1. Introduction
Phasianidae is a family of non-migratory birds belonged to partridges family. One of the well-known species of partridge is Alectoris chukar, a medium-sized bird larger than quails and smaller than pheasants. They are native to Asia, Africa, Europe and the Middle East and belong to the category of gallinaceous birds [1]. 
Knowing the anatomy and histology of the lymphoid tissues is the key to understand the immunology and physiology. Epithelial anlages are the origin of bursa of the Fabricius and thymus. Moreover, mesenchymal anlages will develop into the spleen, lymph nodes and bone marrow [2]. Spleen is regarded as ‘peripheral or secondary’ lymphoid tissues. It filters blood and mediate the immune responses. In addition, phagocytosis of foreign agents, removal of old blood cells, and storage of platelets are other spleen functions [3, 4]. Spleen is round to oval in gallinaceous birds, ducks and psittacines; whereas, spleen in charadriiformes and passeriformes is more elongated. In proportion to the body weight, the spleen is smaller in birds than mammals. Diameter of a normal chicken spleen is approximately one quarter the length of the proventriculus [5]. The trabeculae in the dove [6] and goose [7] spleens were poorly formed and the white and red pulps could not be distinguished from each other as is also the case in the chicken spleen. Review of literature reveals no information about prenatal development of partridge spleen. Therefore, the present study was designed to investigate the anatomy and histology of the spleen of partridge (Alectoris chukar) during their prenatal stages of development.

2. Materials and Methods
Embryos
In this study, 60 fertilized eggs from the healthy Alectoris chukar birds were used. The following steps were performed: At first, very light weight (small) and very heavy (large) eggs were excluded; The eggs were kept for 6 days at 15°C; The eggs were disinfected with formaldehyde gas and incubator supplied with 55% relative humidity at 37.5°C (relative humidity in 3 final days was 60% at 36.5°C); Then, four partridge eggs were randomly used of incubation per day. Eggs were opened and the spleens of Embryonic Day (ED) 10 to 22 were dissected by dissection microscope. In the present study, a total of 52 partridge embryos were used.
Evaluations of morphology were carried out in accordance with the guidelines laid down by the National Institute of Health (NIH) in the USA and in accordance with Iran Veterinary Council (IVC) regulations.

Tissue preparation for histomorphological studies
The spleen samples were fixed in Bouin’s solution. After tissue processing (dehydration, clearing, infiltration), spleens were embedded in paraffin (52°C-58°C). Serial sections, 5 to 6 μm thickness, were cut. After being deparaffinised and hydrated, the sections were stained with method of Hematoxylin and Eosin (H&E) for histomorphological studies. The figures were prepared using digital Dino-Lite lens and Dino-capture 2 software.

3. Results
Microscopic studies
In this study, the defined structure was not observed in the partridge embryos before the 10th day. Histological evaluation of the spleen at ED 10 revealed that the repertoire of undifferentiated mesenchymal cells act as a cellular basement. In this stage, squamous mesothelial cells were seen around of the spleen. They will probably be the precursor cells of splenic capsule (Figure 1a). More proliferation of undifferentiated mesenchymal cells was seen at ED 11 and 
 

 

12. At ED 12, the capsule of spleen was observed as a thin and single layer of the mesothelial cells. The presence of abundant sinusoids was one of the prominent features of the spleen development at this time. Macrophages were observed beneath the endothelium of sinusoids for the first time at ED 12 (Figure 1b). The early distinction between the red and white pulp had been obvious at ED 14. Red pulp with sinusoids along with abundant red blood cells was observed. The presence of the large blood vessels was one of the prominent features at this stage (Figure 1c). At ED 16, the development of splenic capsule was seen completely. The splenic capsules were formed by mesothelial cells multilayer (Figure 1d). With aging, final structure of the spleen was appeared. In partridge embryos, trabeculae had not been obvious in the spleen. The central artery was seen in white pulp of the spleen at ED 20 (Figure 2a). Reticular cells with large euchromatin nucleus and specified nucleolus was observed in the white pulp (Figure 2b). At ED 22, there were specified Billroth cords in the spleen (Figure 2c). Also, trabecular artery was observed in spleen of the partridge embryos at this time (Figure 2d).

Macroscopic studies
Macroscopically, spleen was not observed at ED 10–13. It can be seen as a light red bud at ED 14 or 15. Furthermore, as a pear-like light red, spleen showed clearly with 1 mm of length which lies beside the left lobe of liver between the 
 

 

proventriculus and gizzard at ED 16. At this time, spleen of the partridge was enclosed by a thick capsule (Figure 3).

4. Discussion 
The spleen is the major secondary lymphatic organ in body, which involves in filtering the blood and mounting immune responses against blood antigens. Erythropoiesis is a major function of the fetal spleen [8]. The present study revealed that spleens were covered with mesothelium at 10th to 12th days of incubation in partridge spleen. Although it has been reported that capsule was formed at the 10th to 14th days of incubation in native chickens (Gallus domesticus) of Bangladesh [3] and spleen was covered with mesothelium at the 10th day of incubation in quail [5]. On day 12 of incubation, abundant venous sinusoids were lined with endothelial cells and red blood cells were observed in the sinusoids. Macrophages were seen beneath the endothelium of sinusoids. In contrast to quail, granulopoiesis and erythropoiesis could be activated on the 12th day of incubation in partridge spleen, this result is in agreement with findings of Ogata and associates [9]. It has been reported that the erythropoiesis and granulopoiesis began before the 10th day of incubation in quail [5]. 
On day 14 of incubation, the large blood vessels were seen and capsule was observed in two layers in partridge spleen. Trabeculae were not observed on the 14th day, but there is relatively loose connective tissue around some large blood vessels. It has been reported that trabeculae is formed in native chickens (Gallus domesticus) of Bangladesh on the 14th day of incubation [3]. Islamkhan et al. showed that broiler spleen is enclosed by a thick capsule and a few number of trabeculae. [10]. On the 15th day of incubation, walls of the trabecular were quite distinct in quail spleen [5]. Red pulp and white pulp were distinctive and cells were proliferated in white pulp on the 14th day of incubation in partridge spleen. These results are inconsistent with findings of Seres and associates [4]. They reported that white pulp was appeared as diffuse lymphatic tissue; whereas, the splenic red pulp com
 

 

posed of venous sinuses and anastomosing cord of reticular cells, macrophages, lymphocytes and blood cells [11]. The white pulp in indigenous ducklings of Bangladesh spleen is a normal site of the network of reticular cells and reticular fibers, with a number of lymphocytes and plasma cells [12]; these results are in agreement with our study results. 
In consistent to quail, sinusoids were grown and greater influxes of blood cells were observed on the 16th day of incubation in partridge spleen [5]. In the present study, large artery and vein in hillus of spleen and reticular cells with euchromatin and prominent nucleolus were seen on the 18th to 20th day of incubation. The central artery was covered by a layer of squamous cells, for the first time. Splenic cords were formed in white pulp on the 21st to 22nd day of incubation. At this time, the capsule is composed of dense irregular connective tissue, which is enclosed by mesothelium cells. The cords of Billroth (splenic cords) are found in the red pulp. All results of the present study agree with the results of other studies in the last days [4, 5]. In the present study, spleen was not seen macroscopically until ED 13. It was observed at ED 14 or 15 and clearly lies between the proventriculus and gizzard at ED 16. Macroscopic observations of the current study are similar to the study of Akter and associates [11].
In conclusion, this study constitutes the first study of histogenesis and histological spleen performed in prenatal partridge. It is expected that the results of our research help further studies on prenatal birth lymphoid organs.

Acknowledgments
The authors wish to express their gratitude to the research Council of Science and Research Branch, Islamic Azad University of Tehran, for their financial support. The authors would like to thank Dr. Hamid Reza Moradi for his help in editing this manuscript.

Conflict of Interest
The authors declared no conflicts of interest.


References