Deakin University Report: Chicken Sex Determination by DNA Analysis

This report presents a study on determining the sex of domestic chickens (Gallus gallus) using DNA extracted from feather, blood, and muscle samples, representing non-invasive, invasive, and destructive collection methods, respectively. The research aimed to compare DNA quality and quantity across different sources and to identify the best DNA collection method for sex determination. DNA was extracted using standard techniques, followed by PCR amplification of CHD1Z and CHD1W gene variants, and visualization via gel electrophoresis. The results indicated that muscle samples provided the best DNA quality, while blood samples yielded the highest DNA concentration. Sex determination was successful in most samples, with the exception of one, demonstrating the potential for chicken sex determination using DNA from various sources. The study highlights the impact of DNA source and extraction methods on DNA quality and quantity, emphasizing the importance of proper storage conditions. Limitations include a small sample size and the potential for human error. The conclusion supports the use of blood or feather samples for non-destructive sex determination, though muscle samples may provide the highest DNA quality.

INTRODUCTION
This work presents research on DNA samples, collected from feather, blood, or
muscle of a domestic chicken (Gallus gallus). Each source of DNA represents
non-invasive, invasive, and destructive DNA collection methods, respectively.
In each case DNA quality and quantity are different because factors like source
(Alhaddad et al., 2019), collection conditions and storage conditions, like
storage temperature, humidity and length of DNA samples storage after
collection affect the DNA quality (Huang et al., 2017). DNA breaks down
quicker at higher temperatures, or if stored in a wrong medium or for long time
(Al-Griw et al., 2017). Choice of collection method depends on research type.
When good quality DNA is needed, then destructive or invasive collection
methods used, when DNA quality is not so important, then non-invasive
methods may be applied.
In this research, DNA, collected by destructive, invasive, and non-invasive
methods was used to determine sex of domestic chicken. By application of PCR
and electrophoresis gel methods DNA from different sources was compared to
heterogametic sex markers. Heterogametic sex determination depends on ability
to create two different sex chromosomes. In case of chicken, sex determined by
chromosomes Z and W, and gene variants CHD1Z and CHD1W, consequently.
Males have ZZ genotype and females have ZW genotype (Hirst, Major and
Smith, 2018).
AIMS
This research has two primary aims. First is to determine the sex of a domestic
chicken (Gallus gallus) using DNA, extracted from feather, muscle, or blood of
an animal, using gel electrophoresis method. The second aim is to compare
DNA quality between three different collection methods to identify differences
and to choose the best DNA collection method for purposes of sex
determination.
HYPOTHESIS is that source of DNA affects its quality. PREDICTION is that
destructive DNA collection method gives the best quality DNA, compared to
invasive or non-invasive DNA sources.

METHODS
DNA extraction
20 mg of chicken muscle placed into a sterile microcentrifuge tube, 180μL of
Buffer ATL was added and vortexed. 20 μL of proteinase K was added,
vortexed and tube incubated at 56°C for 30 min. Then 4 μL RNase A (100
mg/mL-1) placed into tube, vortexed and incubated for 2 min at room
temperature. Then 200 μL Buffer AL added, vortexed, 200 μL of 96–100%,
analytical grade ethanol added and vortexed.
Tube content was pipetted into the DNeasy Mini spin column unit and
centrifuged at 6000 x g (8000 rpm) for 1 min. After 500 μL of Buffer, AW1 was
added and tube centrifuged for 1 min at 6000 x g (8000 rpm).
The content was transferred into a new 2 mL collection tube, 500 μL of Buffer
AW2 was added and centrifuged for 3 min at 13-14,000 rpm. Content then was
relocated to 1.5 mL microcentrifuge tube. 100 μL Buffer AE was added to
DNeasy membrane and tube incubated at room temperature for 1 min and then
centrifuged for 1 min at 6000 x g (8000 rpm). Additional 100 μL Buffer AE was
added, and the tube was centrifuged. DNA was collected by pipette from tube
bottom, transferred into a new tube and stored on ice at -200C.
The method of DNA extraction from other tissue types varied slightly. Full
details are given in Hogan et al. (2017).
PCR
A negative control, male control, and female control tubes were prepared by
mixing 40 μL of a master mix of DNA and 10 μL of diluted DNA into each 0.2
ml tube (Desjardins and Conklin, 2010).
Samples tubes prepared in the same way, each with relevant sample DNA. All
samples put into the PCR machine to run an optimized program to amplify
CHD1W and CHD1Z gene variants with the 2550F-2718R primer set:
1 cycle of 3 min at 940C, then 40 cycles of 30 sec at 94°C, 30 sec at 48°C and 1
min at 72°C, followed by 1 cycle of 10 min at 72°C (Ponti et al., 2018).
GEL ELECTROPHORESIS
1% agarose gel used. Gel run at 100 V for 45 min. To highlight DNA colored
indicator dyes and molecular weight marker in loading dye used.

RESULTS
Section 1
Picture 1. Summary of all types of gel electrophoresis.
In this picture: Lane 1 is loaded with λ Hindi III ladder, lane 12 is loaded with the 100 bp
ladder. Blood samples (2,8,9), muscle samples (4,5,7), and feather samples (3,6). Lanes 2
and 8 demonstrate smeared bands, lane 9 is very smeared and not distinct. Line 6
demonstrates distinct thin band along with a smeared trace. Lane 3 shows a very weak
band. Lanes 4,5 and 7, belonging to muscle samples, demonstrate clear and distinct bands.
Table 1. The DNA concentration and quality in samples from different tissue
types
DNA from all samples is of good quality with 260/280 ratio above 1.7. Best
quality DNA derived from muscle samples and the worst quality DNA from
blood samples. Quality of DNA from feather is average. The highest yield of
DNA is from blood samples, and the lowest is from muscle samples. Yield from
feather samples is slightly lower than from blood samples.
Total Samples Mean DNA
concentration
(ng/μl)
Average 260/280
ratios
Blood 11 30 1.7
Feather 2 22 1.75
Muscle 11 14 1.8

Section 2
Picture 2. Gel electrophoresis results-sex determination.
Lane 1 is loaded with λ Hindi III ladder (purple frame), lane 12 (indistinct) is
loaded with the 100 bp ladder (last white frame). Positive male control (number
9, yellow frame) and female control (number 8, green frame) derived from
DNA from individual chickens. Negative control (number 10, blue frame)
loaded with no DNA — the checked sample from muscle tissue marked by red
frame (number3).
Sample 2, and sample 4 belonging to chicken 5 belong to male chickens.
Sample 3, sample 5, and sample 7 belonging to chicken 6, belonging to the
female chicken
Sample 6 did not amplify; therefore, did not show any results.
Sample 2 Male
Sample 3 Female
Sample 4 Male
Sample 5 Female
Sample 6 Unclear
Sample 7 Female
Sample 8 Female control
Sample 9 Male control

Gel results demonstrate the approximate size of CHD1Z male gene of 800bp,
and size of CHD1W female gene is about 700bp. Two samples were male, and
three samples were female
Negative control amplification detected only in sample 10 (see Picture 2). All
the W and Z bands amplified in all samples, besides sample 6. The results of
sex determination by PCR and gel electrophoresis correlate with the actual sex
of individual chickens.
DISCUSSION
The study aimed to determine the sex of chicken from conducting amplification
of DNA, derived from blood, feather or muscles of individual chickens, by PCR
methods. Visualization of results was done by DNA gel electrophoresis. By
conducting the experiment, DNA quality was checked by NanoDrops, and the
results demonstrate the best DNA quality was in muscle samples, while the
highest yield was from blood samples. Feather samples demonstrated average
yield and quality of DNA. These results were consistent with expectations.
There was no band visible on one gel. It is possible that the extraction process
was unsuccessful (Alhaddad et al., 2019).
Source of DNA is thus a factor, affecting both quality and quantity of DNA that
can be derived from the samples. Potentially, both source and extraction
methods play a role in the final DNA quality. Storage conditions may have also
contributed to the quality of DNA in each sample. It is important to store
correctly any genetic samples that are intended for analysis. Incorrect storage
can result in DNA degradation. This is evident in DNA from feather tissue that
was used in sample 6 (Ponti et al., 2018).
Sex determination was successful in all samples, except sample 6, which
demonstrated negative control amplification. In general, the results demonstrate
that the determination of sex may be done using DNA from either blood,
feather, or muscle. DNA extracted from a blood spot on the feather will provide
DNA that is of lower quality as compared to DNA extracted from the tip. Due
to differences in source and extraction methods, muscles are the best source of
DNA, followed by feather and blood (Hirst, Major and Smith, 2018).
Study limitations include a statistically small number of samples (24), and
multiple operators, handling samples and DNA, which increased the risk for
human error (Ito, Sudo-Yamaji, Abe, Murase and Tsubota, 2003).

CONCLUSION
Chicken sex determination may be conducted on DNA, derived from blood,
feather, and muscle tissues. Choice of DNA source should be made in line with
research aims and DNA quality and quantity requirements. While muscles may
provide high-quality DNA, the yield from muscle samples is moderate, and it is
a destructive method of DNA extraction. Blood or feather samples, on the other
hand, are non-destructive and may provide good yield and quality DNA,
sufficient for sex determination on chicken by gene amplification and gel
electrophoresis methods.
References
Al-Griw, H., Zraba, Z., Al-Muntaser, S., Draid, M., Zaidi, A., Tabagh, R., and
Al-Griw, M. (2017). Effects of storage temperature on the quantity and integrity
of genomic DNA extracted from mice tissues: A comparison of recovery
methods. Open Veterinary Journal, 7(3), p.239.
Alhaddad, H., Maraqa, T., Alabdulghafour, S., Alaskar, H., Alaqeely, R.,
Almathen, F., and Alhajeri, B. (2019). Quality and quantity of dromedary camel
DNA sampled from whole-blood, saliva, and tail-hair. PLOS ONE, 14(1),
p.e0211743.
Desjardins, P. and Conklin, D. (2010). NanoDrop Microvolume Quantitation of
Nucleic Acids. Journal of Visualized Experiments, (-1).
Hirst, C., Major, A., and Smith, C. (2018). Sex determination and gonadal sex
differentiation in the chicken model. The International Journal of
Developmental Biology, 62(1-2-3), pp.153-166.
Hogan F, Loke S, and Sherman C (2017) SLE254 Genetics practical
manual 2017. Deakin University.
Huang, L., Lin, P., Tsai, K., Wang, L., Huang, Y., Kuo, H., and Li, S. (2017).
The effects of storage temperature and duration of blood samples on DNA and
RNA qualities. PLOS ONE, 12(9), p.e0184692.

Ito, H., Sudo-Yamaji, A., Abe, M., Murase, T., and Tsubota, T. (2003). Sex
Identification by Alternative Polymerase Chain Reaction Methods in
Falconiformes. Zoological Science, 20(3), pp.339-344.
Ponti, G., Maccaferri, M., Manfredini, M., Kaleci, S., Mandrioli, M., Pellacani,
G., Ozben, T., Depenni, R., Bianchi, G., Pirola, G. and Tomasi, A. (2018). The
value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the
quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate
cancer patients. Clinica Chimica Acta, 479, pp.14-19.
11