Digits Angle and Digits Length Ratio in Japanese Quail (Coturnix coturnix Japonica)

Quails (Coturnix coturnix japonica) are used in poultry for both, meat and egg production. It belongs to the Phasianidae family. It is difficult to determine gender according to its morphological features at a young age. Sexual maturity varies from four to six weeks (Alkan et al., 2008). After this period, ovulation period comes. After the sixth week, the female birds can be separated due to the larger body structure (Brunström et al., 2009). It has been reported that sex discrimination can be made using features such as wing length, weight and chest width (Rathert et al., 2017).
       
It has been reported in previous studies that finger lengths are dimorphic in some primates, including humans (Brown et al., 2002; Leoni et al., 2005; McFadden and Shubel, 2002; Saino et al., 2007). In addition to sexual dimorphism, some of its hormones have been shown to be directly related to this ratio (Lutchmaya et al., 2004; Van Anders et al., 2006). This ratio between genders was observed especially between the second digits (D2) and fourth digits (D4) and lengths of the foot (McIntyre et al., 2005; Peters et al., 2002). It has been reported that D2:D3 ratio is higher in female samples exposed to high androgen hormone (Romano et al., 2005). Estradiol is said to decrease the D2:D3 ratio in male (Saino et al., 2007). In addition, asymmetries between the right and left hands were examined using these ratios and it was observed that this asymmetry was larger in male (Williams et al., 2000). Quails as other domesticated birds have four toes. The first toe oriented in a medial and plantar direction while the other three are spread out and points forwards (Nickel et al., 1977) (Fig 1).
 

       
Geometric morphometric analysis is made with the data taken from the photograph, unlike normal morphometry. The points on the photo are determined and the positions of these points relative to each other are examined on the x-y coordinates and the shape variations are revealed. With this method, which is used especially in the field of osteology, the differences of bone shapes are revealed both between sexes and between species (Gündemir et al., 2020; Gürbüz et al., 2020; Þahiner and Yalçýn, 2007).
       
Various anatomic, genetic and breeding studies of this bird species used for breeding are available in reference information (Kumari et al., 2008; Hertamawati et al., 2019; Demiraslan et al., 2014; Demircioglu et al., 2019; Demircioglu et al., 2020; Monika et al., 2020). In this study, we analyzed digits angle and digits length ratio of Japanese quail using geometric morphometric analysis. With this method, we examined whether male-female distinction can be made.

Samples
 
In the study, 60 (30 female, 30 male) adult quail (60 days-old) were used. Weights of the samples were taken. The application was carried out in the Harran University Anatomy Laboratory from January 2020 to August 2020. Approval was obtained from the ethics committee of the University of Harran University, Faculty of Veterinary Medicine (approval no: 2020/004). In the study, only the left foot of each bird was used. Os tarsometatarsus was allowed to surface on a thin glass surface at an angle of 90 degrees to obtain foot print pictures of all digits. Then, photographs were taken with the camera prepared 10 cm away on the other side of the glass. Angle and length measurements were made on two-dimensional photographs. Rates were determined from these measurements. At the same time, these photographs were analyzed by geometric morphometric method.
 
Geometric morphometric analysis
 
In the first stage, the photographs were converted into a TPS file where the landmarks can be placed using the TpsUtil (version 1.74) program. Photographs taken from 60 samples were used as a single file in tps Dig (version 2.30) program and landmarks were made and their coordinates were determined. Morphoj (version 1.07a) program was used to visually show the changes in the points after geometric analysis.We have used eight landmarks (Fig 1):

1: The point between claw and distal phalanx of the second finger.
2: The point between claw and distal phalanx of the third finger.
3: The point between claw and distal phalanx of the fourth finger.
4: The point between claw and distal phalanx of the first finger.
5: The midpoint of Tela interdigitalis medialis.
6: The midpoint of Tela interdigitalis lateralis (Baumel et al., 1993).
7: Te point between base of the first finger and torus metatarsalis.
8: The most caudal point of torus metatarsalis where the foot is touching the ground (Ghetie, 1976).
 
Ratio and angle analysis
 
The measurements of three lengths from the pixel value were taken using Image J (version 1.52a) program on each photograph. Then, using these measurements, ratios between live finger surfaces contacting the ground were obtained (D2:D3 and D3:D4). It was also recorded at angles between the reference points used for lengths taken using this program (Fig 1).

D2: The distance between the first  and eighth landmarks.
D3: The distance between the second  and eighth landmarks.
D4: The distance between the third and eighth landmarks.
a1: Angle between reference points used for D2 and D3 measurements.
a2: Angle between reference points used for D3 and D4 measurements.
 
Statistical analysis
 
Principal component analysis (PCA) was used for geometric morphometric analysis. For this, the PAST program (IBM SPSS for Windows, Ver. 23) was used. The variations obtained in the PCA analysis were determined. ANOVA test was performed to statistically determine whether there are geometric differences between groups with eight landmarks used in the study. SPSS (version 22) program was used to examine the statistical difference between the female and male ratio and angle values taken from the photograph and ANOVA test was performed. In addition, the correlation values between these ratios and angles were examined.

As a result of the geometrical analysis, 16 principal components were obtained using eight landmarks. The geometric difference between genders is shown in Fig 2A. According to this figure, the biggest difference is seen in the landmark number one. It was observed that the second finger (D2) of male birds was more introverted than in females. In Fig 2B, distribution of female and male birds according to PCA1-PCA2 is given as a result of principal component analysis. It was seen that PCA1 explained 53.07% of the total variation between female and male groups. PCA2 announced 17.60% of the total variation and PCA3 announced 11.83%. In the 95% confidence ellipse obtained using PCA1 and PCA2, it was observed that the female and male samples did not separate (Fig 2B). In addition, Anova test result showed that the difference between male and female was statistically insignificant in geometric analysis.
 

       
Table 1 gives the ratio and angle values of female and male individuals using finger measurements. Using this ratio and angles, no statistically significant difference was observed between male and female. D2:D3, D2:D4 and D3:D4 ratios were higher in males. It was observed that the lateral angle was higher than medial angle in both genders. This difference was statistically significant (p<0.001). While the average of the lateral angle was 50.89% in all individuals, the average of the medial angle was 42.81%. The lengthiest in all individuals belonged to the third finger. Then the longest measurement value belonged to the fourth finger. 
 

       
In Table 2, the correlations of the angle and ratio values related to each other and the weights of the individuals are given. While the D2:D4 ratio was directly related to the D2:D3 and D3:D4 ratio, the correlation between the D2:D3 ratio and the D3:D4 ratio was found to be statistically insignificant. While there was a negative correlation between the finger length ratios of the a1 angle, there was a positive correlation between the finger length ratios of the a2 angle.
 

       
We planned to use this measurement method, which can be obtained from radiographic measurements (Leoni et al., 2008) or, from the external appearance of the foot. In this study, no statistically significant difference was found between adult quails D2:D3, D2:D4 and D3:D4 finger length ratios. In the study, only the photos of the normal posture position of the fingers were taken and the proportions were made on these photos. No osteometric or radiological measurements were taken. It has already been said that there is a consistency between these rates taken from both radiological and external appearance (Ruuskanen et al., 2011). However, in the study of Ruuskanen et al., (2011), it was observed that the ratio of D2:D3 and D2:D4 is sexually dimorphic and these rates are higher in male than in female.
               
Finger lengths were previously measured osteometrically in some birds and the D2:D4 ratio between the female and male was examined (Lombardo et al., 2008). In the study by Lombardo et al., (2008), the left toe length ratios are house sparrows (male 0.919, female 0.895), tree swallows (male 0.965, female 0.961), budgerigars (male 0.761, female 0.685) and chicken (male 0.869, female 0.853) also taken. However, the D2:D4 ratio difference between the male and female was not statistically significant as in this study.

In this study, the left foot D2:D4 ratio was found to be 0.867 in females and 0.877 in males. It was observed that the D2:D4 ratio was higher in all birds used by Lombardo et al., (2008) in his studies, as in this study. In addition, another study reported that males have higher D2:D4 ratios than females (Chang, 2008).
       
Dimorphism studies were carried out in birds using digit ratio. There are studies suggesting that digits parameters length are related to the hormone level in the body (Romano et al., 2005; Saino et al., 2007). However, in another study, it was stated that androgen hormone was not associated with digit ratio (Ruuskanen et al., 2011). When these differences in results are evaluated, it makes us think that it is important to carry out more studies on this subject. In addition, digit ratio was also examined in terms of right and left foot. The hypothesis of using one particular limb more than another was mentioned (Robins et al., 1998). For example, in a study conducted in White Stork, it was reported that there was no significant difference between the digit ratios between the right and left foot (Kazimirski et al., 2019).
       
In this study, it was investigated whether there is a shape difference between the genders by using geometrical analysis. Although it is observed that the second finger (D2) looks more inward in males than females, even this difference was found to be statistically insignificant. Geometrical analysis studies on the foot structure of birds have not been encountered. However, there is a geometrical analysis study in cattle (Parés-Casanova et al., 2020) to determine the asymmetries of the fingers. In addition, sexual dimorphism with skull bones was demonstrated in birds using geometrical analysis (Gündemir et al., 2020). In a study conducted in bats, the geometrical analysis of wing shapes was carried out and the shape relationships between the species were examined (Schmieder et al., 2015).

Different bird species have been studied with the ratios of finger lengths to each other and differences between the female and male have been tried to be revealed. These rates provide information about the development of the fingers of males and females. As stated in the literature studies using this method, the effect of hormones is said to have a direct effect on this finger morphometry. In this study, in contrast to the literature information, in addition to finger length ratios, geometric analysis of finger shape caused by body mass pressure was performed. We think that the geometrical analysis of the way birds hit the ground can be a helpful analysis in determining the taxonomy of birds in addition to the gender gap.