Craniometric Analysis of European Rabbit (Oryctolagus Cuniculus) Breeds to Trace out Intraspecific and Inter Gender Morphometric Variations

The family Liproidae expresses the significant variation of morphometric features at species level. In already available literature on the European rabbit (Oryctolagus cuniculus) the idea is proposed that there can be variation in the morphometric character of different breeds of Oryctolagus cuniculus but there are negligible scientific research base evidences for it. So in this study interbreed craniometric comparison of the European rabbit is carried out to trace out their intergender and intraspecific morphometric variations. The study material consists of 32 sexually mature rabbits belong to two breeds of European rabbit that are New Zealand rabbit (eight males and eight females) and American Dutch rabbit (eight males and eight females) collected from different districts of the Punjab, Pakistan. There were 29 different measurements taken from the skull and mandible of both breeds by using digital and manual verniar calipers. The results of this study have revealed that there is significant interbreed variation in the nasal length measurement of the New Zealand and American Dutch rabbit. This significant interbreed difference in nasal length is observed in both female as well as male rabbits (p=0.0059 and 0.0069 respectively). The current study also depicts that inter gender craniometric variations exist in the New Zealand as well as American Dutch rabbit. The standard deviation of the foramen magnum index of the both included breeds is comparatively higher than all the other included morphometric features. This study provides the baseline data on interbreed craniometric variations in the Oryctolagus cuniculus.


INTRODUCTION
The European rabbit is the rodent animal from family Liproidae (Demirsoy et al., 1992). The animals of family Liproidae are differ from that of family Octinidae (pikas) in that they have hind legs (longer than pikas), elongated ears and short, furry tail. Rabbit became popular as a pet because of its wide usage in research studies. Rabbits are frequently in use as a model in laboratories because they are mild and easily to handle and breed. Rabbits have been used as an experimental model for various human diseases, like cardiovascular disease, cancer and ADIS (Acquired Immunodeficiency Syndrome). Rabbit is also used as a bioreactor for the production of protein in pharmaceutical industries. The rabbit is the breed of choice for polyclonal antibody production. Rabbit is commonly used for testing toxicity and safety of substance such as drugs, chemical and medical devices. In past rabbit were used for the pregnancy testing.
There are some preceding morphometric studies implemented on the skulls of various animal species (Onar et al., 2002;Onar et al., 2005;Olopade et al., 2005;Uddin et al., 2013;Çakır et al., 2012). Even though there are also morphometric studies conducted on rabbits (Özkan, et al., 1997;Kahvecioğlu et al., 2000;Abreu et al., 2006). Some previous study show that the difference in the skull measurement is caused not only due to difference between the species and genera but also the age and gender difference between different individuals of the same species (Gürbüz et al., 2015). The comparative macro anatomic studies in rabbits conducted at gender level are limited (Taylor et al., 1977;Özkadif, 2011;Akbulut, 2014). In spite of the role of European rabbit (Oryctolagous cuniculs) in the experimental medical science all the previous inter gender craniometrics studies are at only the species but not at the breed level. As per the American Rabbit Breeders Association (ARBA) there are 48 known breeds of Oryctolagous cuniculs. This shows that the inter gender craniometric data should also be available to the zoologist and veterinary sciences researchers as a primary information on the breeds of rabbits but no such data is available up to best of our knowledge. This study is planned to conduct the inter gender craniometric analysis in two different breeds of Oryctolagous cuniculs that are New Zealand rabbit and American Dutch rabbit.
"There should be recognized that there is a great variety in shape and structure of rabbit skulls depending on breed." This statement is given in Meredith (2007) but no any craniometric comparative analysis has been conducted in any of the 48 known breeds of rabbits.
Another aim of our study is to fill up this gap by evaluate the interbreed variation of skull morphometric features by using two different breeds of rabbits including New Zealand rabbit and American Dutch rabbit. The studied samples belong to the New Zealand rabbit breed are of two types; pure white in color with red eyes and pure black in color with black eyes. The New Zealand rabbit exist in five different colors according to ARBA. The two colors used in current study are common in Pakistan and preferred for laboratory use due to their suitable size and docile and calm nature. On the other hand, the second breed used in this study is American Dutch rabbit recognized by their beautifully patched body.

Study Material
The specimens selected for the research purpose are elected from different villages and farms of the three districts of the Punjab, Pakistan; Okara, Sahiwal and Kasur. In the collected rabbits from these areas, are the animals that have the weight less than one kilogram or appearently not healthy were excluded from the study. One of the pragnent female rabbit was also there in the colletion and was excluded. The 32 sexually mature rabbits were selected for the current craniometric analysis. In these 32 rabbits; 16 belong to New Zealand rabbit breed (eight male and eight female rabbits) and 16 belong to American Dutch rabbit breed (eight male and eight female rabbits). Google lens and ARBA website are used for identification of breed of rabbits. Most of the specimens were collected from different villages, so the feed of these rabbit are grasses, herbs, fruits and vegitables.

Craniometric Analysis
The rabbits having age of 6 to 8 month and body weight between 1-1.5 kg were used for the skull prepration. Slaughtering is the process used for collecting skulls of rabbits. After removing meat and gluteus material from the skull, classical steeping process was applied. The skull was prepared by boiling it in calciun carbonate and calcium bicarbonate and after boiling, the skulls were dunked in hydrogen peroxide (H 2 O 2 ) to make them more apropriate for craniometric analysis. In this study 23 different measurement are taken from the skull (cranial and maxillary) of the New Zealand and American Dutch rabbit according to the morphometric features given in (Özkadif et al., 2016;Gürbüz et al., 2015;Monfared, 2013) and six measurements of mandible of New Zealand and American Dutch rabbit according to Pintur et al., (2014). All measurements are in mm and were taken by digital and manual Varner calipers. Indexes were calculated by using the method introduced by Onar et al., (2001).

Skull Measurements
L1. Skull length: From the dorsal lateral nasal cartilages to the external occipital protuberance, L2. Nasal length: Distance from the central point of the frontonasal sutura to the dorsal lateral nasal cartilages, L3. Cranial length: Distance from the central point of the frontonasal sutura to the external occipital protuberance, L4. Skull width: Distance between two zygomatic arches, L5. Cranial width: Distance between two external auditory meatus, L6. Nasal width: Maximum distance across the nasal bones or maximum distance between the nasomaxillary sutures, L7. Facial length: Distance from the frontonasal structure to the centre of the incisive bone, L8. Facial width: Distance between the caudal extents of the orbital rims, L9. Foramen magnum height: The distance between the midpoints of the dorsal ventral rims of the foramen magnum, L10. Foramen magnum width: The maximum distance between the two occipital condyles, L11.

Statistical Analysis
Standered daviation and Mean value of the measurement were analysed and Independant t-test was applied to determine the level of significance of the difference of the craniometric measurements between gender and breeds by using SPSS (20.0 version) programm.

RESULTS
The table 1 illuminates the standered daviations,mean value and level of significance of the skull of New Zealand and American Dutch female rabbit in which the nasal length of New Zealand female is 32.04±1.16 and female of American Dutch is 26.52±3.7 his difference in the nasal length of the female of the both included breeds is statistically significant (p < 0.05).

DISCUSSION
The current comparative analysis of measurements in the skull size of male and female rabbits of different breeds indicates a greater degree of variations in skull size in the different breeds. While there are no significant interbreed differences in the mandible morphometric features of female and male rabbits of the included two breeds. This depicts that the mandible measurements are almost same but interbreed difference is there in the skull of the New Zealand and American Dutch rabbit. The results of our measurements (table 3) indicate that the significant differences are present in the basal length, dental length (postdentaleprosthion) and largest nasal length between the male and female of the New Zealand rabbit. In this study the skull length and nasal length are matched with the result of Özkadif et al., (2016) rabbit craniometrics analysis and skull length, nasal length observed in current study are also matched with the results of Gürbüz et al.,(2015).    Length from angle to tip and length of cheek teeth row show statistical significant difference on the mandible portion of the New Zealand rabbits. The table 4 elucidates that skull length, skull width, foramen magnum height, diastema length, maximum neurocranium width and skull width (distance between temporal fossae) values have statistically significant difference between male and female of American Dutch rabbit ( 0.05). Likewise, the values of mandible portion of American Dutch rabbits provide a strong evidence about the skull size difference in males and female rabbits (table 4) in which the difference in the length aboral border of the alveolus of M3-Infradentale to tip (excluding incisors) is statistically significant between the mandibles of the males and females of American Dutch rabbit breed.
In this study we also have the statistical results for difference of skull index, cranial index, nasal index, facial index and foramen magnum index value (table 5, 6, 7 and 8). Our results are different with the previous studies. In previous study skull index Özkadif et al., (2016) and facial index Gürbüz et al., (2015) have statistical significant difference but in our study there is no statistically significant difference present at gender level in both New Zealand rabbit (table 7) as well as American Dutch rabbit (table 8) but in our results cranial index have statistically significant difference at breed level in the male rabbits (table 6). These differences in the current result from the craniometric results of the other studies on the rabbits might be due to difference in the breed of the rabbits included in the study as the breeds of the rabbits are not mentioned in these studies.
The skull of female rabbit is bigger than male as per the already available information in the literature, Özkadif et al. (2016) and Gürbüz et al., (2015) but in our study some craniometric values of female are bigger than male and some value of male are bigger than female. The facial length (40.31±2.02 mm) for the male of New Zealand rabbit are bigger then facial length (38.40±4.96 mm) of female. Similarly, the skull length for male (78.77±3.16 mm) of American Dutch rabbit is bigger than the skull length (70.96±7.89 mm) of female.

Graphical Representation
Graphical representation of the current inter gender and interbreed craniometric comparison (figures 5, 6, 7 and 8) and indexes of the measurements comparison (figures 9, 10, 11, 12) shows that the foramen magnum index has the highest standard deviation as compare to all other measurements used in this study. This indicates that there can be high variation in the foramen magnum index values at the individual level in the New Zealand as well as American Dutch rabbit.

C R A N I O M E T R I C A L VA L U E S O F A M E R I C A N D U T C H R A B B I T S
Female Male

CONCLUSION
This study gives a research based evidence for the existence of craniometric variations in two breeds of rabbit that are the New Zealand and the American Dutch rabbits. This study is limited to these breeds due to the unavailability of other rabbit breeds. This is a baseline finding on interbreed craniometric variations in rabbits so this same study is recommended to be replicated on the different other breeds of Oryctolagous cuniuclus depending on their availability. This was not clear previously that the inter gender variations exist in specific morphometric features in all rabbit breeds or not. This study has cleared this confusion as the results of current study has shown that the craniometric features having significant inter gender variations are different in the New Zealand and American Dutch rabbit. This indicates that the features having inter gender variations can be different in different breeds of rabbits.

Female
Male