatae species (Itoh et al., 2001). The zinc finger SWIM domain containing protein 6 like (SWIM), is located on W-chromosome; therefore serves as specific marker for female. However, comprehensive investigation of the W chromosome has yet to be undertaken in birds due to the presence of smaller and highly hetero-chromatic genomic regions in the W chromosome. The W chromosome has recently been shown to have several roles in crucial sex-specific evolutionary processes and sex determination (Moghadam et al., 2012). The PCR amplification of SWIM gene on W chromosome is the simple, robust method for sex determination in chicken.In an attempt to determine the sex of the chicken embryos using the primers reported by He et al. (2019), there appeared non-specific bands in male and female samples and specific bands in the females. Previous research has demonstrated the significance or specificity of SWIM primers in detecting the sex of birds.The present investigation was undertaken to study the presence of non-specific bands in male and female chicken and to establish an even more specific primer for sex determination.

Experimental birds

The present study was carried out in Kadaknath chicken that is being bred and maintained at ICAR-Directorate of Poultry Research in Rajendranagar, Hyderabad. To conduct the experiment, female Kadaknath chicken were artificially inseminated with Kadaknath semen. Subsequently, the laid eggs were carefully collected and placed in an incubator, maintaining a constant temperature of 99.5°F and a relative humidity ranging between 55-60%. On the 18^th day of incubation, the eggs were subjected to candling. The infertile eggs were not processed further and only fertile eggs were selected for the experiment.

Collection of sample and Isolation of Genomic DNA

The fertile eggs of Kadaknath birds were chosen at random on the 18.5 day of incubation. A total of 10 liver tissue samples were then collected under sterile conditions, rinsed with saline to remove blood and placed in 1.5 ml centrifuge tubes containing 1X PBS and stored at -80°C for subsequent processing. Using a mortar and pestle, the liver tissue from the individual samples were homogenised. One millilitre of 1X PBS was then added and the mixture was centrifuged at 2000 rpm for 10 minutes and supernatant was decanted. In order to further break down the cells in the pellet, 1 ml of cell lysis solution was added, incubated for 20 minutes and centrifuged at 2000 rpm for 10 minutes. Later, genomic DNA was recovered with a few minor alterations utilising the phenol-chloroform extraction method (Sambrook et al., 1989). The extracted DNA was then stored at 4°C till use. Further, purity of DNA was evaluated by measuring the optical density (OD) at wave lengths 260 nm and 280 nm in a UV-spectrophotometer. Genomic DNA quality was checked to ensure intact DNA without any shearing using horizontal agarose gel electrophoresis.

Polymerase chain reaction (PCR) and sequencing

The composition of PCR reaction mix for amplification of SWIM primers were 10X Taqbuffer, DNTPs (0.4 Mm), Primer-Forward (4 Picomoles), Primer-Reverse (4 Picomoles), Taq Polymerase (1 unit/µl), Autoclaved Mille Q water with a final volume as 11.5 ul and 1 ul DNA as template. The PCR reaction conditions for SWIM primers includes, initial denaturation at 94°C for 5 min, cyclic denaturation at 94°C, Primer annealing at 55°C, extension at 72°C repeating over 36 cycles and Final extension at  at 72°C and hold at 4°C.The amplification of the SWIM gene with specific primers (Table 1) was carried out in 0.2 ml capacity PCR tubes, using a thermal cycler (Himedia).



The amplified product was checked in gel electrophoresis using UV transilluminator. The specific and non-specific PCR products were purified using GeneJET gel extraction kit (ThermoFisher) following user manual.The purified products were custom sequenced for Sanger sequencing. The obtained DNA sequences were subjected to multiple sequence alignment using CLUSTALW and BLAST (Samal et al., 2021) analysis using NCBI BLAST tool.

In the breeding industry, the most reliable way of establishing the gender of bird species is PCR analysis. The number of studies on sex-linked markers and PCR-based approaches for detecting gender in bird species is continuously increasing. The bulk of PCR-based methods for detecting the birds sex concentrate on sex chromosome genes. PCR-based sex determination methods have steadily improved in terms of accuracy, speed and high-throughput applicability (Dhanasekaran et al., 2016). The extracted DNA from different liver samples has shown purity level ranging from 1.8 to 1.9 in spectrophotometer. The PCR products were further resolved in 1% gel electrophoresis that revealed a 212 bp which is specific to female birds and a  non specific band in both male and female birds at 900 bp (Fig 1).



The CLUSTAL W (Fig 2) and BLAST analysis of the sequences belonging to the 900 bp PCR product revealed its homology in female and male embryos (Fig 3) and 212 bp is unique to females.




The non-specific bands in females (FLC 1,2,4 and 5) has same base alignment with upper bands of both sexes. BLAST analysis confirmed the sequences of the upper extra band appearing in both females and males due to the sequence similarity with autosomes and W chromosome. However, SWIM primers can be used to differentiate male and females sexes through PCR.

In this study, the primers that was used for amplification of SWIM gene for sex determination in Chicken, that was previously reported to amplify the SWIM gene on chromosome W was also found to amplify a region in autosomes on chromosome 9.

The authors would like to thank the Director, ICAR-DPR, Hyderabad and P.V.Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad for providing all the facilities and support to carry out the work.

The authors declare that there is no conflict of intrest.